Disrupting the physical interaction between serotonin transporter and soluble guanylate cyclase produces a fast–acting antidepressant activity

Disrupting the physical interaction between serotonin transporter and soluble guanylate cyclase produces a fast–acting antidepressant activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Disrupting the physical interaction between serotonin transporter and soluble guanylate cyclase produces a fast–acting antidepressant activity Xingyu Huang, Chan Li, Jiayan Lin, Xintong Zhang, Yanhong Xu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8689963/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Major depressive disorder (MDD) is a refractory neurological disorder often linked to dysregulated 5–hydroxytryptamine (5–HT) neurotransmission. Conventional selective serotonin reuptake inhibitors (SSRIs) face criticism due to their delayed therapeutic onsets and severe adverse effects. The present study aims to illustrate the molecular mechanism by which serotonin transporter (SERT) is regulated by its physical interaction with soluble guanylate cyclase (sGC) for exploring a new therapeutic target with a novel mechanism of action. Methods RBL-2H3 cells and synaptosomes isolated from the dorsal raphe nucleus (DRN) were employed to investigate the SERT–sGC association under physiological conditions or in response to various treatments by using the molecular and cellular approaches. The functional consequences of the SERT–sGC interaction were assessed by biotinylation of membrane proteins and 5–HT uptake assay. GST-tagged intracellular fragments were produced to map the structural motif of SERT responsible for the interaction with sGC and the forth internal loop (IL4)–mediated SERT–sGC association was further confirmed by mutagenesis. Finally, a peptide to dissociate SERT from sGC was synthesized and its antidepressant activity was evaluated with chronic unpredictable mild stress (CUMS) mouse models. Results A stable SERT–sGC complex was identified. Augmentation of the SERT–sGC interaction decreased SERT cell surface expression by interfering with its trafficking, thereby reducing 5–HT uptake. The specific SERT–sGC association was mediated by the IL4 motif with a unique structural folding in SERT and exerted a unilateral modulation of SERT without affecting sGC. Notably, the SERT–sGC interaction was altered in response to treatment of a variety of susceptibility factors in both cells and animal models. Two hours after administration, SERT–IL4 peptide reversed CUMS–induced enhancement of the SERT–sGC interaction and normalized 5–HT neurotransmission from the DRN to hippocampus, exerting a fast-onset antidepressant activity. Conclusion This study revealed the mechanism by which SERT is under the regulatory control by the SERT–sGC association and provided insights into a novel target toward the SERT–sGC interaction in developing rapid–onset agents in the treatment of MDD. Serotonin Serotonin transporter Soluble guanylate cyclase Regional regulation Antidepressants Rapid-onset antidepressants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Serotonin (5–hydroxytryptamine, 5–HT) signaling plays a central role in the modulation of mood, cognition, appetite, and motor behavior. It is generally acknowledged that abnormality in 5–HT signaling is a major risk in the pathophysiology of several psychiatric disorders, including major depressive disorder (MDD) ( 1 – 3 ). Serotonin transporter (SERT) precisely regulates synaptic 5–HT signaling through the reuptake of 5–HT after its release from presynaptic neurons. Notably, agents that specifically inhibit SERT, so–called selective serotonin reuptake inhibitors (SSRIs), are widely used to treat MDD and other psychiatric disorders ( 4 – 6 ). However, these conventional antidepressants have many limitations ( 7 – 9 ), supporting the development of fast–acting and more effective agents with a novel mechanism of action. Multiple signal transduction pathways have been demonstrated to modulate 5–HT signaling by regulating SERT subcellular localization or catalytic function ( 10 – 14 ). Thus, understanding of these signal systems and molecular mechanisms underlying SERT regulation has recently become a major research focus ( 15 – 17 ). Evaluation of physiological significance and clarification of the specificity of key molecules involved in SERT regulation may provide insights into potential drug targets in the treatment of 5–HT–linked psychiatric disorders. The cGMP signal cascade is one of the most striking transduction pathways in SERT regulation because it has been indicated to be implicated in the pathophysiological states of several psychiatric disorders ( 13 , 18 – 20 ). Interestingly, several components in the cGMP signal cascade, such as neuronal nitric oxide synthase (nNOS) and cGMP–dependent protein kinase (PKG), were previously shown to form complexes with SERT, respectively ( 21 , 22 ). Formation of these regulatory complexes was proposed to be effective locally for production of the second messengers NO and cGMP so as to rapidly activate cGMP signaling in SERT phosphorylation ( 23 ). However, these individual physical associations by themselves were also shown to exert specific effects on SERT activity ( 12 , 21 ). For example, enhancement of the SERT–nNOS association was indicated to reduce SERT cell surface expression and subsequently to decrease 5–HT uptake ( 21 ). Furthermore, disruption of the SERT–nNOS interaction has recently been demonstrated to produce a fast onset antidepressant activity in mice ( 24 ). Soluble guanylate cyclase (sGC), another indispensable component in the cGMP signal cascade, is a heterodimeric protein consisting of α and β subunits and catalyzes synthesis of the messenger cGMP upon binding of NO to a prosthetic heme group in sGCβ1 subunit ( 25 , 26 ). sGC is prevalently expressed in the somatodendritic compartment of neurons in most brain regions and plays an important role in neuroplasticity and memory formation ( 27 – 29 ). In the present study, we focus on sGC to establish the molecular basis and functional consequence for the interaction between SERT and sGC as well as biological responses of the SERT–sGC association to the pathophysiological factors of psychiatric disorders. Because SERT is predominantly expressed in serotoninergic neurons in the dorsal raphe nucleus (DRN) ( 24 , 30 ), its specific localization provides an opportunity to selectively manipulate the SERT–sGC interaction in the DRN for modulating 5–HT signaling in other brain regions, such as hippocampus that plays a major role in modulating mood ( 31 ). Our results may lead to the development of a novel drug target in the treatment of 5–HT–linked mental disorders. Materials and Methods Plasmids The lentiviral plasmid (Lenti–EF–1α–SERT–BSD) encoding C–terminally Flag–tagged SERT for generating stable cell line was constructed using the Mut Express II Kit (Vazyme). The plasmids for SERT, DAT, NET, sGCα1 and sGCβ1 in pcDNA3.1 were from Dr. Gary Rudnick’s Laboratory at Yale University School of Medicine. The plasmids for SERT mutants, SERT–IL1, and SERT–IL4 in pcDNA3.1 were generated by PCR amplification of the sequences using the Mut Express II Fast Mutagenesis Kit V2 (Vazyme) and confirmed by full–length DNA sequencing. The prokaryotic expression plasmids were generated by amplifying the sequences encoding N–terminally GST–tagged cytoplasmic regions of SERT and inserting them into an E. coli expression vector pGEX under the control of the tac promoter using the ClonExpress II One Step Cloning Kit (Vazyme). Immunocytochemistry Immunocytochemistry was performed according to a previous protocol ( 22 ). Briefly, cells grown on circular poly–D–lysine–coated coverslips in 12–well plates were fixed with 4% paraformaldehyde at room temperature for 20 min, permeabilized by 0.1% Triton X–100, and blocked with 10% goat serum, followed by application of a primary antibody overnight at 4°C. The primary antibodies used were rabbit anti–SERT (Synaptic Systems, 1:500), mouse anti–sGCβ1 (Santa Cruz, 1:50), mouse anti–sGCα1 (Santa Cruz, 1:50), rabbit anti–calnexin (Huabio, 1:500), and mouse anti–calnexin (Huabio, 1:500). In the next day, cells were treated with a secondary antibody conjugated with either fluorophores Alexa Fluor 488 or Alexa Fluor 594 at room temperature for 1 h. Immunofluorescence images were captured using a LSM 900 confocal microscope (Zeiss) and quantified using Image J. Fluorescence intensity correlation quotient (ICQ) values were calculated as evidence for dependent staining or colocalization when the ICQ value falls between 0 and + 0.5. Independent staining or lack of colocalization was indicated when ICQ values were not different from 0 or were between 0 and − 0.5. Co–immunoprecipitation and pull–down experiments Co–immunoprecipitation and pull–down experiments were carried out as described previously ( 32 , 33 ). Briefly, cell lysates in RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 1% Triton X–100) were centrifugated at 15,000 x g for 20 min at 4°C and the resulting supernatants were incubated with anti–Flag agarose gel, Ni–NTA beads, or antibodies such as anti–SERT (Synaptic Systems, 1:1000), anti–sGCα1 (Santa Cruz, 1:200) or anti–sGCβ1 (Santa Cruz, 1:200), with protein A/G agarose beads, respectively, overnight at 4°C with gentle rotation. After washed three times with ice–cold RIPA buffer, proteins bound to the beads were eluted into 100 µL SDS–PAGE sample buffer. Escherichia coli BL21 (DE3) cells were transformed with an expression plasmid for a GST–tagged SERT cytoplasmic region (GST–SERT–X, X refers to a cytoplasmic region of SERT) and cultured in 25 mL LB broth at 37°C. When the OD 600 reached to 0.6–0.8, 1 mM IPTG was added to induce expression of GST–tagged SERT–X and the cells were further grown at 16°C overnight. IL1, IL3, IL4, IL5, N86, C36, or C15, transformed with a plasmid encoding N–terminally GST–fused SERT–IL1, IL3, IL4, IL5, N–terminal region comprising 1–86 residues, C–terminal tail comprising 36 residues from 594–630, or C–terminal region comprising 15 residues from 615–630, respectively. The bacterial cells were lysed in PBS buffer using a combination of lysozyme and a gentle sonication step, and the supernatants of cell lysates prepared from a centrifugation at 15,000 x g for 20 min at 4°C were incubated with 30 µL of glutathione agarose beads at 4°C for 4 hours. After washed three times with cold PBS, the beads were further incubated overnight at 4°C with 500 µL of cell lysates prepared from 2 x 10 6 HEK–293 cells transiently transfected with an expression plasmid, pcDNA3.1–sGCβ1 or pcDNA3.1–sGCα1. After washing 4 times with PBS, proteins bound to the beads were eluted by incubating with 100 µL of 50 mM reduced glutathione in PBS buffer. [H]5–HT or APP uptake assay [ 3 H]5–HT (27.1 Ci/mmol, PerkinElmer) uptake assay was performed by incubating cells expressing SERT or synaptosomes isolated from the DRN of mice with 20 nM [ 3 H]5–HT in KRH buffer containing 20 mM HEPES, pH 7.4, 120 mM NaCl, 1.3 mM KCl, 2.2 mM CaCl 2 , 1.2 mM MgSO 4 , and 0.1% (w/v) glucose at room temperature for 10 min, and [ 3 H]5–HT accumulation into cells or synaptosomes was measured by a MicroBeta2 microplate counter (PerkinElmer), as described previously ( 34 ). For kinetic analysis, cells expressing SERT or its mutants were incubated with 5–HT at various concentrations generated by adding unlabeled 5–HT to a constant concentration of [ 3 H]5–HT. Nonspecific [ 3 H]5–HT uptake was determined in the presence of 100 µM fluoxetine. For APP + uptake assay, cells expressing SERT, DAT, or NET were incubated with 2 µM APP + at room temperature for 5 min, and the extent of APP + accumulated in the cells was measured by fluorescence spectrometry with an Infinite 200 Pro microplate reader (Tecan) as described previously ( 35 ). Nonspecific APP + uptake was determined in the presence of 100 µM fluoxetine, GBR-12909, or desipramine, respectively. Cell surface biotinylation Cell or synaptosome surface biotinylation was performed using a membrane–impermeant biotinylation reagent sulfo–NHS–SS–biotin, as described previously ( 36 ). In brief, cells expressing SERT or synaptosomes were labeled with sulfo–NHS–SS–biotin, and biotinylated proteins were captured by streptavidin–agarose beads. After washed, biotinylated proteins were eluted into 100 µL SDS–PAGE sample buffer. Samples were separated on a 10% SDS–polyacrylamide gel and visualized by immunoblot analysis using an eBlot touch imager (eBlot). Microdialysis Microdialysis was performed according to a procedure described previously ( 37 ). Mice were anesthetized with isoflurane and fixed on stereotaxic apparatus. A positioning needle was inserted into the ventral hippocampus (AP = − 2.9 mm, ML = ± 2.8 mm, DV = − 3.6 mm, from bregma) ( 24 ). A probe cannula was implanted to a depth of 3.6 mm using a probe holder. Dental cement was prepared by mixing dental powder with water to fix the probe cannula. After dental cement was solidified, the holder was removed. Mice were singly housed and allowed to recover for 1 day before microdialysis. On the day of dialysis, a microdialysis probe was inserted through the guide cannula. The probe was perfused at a constant flow rate of 1.0 µL/min with an artificial cerebrospinal fluid containing 2.4 mM KCl, 125.9 mM NaCl, 1.1 mM CaCl 2 , 0.85 mM MgCl 2 , 27.5 mM NaHCO 3 , 0.5 mM Na 2 SO 4 , 0.5 mM KH 2 PO 4 , and 0.2 mM ascorbate, pH 7.4. After a 90–min equilibration period, the baseline dialysate samples were collected for consecutive 60 min from all mice. Then, a subset of mice received an intraperitoneal injection of Tat–IL4 (100 µg). Starting 90 min post–administration, additional dialysate samples were collected for 60 min from the Tat–IL4 group. The concentrations of 5–HT in the dialysates were determined by high–performance liquid chromatography (HPLC) with electrochemical detection (Sykam). Separation was performed on a C 18 reverse–phase column (Thermo Scientific, 250 × 4.6 mm) with a mobile phase consisting of 100 mM NaH 2 PO 4 , 0.2 mM ascorbate, 0.74 mM sodium octane sulfonate, 0.027 mM EDTA, 2 mM KCl, and 10% methanol, pH 3.0, at a flow rate of 0.2 mL/min. 5–HT was detected using an electrochemical detector with the electrode set at + 0.70 V. Quantification was performed according to an external standard curve (1, 5, 10, 50, and 100 ng/mL), which was linear over this range of 5–HT concentrations with a correlation coefficient (R²) of 0.99972. Behavioral tests C57BL/6 male mice (6–8 weeks old) were obtained from the Experimental Animal Center at Southern Medical University (Guangzhou, China). Mice were maintained in a standard condition with a 12–hour light/12–hour dark cycle and ad libitum access to diet during the experiments. Animal use and procedure were approved by the Animal Use and Care Committee of Southern Medical University. Synaptosomes were isolated and purified from the DRN regions according to the work described previously ( 14 ). Behavioral tests were conducted after a 4–week chronic unpredictable mild stress (CUMS) procedure ( 38 ). Mice were subjected to three behavioral tests, including sucrose splash test (SST), tail suspension test (TST), and forced swim test (FST), in 3 consecutive days, according to a previous work ( 39 ). For SST, mice were placed in a cage and sprayed with a 10% sucrose solution on their backs. Grooming time was measured for 5 min during 2–6 min. In TST, the tail tips of mice were fixed at approximately one–third of its length, and mice were suspended with their heads facing downward. Immobility time during 2–6 min was measured. In FST, mice were placed in a glass cylinder filled with water (18 cm depth, 24 ± 2°C) to swim for 6 min. Immobility time during 2–6 min was measured. Statistical analysis All data were derived from experiments replicated a minimum of three times. Values are expressed as mean ± SEM. Statistical analysis was performed using one–way ANOVA followed by Tukey’s post hoc tests, and statistical significance was set at p < 0.05. Results SERT associates with sGC to form a stable complex To analyze sGC gene products engaged in SERT regulation, we examined the expression of sGC isoforms in RBL–2H3 cells, which have been used as a cell model for exploring the cGMP signaling–mediated regulation of SERT ( 31 , 40 – 42 ), by using qualitative RT–PCR on total RNA with oligonucleotide primers specific for all sGC subunits (sGCα1, sGCα2, sGCβ1 and sGCβ2). Compared to mice midbrain RNA, where we identified expression of sGCα1 and sGCβ1 as predominant isoforms with a smaller amount of sGCα2 estimated by an additional quantitative RT–PCR (Fig. S1 A), RBL–2H3 RNA only yielded amplification of sGCα1 and sGCβ1 (Fig. S1 B). In the present study, we, thus, focus on the predominant sGCα1/β1 heterodimeric isoforms in the model cells or mice midbrain. To examine the association between endogenous sGC with SERT, we performed co–immunoprecipitation of protein complexes from 1% Triton X–100 extracts from RBL–2H3 cells by using antibodies specific for SERT or sGC (sGCα1 or sGCβ1). Anti–sGCα1 or –sGCβ1 immunoblot demonstrated the existence of sGCα1 or sGCβ1 immunoreactivity in the SERT immunoprecipitates, respectively (Fig. 1 A). Similarly, an additional anti–SERT blot also indicated the presence of SERT immunoreactive bands in the sGCα1 or sGCβ1 immunoprecipitates (Fig. S1 C). These results provided evidence that a detergent \(\:-\) resistant complex of SERT with sGCα1/β1 is formed in RBL–2H3 cells. For subcellular colocalization studies of SERT and sGC, RBL–2H3 cells were probed with antibodies for SERT and sGC (sGCα1 or sGCβ1) under permeabilized conditions. Double labeling experiments revealed evidence of significant colocalization of SERT with either sGCα1 or sGCβ1 throughout the cell soma and plasma membrane (Fig. 1 B). To assess SERT and sGC colocalization, we calculated the intensity colocalization quotient (ICQ), which represents a measure of the extent of correlation of intensity values in space for two separate fluorophores ( 22 ). Both SERT/sGCα1 and SERT/sGCβ1 ICQ values fall significantly above zero (Fig. 1 B), suggesting that a remarkable colocalization of SERT/sGC occurs in both the cell soma and plasma membrane. These results are supported by our co–immunoprecipitation analyses for the subcellular association between sGC and SERT. SERT or sGCβ1 (as a representative subunit of sGC) immunoreactivity was detected in anti–sGCβ1 or anti–SERT immunoprecipitates of both the subcellular membrane and plasma membrane fractions, respectively (Fig. 1 C and S1D). To further examine sGC translocation to associate with SERT in the plasma membrane, we performed transient transfection of HeLa or stable HeLa–SERT cells with a plasmid encoding sGCβ1, respectively. Our immunofluorescence analysis indicated that sGCβ1 immunoreactivity was observed intracellularly, with a pattern similar to that seen for the endoplasmic reticulum (ER) marker calnexin in the host cells lacking SERT expression, consistent with the fact that sGC is a cytosolic protein (Fig. S2A). By contrast, a strong sGCβ1 immunoreactivity was detected in the plasma membrane, while little overlap with calnexin occurred in sGCβ1–transfected HeLa–SERT cells, indicating sGC translocation to the plasma membrane (Fig. S2B). In addition, SERT immunoreactivity was mainly observed in the plasma membrane of the cells co–expressing SERT and sGCβ1 (Fig. S2C), thereby exhibiting extensive overlap with the immunoreactivity of sGCβ1 in confocal images (Fig. S2D). These results support the proposal that the mammalian cells maintain a stable association of sGC with SERT. IL4 in SERT is responsible for the association with sGC To map structural region of SERT responsible for the interaction with sGC, we constructed several prokaryotic expression plasmids that produce various N–terminally GST–tagged cytoplasmic regions of SERT in Escherichia coli cells (except for IL2 because it is too short, Fig. 2 A) and performed pull–down experiments using glutathione affinity beads (Fig. 2 B). Glutathione Sepharose–bound recombinant GST–fused SERT fragments were incubated with total lysates of HEK–293 cells transiently transfected with a plasmid encoding sGCα1 or sGCβ1, followed by elution of GST fusion proteins from the beads by reduced glutathione displacement. Immunoblot analysis using sGC antibodies showed that specific sGCα1 or β1 immunoreactivity was only observed in eluates from the glutathione beads bound with GST–tagged internal loop 4 (IL4) of SERT (Figs. 2 C and S3), indicating that sGC associates with the affinity–purified SERT–IL4 fragment. To confirm the role of IL4 motif in the SERT–sGC association in mammalian expression systems, we performed anti–Flag pull–down experiments with HeLa–SERT (C–terminally Flag–tagged) stable cells transiently transfected with plasmids encoding sGCβ1 or sGCβ1 + IL4, respectively. In comparison to a low immunoreactivity of endogenous sGCβ1 detected with the cells without exogenous sGCβ1 expression, overexpression of sGCβ1 by transient transfection significantly increased sGCβ1 immunoreactive intensity in elutes from anti–Flag agarose beads (Fig. 2 D). On the other hand, co–expression of the SERT–IL4 fragment effectively reduced sGCβ1 immunoreactivity, suggesting that the IL4 fragment competitively displaced SERT from the SERT–sGC complex. The SERT–sGC association decreases SERT transport activity and cell surface expression by interfering with SERT trafficking To examine functional consequences of the SERT–sGC association, we carried out transport assay with HeLa–SERT stable cells transiently transfected with plasmids encoding sGCβ1, sGCβ1 + SERT–IL1, or sGCβ1 + SERT–IL4. Compared to the control cells, exogenous expression of sGCβ1 significantly decreased SERT ability to transport 5–HT (Fig. 2 E). Co–expression of SERT–IL4 but not SERT–IL1 fragment effectively reversed sGCβ1–induced reduction in SERT activity, indicating that SERT–IL4 exerts a specific effect on SERT transport activity. In addition, we investigated SERT–IL4’s influence on the cell surface expression of SERT. As shown in Fig. 2 F, sGCβ1 overexpression had little effect on total SERT expression but significantly reduced SERT expression on the cell surface. Co–expression of SERT–IL4, however, effectively reversed sGCβ1–induced decrease in SERT cell surface expression. The SEC23/SEC24C complex as an essential component of the coat protein complex II has been demonstrated to play a critical role in SERT trafficking by binding with SERT ( 43 , 44 ). To examine the effect of the SERT–sGC association on the interaction of SERT with SEC23/SEC24C, we performed transient co–transfection of HeLa–SERT stable cells to express SEC24C at a constant amount and sGCβ1 with a gradually increasing level. As sGCβ1 expression was increased, SERT activity to uptake 5–HT was decreased, consistent with the proposal that augmentation of the SERT–sGC association reduces SERT cell surface expression (Fig. 2 G). Strikingly, a sGCβ1 expression level–dependent decrease in SERT-Flag immunoreactivity was observed in a His-tagged SEC24C protein complex bound to Ni-NTA beads, suggesting that the SERT–sGCβ1 association inhibits the interaction of SERT with SEC24C. Hence, we assume that enhancement of the SERT–sGC association leads to an impairment of SERT trafficking by decreasing the binding of SEC24C to SERT, thereby reducing SERT expression on the cell surface. The unique SERT–sGC association SERT belongs to the monoamine transporter family, which also includes transporters for dopamine (DAT) and norepinephrine (NET). Figure 3 A shows an amino acid sequence alignment of the IL4s of human monoamine transporters, in which the residues are highly conserved between DAT and NET but less with SERT. Interestingly, there is a potent α–helix–breaking residue, Pro455, in SERT–IL4 but not in either DAT or NET. The high–resolution structures show that the IL4 is packed as a full or partial α–helix in NET or DAT ( 45 , 46 ). By comparison, a pronounced kink that leads to IL4 unwinding is observed in the SERT structures (47) (Fig. 3 B), suggesting the presence of a unique folding in SERT–IL4. To learn if sGC also associates with DAT– or NET–IL4, we performed pull–down experiments by using N–terminally GST–tagged DAT– or NET–IL4 produced in prokaryotic expression systems. Unlike SERT–IL4, neither DAT– nor NET–IL4 was able to associate with sGCβ1 (Fig. 3 C). Additionally, transport assay with a fluorescent substrate APP + for all three monoamine transporters showed that sGCβ1 co–expression remarkably reduced APP + uptake by SERT but not by DAT or NET (Fig. 3 D), suggesting that functional regulation by the sGC–IL4 association is specific for SERT. To further investigate the effects of SERT–IL4 residues on the SERT–sGC interaction, we performed alanine–scanning mutagenesis (except for Ala459), one at a time, and assessed the association between sGCβ1 and individual N–terminally GST–tagged SERT–IL4 alanine mutants by glutathione affinity pull down experiments. As shown in Fig. 3 E, compared to SERT–IL4 WT, six of seven mutants, F454A, P455A, H456A, W458A, K460A, and R461A, exerted significant changes in their ability to interact with sGCβ1; of those, one mutant, P455A, showed a > 3–fold increase in its interaction with sGCβ1, whereas other five mutants exhibited a remarkably reduced interaction with sGCβ1, suggesting that the entire SERT–IL4 region plays a critical role in the SERT–sGC association. We then mutated two residues critical for maintaining the sGC–SERT–IL4 association, His456 or Trp458 by alanine, one at a time, in the C–terminally Flag–tagged full–length SERT and examined the effects of these substitutions on the SERT–sGC association and SERT cell surface expression and transport activity, respectively. Anti–Flag agarose pull–down experiments demonstrated that replacement of His456 or Trp458 with alanine decreased the SERT–sGC association (Fig. 3 F), consistent with those observed with GST–tagged SERT–IL4 mutants produced in our prokaryotic expression systems. Furthermore, although the alanine mutants, H456A and W458A, showed slight decreases in their cell surface expression (~ 87% of SERT WT for H456A and ~ 72% for W458A, respectively), possibly due to the impacts of these mutations themselves, co–expression of sGCβ1 significantly reduced the cell surface expression of SERT WT but had little effect on these mutants (Fig. 3 G). Correspondingly, transport assay indicated that sGCβ1 co–expression significantly decreased transport activity of SERT WT but not H456A or W458A mutant (Fig. 3 H). Our kinetic analysis also showed that sGCβ1 co–expression decreased V max of 5–HT transport with a similar K m for the substrate in WT but had little effect on kinetic parameters in these alanine mutants (Figs. S4A, 4B, 4C, 4D). These results suggest that the SERT–sGC association modulates SERT activity by alternating its subcellular distribution not catalytic function. Furthermore, HeLa cells were transiently co–transfected with equal amounts of cDNAs encoding sGCβ1, His-tagged SEC24C, and SERT or its mutants and then used for examining the effects of the two mutations, H456A and W458A, on the interaction between SERT and SEC24C. The cells were lysed and the resulting lysates were then incubated with Ni–NTA beads. Immunoblot analysis indicated a remarkably increased SERT immunoreactivity in the imidazole elutes with H456A or W458A mutant, compared to that with SERT WT (Fig. 3 I), consistent with our previous observation that sGCβ1 competes with SEC24C to interact with SERT (Fig. 2 G). The SERT–sGC interaction is responsive to susceptibility factors in both RBL–2H3 cells and mice Inflammation and oxidative stress have been demonstrated to be the primary risk factors in the pathology of depression and other psychiatric illnesses ( 48 – 51 ). To learn if the SERT–sGC association is altered by exposure to these susceptibility factors, we examined the SERT–sGC association under the treatment with a potent activator of inflammation, lipopolysaccharides (LPS), pro–inflammatory cytokines, tumor necrosis factor alpha (TNF–α) and interleukin–1 beta (IL–1β), or an oxidative agent, hydrogen peroxide, in RBL–2H3 cells. Exposure to these agents in culture medium led to an increase in 5–HT uptake by SERT, compared to that with the control cells (Fig. 4 A). The enhancement of SERT transport activity is apparently due to an increased SERT expression on the cell surface (Fig. 4 B). Importantly, our co–immunoprecipitation experiments indicated that exposure to these agents impairs the SERT–sGC interaction in RBL–2H3 cells (Fig. 4 C). Then, we asked if the SERT–sGC association influences sGC activity or cGMP–mediated phosphorylation of SERT. To this end, we assessed sGC activity in response to various treatments in the absence or presence of a sGC stimulator, S–nitroso–N–acetyl penicillamine (SNAP), which breaks down spontaneously to produce NO in an aqueous medium, by measuring cGMP production in RBL–2H3 cells. As shown in Fig. 4 D, SNAP stimulated sGC activity to produce significantly increased cGMP, compared to those detected in the absence of SNAP. However, exposure to various agents had little effect on cGMP production either in the absence or presence of SNAP, although these treatments have been indicated to reduce the SERT–sGC interaction in RBL–2H3 cells. Additionally, we evaluated cGMP–mediated phosphorylation of SERT in response to these treatments in RBL–2H3 cells. Compared to the control, no statistical difference in phosphorylation level of SERT–Thr276 was detected under any treatment tested (Fig. 4 E), supporting that the SERT–sGC association does not alter sGC activity. Moreover, as shown in Fig. 4 F, our results also demonstrated that incubation of RBL–2H3 cells with 5–HT at various concentrations had little effect on cGMP production, indicating that 5–HT transport does not affect sGC activity, a different action from the SERT–nNOS association on sGC activity ( 21 ). Next, we examined the SERT–sGC association in response to stressful events in mice by using a CUMS procedure (Fig. 5 A). Exposure to CUMS for 4 weeks led to depression–like behaviors including a decrease in the grooming time in SST and increases in the immobility time in both TST and FST in our animal models (Fig. 5 C). We then isolated synaptosomes from the DRN of mice and measured 5–HT uptake by somatodendritic SERT in the serotonergic neurons. Compared to the control, CUMS treatment resulted in an impaired 5–HT uptake, reflecting an increased extracellular level of 5–HT in the DRN (Fig. 5 D). Immunoblot analysis for the biotinylated SERT indicated that CUMS exposure caused a significant reduction in SERT distribution on the synaptosomal surface (Fig. 5 E). Co–immunoprecipitation experiments further revealed that the CUMS procedure led to a stronger immunoreactivity of sGCβ1 than that detected with the control group in anti–SERT immunoprecipitates, indicating that the SERT–sGC interaction in the DRN is augmented by the CUMS exposure. Disrupting the SERT–sGC association in the DRN produces a fast–acting antidepressant–like activity in mice We synthesized a N–terminally Tat–fused peptide that links the HIV Tat protein transduction domain to SERT–IL4 (Tat–IL4, YGRKKRRQRRRGGGFPHVWAKR), allowing Tat peptide to deliver the SERT–IL4 into cells, and then examined its effects on 5–HT uptake by incubating HeLa–SERT stable cells transiently transfected with a plasmid encoding sGCβ1 with Tat–IL4. Our results showed that Tat–IL4 treatment significantly reversed sGC–induced decrease in 5–HT uptake, indicating Tat–IL4 effectively dissociated SERT from sGC (Fig. S5). To learn behavioral and functional consequences of Tat–IL4 disruption of the SERT–sGC association, mice were administered by intraperitoneal injection (i.p.) of Tat–IL4 after exposure to a 4–week CUMS procedure (Fig. 5 A). At 2 hours after administration, Tat–IL4 peptide labelled with Cy7.5 fluorophore was detected in the midbrain of mice by in vivo imaging, indicating Tat–IL4 crossed the blood–brain barrier into the brain (Fig. 5 B). Animal behavioral tests showed that a single injection of Tat–IL4 significantly attenuated CUMS–induced behavioral abnormalities in SST, FST and TST 2 hours after administration, exerting rapid onset antidepressant–like effects in our animal models (Fig. 5 C). The DRN synaptosomes were then subjected to various biochemical analyses. Administration of Tat–IL4 remarkably reversed CUMS–induced alterations of 5–HT uptake, SERT distribution, and the SERT–sGC association (Fig. 5 D– 5 F). Furthermore, we measured the extracellular 5–HT levels in the hippocampus by microdialysis combined with HPLC. As expected, exposure to CUMS resulted in markedly reduced hippocampal 5–HT concentrations. By contrast, Tat–IL4 rapidly elevated 5–HT to the levels detected in control mice 2 hours after injection (Fig. 5 G). We also examined the effects of administration of a control peptide Tat–8A (YGRKKRRQRRRGGGAAAAAAAA) on animal behaviors. Compared to CUMS group, a single injection of Tat–8A had little effect on CUMS–induced depression–like behaviors (Fig. S6). Thus, we propose that disruption of the SERT–sGC association by Tat–IL4 led to an increased 5–HT uptake by SERT in the DRN and an increased 5–HT release from the presynaptic never terminals in the hippocampus, thereby exerting a fast–acting antidepressant activity. Discussion In the present study, we identified a stable protein–protein complex formed by the physical interaction between SERT and sGC and revealed the molecular mechanism by which SERT is under a regulatory control mediated by the SERT–sGC complex. Enhancement of the SERT–sGC interaction reduced SERT trafficking, thereby decreasing SERT cell surface expression and 5–HT uptake, and vice versa . Additionally, our results indicated that alteration of the SERT–sGC interaction has little effect on sGC activity, exerting a unilateral modulation of SERT. Furthermore, we demonstrated that a unique structural folding of the internal loop IL4 renders a specificity for SERT to associate with sGC. Most importantly, the SERT–sGC association was shown to be responsive to a variety of susceptibility factors in the pathophysiology of 5–HT–related psychiatric disorders. Thus, we propose a possible scenario for SERT regulation by the SERT–sGC association (Fig. 6 ). Under physiological conditions, SERT is present in a dynamic equilibrium between the association with sGC and dissociation from sGC, which determines SERT distribution on the cell surface. Alteration of the SERT–sGC interaction affects the equilibrium, which, in turn, changes SERT subcellular localization and its ability to uptake 5–HT. The finding of this phenomenon not only promotes our understanding of the pathophysiology of several 5–HT–linked mental diseases but also provides an opportunity to develop novel therapeutic agents targeting SERT regulation mediated by the SERT–sGC association. Indeed, exposure to CUMS increases the SERT–sGC interaction and then reduces SERT cell surface expression and synaptic 5–HT uptake in the DRN, thereby causing a decreased 5–HT level in the hippocampus and behavioral abnormalities in our models. At 2-hour after administration, however, SERT–IL4 peptide rapidly reverses CUMS–induced enhancement of the SERT–sGC interaction and normalizes 5–HT neurotransmission from the DRN to hippocampus, exerting a fast onset antidepressant activity. The conventional SSRI antidepressants inhibit SERT in both the serotoninergic neurons in the DRN and presynaptic never terminals in other brain regions, leading to a non–selective activation of both 5–HT 1A autoreceptors in the DRN and postsynaptic 5–HT 1A heteroreceptors in the hippocampus and cortex ( 52 ). Activation of the DRN 5–HT 1A autoreceptors has been demonstrated to suppress 5–HT release and firing rate of the serotoninergic neurons by its negative feedback mechanism ( 53 , 54 ). Thus, an additional desensitization of 5–HT 1A autoreceptors is required for the antidepressant effects of SSRIs ( 55 , 56 ). The desensitization process has been proposed to be achieved by sustained activation of 5–HT 1A autoreceptors with chronic exposure to SSRIs, thereby leading to the delayed therapeutic effects and other limitations of the SERT inhibitors ( 57 ). On the other hand, specific suppression of 5–HT 1A autoreceptors by reducing synaptic 5–HT level in the DRN can rapidly stimulate the serotonergic neurons activity and 5–HT release into the hippocampus and cortex without the requirement of 5–HT 1A autoreceptors desensitization and then produce fast onset antidepressant effects ( 58 ). SERT is predominantly expressed in the serotonergic neurons in the DRN ( 24 , 30 ), thus allowing us to selectively control 5-HT 1A autoreceptors by modulating SERT subcellular localization in the DRN with minimal effects on 5–HT 1A heteroreceptors in other brain regions. Hence, we propose that agents such as the SERT–IL4 peptide could specifically decrease synaptic 5–HT level in the DRN by disrupting the SERT–sGC interaction and subsequently deactivate 5–HT 1A autoreceptors, thereby exerting fast–acting antidepressant effects without 5–HT 1A autoreceptors desensitization (Fig. 6 ). The SERT–IL4 motif connecting transmembrane (TM) α–helices 8 and 9 is fully exposed to the cytosolic medium ( 59 ), providing an accessibility to sGC for the SERT–sGC interaction. In addition, this internal loop adopts an extended conformation adjacent to an intercellular gate residue Asp–452, which was proposed to control SERT conformational transitions by forming or breaking a salt–bridge with N–terminal Arg–79 ( 60 ). Comparison of SERT structures in several conformations such as outward–open, occluded, and inward–open conformational states showed that the structural rearrangements in the conformational transitions largely involve a hinge–like movement of the unwound TM1a, which leads to a significant shift of the adjacent N–terminal tail containing Arg–79. By contrast, TMs 8 and 9 in a scaffold region are among several α–helices with minimal structural rearrangements during the conformational transitions ( 59 ) (Fig. S7), consistent with the proposal that the IL4 motif is conformationally insensitive and flexible. Our kinetic analysis showed that augmentation of the SERT–sGC interaction decreases 5–HT transport velocity but with a similar affinity for the substrate to that observed in the basal state, indicating that the IL4–mediated association has a profound impact on the SERT subcellular distribution rather than its catalytic function. In addition, the C–terminal residues of SERT, such as Arg–607, Ile–608, and Lys–610, have been demonstrated to participate in the binding of the SEC23/SEC24C complex, which is critical to form vesicles for transporting proteins from the ER to plasma membrane ( 61 ). Our docking indicated that the interaction between sGC and SERT–IL4 forms a spatial barrier that prevents the C–terminal residues from binding with SEC23/SEC24C (Fig. S8), consistent with our experimental results in showing the effect of sGC on the SERT–SEC24C interaction. Hence, we propose that augmentation of the SERT–sGC interaction impairs SERT trafficking to the plasma membrane, thereby resulting in a reduced SERT cell surface distribution. sGC is a heterodimeric hemoprotein generally activated by NO binding ( 62 ) and each subunit contains four domains, heme NO/oxygen (H–NOX) binding domain, Per–Arnt–Sim (PAS) domain, coiled–coil (CC) domain and catalytic (CAT) domain ( 63 ). Our molecular docking indicated that the SERT–sGC interaction mainly occurs in the interface between the SERT–IL4 and sGCα1 pseudo H–NOX domain with sGCβ1 PAS domain in a sGC heterodimer (Fig. S8). The heme molecule only binds to the sGCβ1 H–NOX domain but not to the sGCα1 pseudo H–NOX domain ( 64 , 65 ). In addition, the PAS domain in either sGCα1 or sGCβ1 is believed to act as an anchor to stabilize each subunit in sGC activation ( 66 ). Our results showed that the SERT–sGC interaction does not alter sGC activity. Thus, it is reasonable to assume that the SERT–sGC interaction does not interfere NO binding in the sGCβ1 and activation of the C–terminal cyclase domains in both subunits to synthesize cGMP from GTP. With the addition of sGC, it seems increasingly likely that all of the components of cGMP signal cascade are associated with SERT ( 21 , 22 , 41 , 67 ). However, functional consequences of these individual associations are not same. While the SERT–nNOS association impaired 5–HT uptake by reducing SERT cell surface expression, SERT was also shown to reciprocally affect nNOS enzymatic activity, which, in turn, modulated NO signaling in the central never system ( 21 ). An agent that disrupts the SERT–nNOS association has recently been indicated to produce fast onset antidepressant–like effects by enhancing 5–HT signaling in forebrain circuits ( 24 ), the reciprocal modulation between SERT and nNOS, however, increases the concerns about its off–target effects due to an altered nNOS activity. It was previously proposed that an allosteric conformational change of nNOS caused by 5–HT transport in the SERT–nNOS complex could increase its affinity for calmodulin, thereby elevating nNOS catalytic activity ( 21 ). By contrast, the SERT–PKG association was proposed to have two effects on SERT, alteration of SERT localization and stimulation of SERT phosphorylation ( 11 ). Alteration of SERT distribution was demonstrated to be independent of PKG activity, because a similar effect on SERT subcellular localization was found with catalytically inactive mutants of PKG ( 12 ). Unlike nNOS and sGC, enhancement of the SERT–PKG interaction was shown to increase SERT cell surface expression ( 12 ). In addition, PKG–mediated phosphorylation of SERT was indicated to elevate 5–HT uptake, which, in turn, further stimulated SERT phosphorylation ( 42 ). Subsequent studies showed that phosphorylation and activation of SERT were blocked by a mutation of Thr276 near the cytoplasmic end of TM5 ( 68 ), consistent with phosphorylation of SERT on Thr276. However, the physiological significance and specificity of the SERT–PKG association have not been determined yet. The protein–protein interactions (PPIs) play critical roles in life processes. Abnormal PPIs have been demonstrated to be associated with many human diseases, supporting the development of drug targeting therapeutics for the PPIs ( 69 ). Many protein–protein interaction modulators such as venetoclax, sarilumab, maraviroc, adagrasib, siltuximab, and sotorasib have been approved for clinical treatment of various diseases ( 70 – 75 ). Although the Tat–IL4 peptide was shown to produce a rapid–onset antidepressant activity in our depressive models, it might suffer from several intrinsic drawbacks as a peptide molecule, including limited stability, poor bioavailability, and low selectivity. Development of small molecule or peptidomimetic agents targeting the SERT–sGC interaction is undoubtedly a future strategy to address these limitations. Nevertheless, understanding the molecular mechanism in the functional modulation of a physiologically important membrane transporter by the interaction with its regulatory proteins could have broader implications for the development of new therapeutics targeting its regulation rather than the transporter itself. Conclusions The conventional SSRI antidepressants are plagued by their significant limitations caused by a global elevation of 5–HT level in the entire brain. By comparison, regional regulation by PPIs can achieve precise, rapid, and fine–tuning modulation of SERT function, while avoiding dysregulation of the essential neurological functions in other brain regions. The present study identified a physical interaction between SERT and its regulatory protein sGC. The SERT-sGC association specifically regulated SERT capability to uptake 5–HT by modulating its membrane trafficking. The SERT–sGC interaction was shown to be mediated by SERT–IL4 motif and exerted a unilateral modulation of SERT but not sGC. Most strikingly, dissociating SERT from sGC by the SERT–IL4 peptide produced a rapid-onset antidepressant activity. These findings open up a promising way for future antidepressant development. Targeting the PPI interface rather than the transporter itself represents a paradigm shift from the conventional pharmacological approaches to a novel strategy for the precise control of the serotonergic system. Abbreviations 5-Hydroxytryptamine 5-HT cGMP–dependent protein kinase PKG Chronic unpredictable mild stress CUMS Dopamine transporter DAT Dorsal raphe nucleus DRN Endoplasmic reticulum ER Forced swim test FST Heme NO/oxygen H–NOX High–performance liquid chromatography HPLC Intensity correlation quotient ICQ Major depressive disorder MDD Neuronal nitric oxide synthase nNOS Norepinephrine transporter NET Selective serotonin reuptake inhibitor SSRI Serotonin transporter SERT S –Nitroso– N –acetyl penicillamine SNAP Soluble guanylyl cyclase sGC Sucrose splash test SST Tail suspension test TST Declarations Ethics approval and consent to participate Animal use was approved by the Animal Use and Care Committee of Southern Medical University (approved code: SMUL2021177, approved date: 12 December 2021). Consent for publication Not applicable. Competing interests Authors declare that they have no competing interests. Author information School of Life Sciences, Guangzhou University 230 Outer Ring West Road Higher Education Mega Center, Panyu District, Guangzhou, Guangdong 510006, China Footnotes Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Funding National Natural Science Foundation of China 32371304 and 32071233 (Y–WZ). Guangdong Basic and Applied Research Foundation 2021A1515012067 (Y–WZ). Innovative Research Program for Guangzhou University Postgraduate Students JCCX2024–017 (XH). Author Contribution Conceptualization: XH, Y–WZBench experiments: XH, CL, JLMolecular docking: XZ, YXVisualization: XZ, YXSupervision: Y–WZWriting—original draft: XH, Y–WZWriting—review & editing: CL, Y–WZResources, project administration and funding acquisition: Y–WZ, XH Acknowledgement We thank all members in Zhang’s Lab for their suggestions in preparation of this manuscript. 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Nat Commun. 2021;12:5492. Pierce BG, Wiehe K, Hwang H, Kim BH, Vreven T, Weng Z. ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers. Bioinformatics. 2014;30:1771–3. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx FigureS1.tif FigureS2.tif FigureS3.tif FigureS4.tif FigureS5.tif FigureS6.tif FigureS7.tif FigureS8.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8689963","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":584367266,"identity":"a55dbeb2-2069-4144-b091-2d712c7fafdd","order_by":0,"name":"Xingyu Huang","email":"","orcid":"","institution":"Guangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xingyu","middleName":"","lastName":"Huang","suffix":""},{"id":584367268,"identity":"4884a069-714a-4538-98e9-fa001d7208a7","order_by":1,"name":"Chan Li","email":"","orcid":"","institution":"Guangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Chan","middleName":"","lastName":"Li","suffix":""},{"id":584367270,"identity":"44be94eb-9063-4df8-aae8-981612b03627","order_by":2,"name":"Jiayan Lin","email":"","orcid":"","institution":"Guangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jiayan","middleName":"","lastName":"Lin","suffix":""},{"id":584367272,"identity":"1062eb1b-7e9a-487d-bbb1-aa90efd0c4a2","order_by":3,"name":"Xintong Zhang","email":"","orcid":"","institution":"Guangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xintong","middleName":"","lastName":"Zhang","suffix":""},{"id":584367274,"identity":"10b522ae-a927-405d-af6d-daaccc232f9c","order_by":4,"name":"Yanhong Xu","email":"","orcid":"","institution":"Guangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yanhong","middleName":"","lastName":"Xu","suffix":""},{"id":584367277,"identity":"68036d22-8efa-4d88-b0b1-e3bd0f8b189a","order_by":5,"name":"Yuan–Wei Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYPACCTBiYKhgYDAA0TzEazlDvBaILgbGNiK0GNxIfvaYp8KCQX5287OHX+fV2ptLJDA+eNvGIG+OU0uauTHPGQkGxjnHzI1ltx1ntpyRwGw4t43BcGcDLi0JZtK5bRIMzBJAhuS2Y2xAETZp3jaGBIMDuLSkf5PO/SfBwCYBZEjOOcYD1ML+G7+WHKAtDRIMPBI5ZpIfG2okQLYw49MieeZNmfSfYxI8EhI5ZdIMxw4YGJx52Cw555yE4QYcWviOp2+TnFFTJyc/A8j4UVNnb3A8+eCHN2U28rhsUYCKgyOCmYfhMJBibGCAxixWIN+AxGH8wVCHU+UoGAWjYBSMXAAARFNVgw54aQEAAAAASUVORK5CYII=","orcid":"","institution":"Guangzhou University","correspondingAuthor":true,"prefix":"","firstName":"Yuan–Wei","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-01-25 03:38:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8689963/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8689963/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102277735,"identity":"3fd80972-53cc-4508-bff4-5afca48df95d","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1724752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociation between SERT and sGC heterodimeric subunits.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Co–immunoprecipitation of protein complexes from RBL–2H3 cell extracts with IgG or SERT antibody, followed by immunoblot analysis using anti–sGCα1 (\u003cem\u003eupper\u003c/em\u003e) or anti–sGCβ1 antibody (\u003cem\u003elower\u003c/em\u003e). \u003cstrong\u003e(B)\u003c/strong\u003e Double immunocytochemistry labeling of SERT and sGCα1 (\u003cem\u003eupper\u003c/em\u003e) or sGCβ1 (\u003cem\u003elower\u003c/em\u003e). Scale bars represent 10 μm. Graph shows quantitative analysis of SERT/sGCα1 or sGCβ1 colocalization expressed by the ICQ values. \u003cstrong\u003e(C)\u003c/strong\u003e Subcellular distribution of the SERT–sGCβ1 association. Proteins in the subcellular membrane (SM) or plasma membrane (PM) were separated by biotinylation of RBL–2H3 cells using a plasma membrane–impermeant reagent sulfo–NHS–SS–biotin. Immunoprecipitation of membrane protein complexes was performed with anti–SERT, followed by immunoblot analysis using anti–sGCβ1 antibody, respectively. Data are representatives of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/ad848f88dc1ccbf353189911.jpg"},{"id":102277737,"identity":"60d85ad9-3a4d-4bb6-afeb-7bed17fc8f9a","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3561091,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSERT–IL4 is responsible for the association with sGC. (A) \u003c/strong\u003eSERT topology.\u003cstrong\u003e \u003c/strong\u003eTransmembrane\u003cstrong\u003e \u003c/strong\u003eα–helices and internal loops (ILs)\u003cstrong\u003e \u003c/strong\u003ewere marked with numbers. \u003cstrong\u003e(B)\u003c/strong\u003eSchematic presentation of identifying the internal region of SERT responsible for the association with sGC by GST pull–down assay. \u003cstrong\u003e(C)\u003c/strong\u003e Immunoblot analysis of elutes from glutathione beads incubated with GST-fused SERT fragments and sGCβ1, using anti-sGCβ1 (upper) or anti-GST (lower) antibodies. Ctrl: \u003cem\u003eE. coli\u003c/em\u003e cells expressing GST without IPTG induction; GST: GST induced with IPTG; IL1, IL3, IL4, IL5, N86, C36, or C15 other lanes (IL1, IL3, IL4, IL5, N86, C36, C15): cells expressing the indicated GST-fused SERT fragments with IPTG induction. (\u003cstrong\u003eD, E, F\u003c/strong\u003e) Effects of SERT–IL4 on the SERT–sGC association, 5–HT uptake, and SERT cell surface expression. HeLa–SERT stable cells were transiently transfected with an empty vector or plasmids expressing sGCβ1, sGCβ1 and SERT–IL4, or sGCβ1 and SERT–IL1, respectively. The SERT–sGCβ1 association was assessed by anti-Flag pull-down and immunoblotting with an anti-sGCβ1 antibody (\u003cstrong\u003eD\u003c/strong\u003e, \u003cem\u003elower\u003c/em\u003e). The SERT–sGC association was assessed by normalizing sGCβ1 immunoreactivity to the amount of SERT pulled down by anti-Flag agarose (\u003cstrong\u003eD\u003c/strong\u003e, \u003cem\u003eright\u003c/em\u003e). 5-HT uptake was measured by incubating cells with 20 nM [³H]5-HT at 22 °C for 10 min and quantifying intracellular accumulation. Data are expressed as a percentage of control (\u003cstrong\u003eE\u003c/strong\u003e). SERT surface expression was assessed by normalizing the level of biotinylated (sulfo-NHS-SS-biotin), Flag-tagged SERT to total SERT via immunoblot analysis (\u003cstrong\u003eF\u003c/strong\u003e). \u003cstrong\u003e(G)\u003c/strong\u003e The effect of sGCβ1 expression on the SERT-SEC24C interaction was tested in HeLa-SERT cells co-transfected with His-SEC24C and graded amounts of sGCβ1 plasmid, followed by 5-HT uptake measurement and examination of the interaction via Ni-NTA pull-down. Data represent mean values ± SEM from at least three independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/5c097860b1274b17251ff31d.jpg"},{"id":102277748,"identity":"a5c90591-f7cb-4cbc-ac5a-ea86b7ab1096","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3119066,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe unique SERT–sGC association. (A) \u003c/strong\u003eSequence alignment of the IL4 motifs from human monoamine transporters. Identical or similar residues are highlighted in blue. \u003cstrong\u003e(B)\u003c/strong\u003e Structural superimposition of the IL4 motifs of SERT (PDB, ID# 7LIA), DAT (9EO4), and NET (8Y94). Residues only in SERT–IL4 were shown.\u003cstrong\u003e (C)\u003c/strong\u003e Pull-down assay showing that sGCβ1 associates with GST-fused SERT-IL4, but not with DAT-IL4 or NET-IL4. Eluates from glutathione beads were immunoblotted with anti-sGCβ1 or anti-GST antibodies.\u003cstrong\u003e (D)\u003c/strong\u003e APP⁺ uptake in HeLa cells stably expressing SERT, DAT, or NET, following transfection with increasing amounts of sGCβ1 plasmid. Uptake was measured after 5 min incubation with 2 μM APP⁺ at 22°C. \u003cstrong\u003e(E) \u003c/strong\u003eEffects of alanine-scanning mutagenesis in SERT-IL4 on sGCβ1 binding, assessed by GST pull-down and immunoblotting. Quantitative analysis normalized to the SERT-IL4-WT (\u003cem\u003eright\u003c/em\u003e). (\u003cstrong\u003eF, G, H\u003c/strong\u003e) Effects of H456A and W458A mutations on the SERT–sGC association \u003cstrong\u003e(F)\u003c/strong\u003e, SERT cell surface expression \u003cstrong\u003e(G)\u003c/strong\u003e, and 5–HT uptake \u003cstrong\u003e(H)\u003c/strong\u003e, as described in Fig. 2. \u003cstrong\u003e(I) \u003c/strong\u003eEffects of two alanine mutations on the SERT–SEC24C interaction. HeLa cells were transiently co–transfected with equal amounts of plasmids encoding His-SEC24C, sGCβ1, and SERT–Flag WT or its mutants and then used for pull–down experiments using Ni–NTA beads. Imidazole eluates were analyzed for SERT immunoreactivity with anti–Flag antibody (top panel, \u003cem\u003eleft\u003c/em\u003e). Total cell lysates were used for immunoblotting analysis for expression of SERT, SEC24C, and sGCβ1 in the cells. The SERT–SEC24C interaction was estimated by normalizing SERT immunoreactivity detected in imidazole eluates to total SERT (\u003cem\u003eright\u003c/em\u003e). Data represent mean values ± SEM from three independent experiments. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, compared to WT.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/c1528de0e475b17ab019adf9.jpg"},{"id":102277736,"identity":"ee0f9215-9687-4d7d-b55a-990d06d5fbf9","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2294513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSERT–sGC association under LPS, cytokine, or H₂O₂ challenge in RBL–2H3 cells.\u003c/strong\u003e RBL–2H3 cells\u003cstrong\u003e \u003c/strong\u003ewere incubated with\u003cstrong\u003e \u003c/strong\u003e1 μM LPS, 1.5 nM TNF–α, 1.5 nM IL–1β, or 100 μM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in culture medium at 37 °C for 24 h and their effects on SERT activity\u003cstrong\u003e (A)\u003c/strong\u003e, SERT cell surface expression \u003cstrong\u003e(B)\u003c/strong\u003e, the SERT–sGC association \u003cstrong\u003e(C)\u003c/strong\u003e, sGC activity \u003cstrong\u003e(D)\u003c/strong\u003e, and cGMP–mediated phosphorylation of SERT Thr276 \u003cstrong\u003e(E)\u003c/strong\u003e were then examined. SERT activity, SERT cell surface expression, or the SERT–sGC association was expressed as 5–HT uptake, immunoreactivity of the biotinylated SERT, or sGCβ1 immunoreactivity in anti–SERT immunoprecipitates relative to those measured with the cells without treatment (Ctrl), respectively. For evaluating the effects of these agents on sGC activity, cells were further incubated with or without 100 μM SNAP for an additional 15 min and then lysed. cGMP concentrations in the lysates were measured by ELISA. Phosphorylation of Thr276 in SERT was examined by immunoblot analysis for total cell lysates with anti–pThr276–SERT antibody. Phosphorylation levels of Thr276 were expressed as p–Thr276 SERT immunoreactivity (\u003cem\u003eupper\u003c/em\u003e) relative to that detected in the cells without treatment after normalization to total SERT immunoreactivity (\u003cem\u003elower\u003c/em\u003e). \u003cstrong\u003e(F)\u003c/strong\u003e Effects of 5–HT uptake on cGMP production. RBL–2H3 cells were incubated with various 5–HT concentrations (0 –10 μM) or co–incubated with 10 μM 5–HT and 100 μM fluoxetine at 37 °C for 30 min and then lysed. cGMP concentrations were measured by ELISA. All data represent mean values ± SEM from at least three independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, compared to the control.\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/eae83eaefdf3ad0195128b55.jpg"},{"id":102298090,"identity":"aa23445d-6391-4989-9fca-83da98f1b93b","added_by":"auto","created_at":"2026-02-10 10:30:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1738281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdministration of Tat–IL4 produces a fast onset antidepressant–like activity in mice. (A)\u003c/strong\u003e Schematic illustration of experimental timeline with mice. \u003cstrong\u003e(B) \u003c/strong\u003e\u003cem\u003eIn vivo\u003c/em\u003e imaging of Cy7.5–labelled Tat–IL4 2 hours after i.p. injection (100 μg). \u003cstrong\u003e(C)\u003c/strong\u003e Animal behavioral tests. SST, FST, and TST were conducted with control and CUMS mice 2 hours after a single injection of saline or Tat–IL4 (100 μg), respectively (\u003cem\u003en \u003c/em\u003e= 7 –12). \u003cstrong\u003e(D)\u003c/strong\u003e 5–HT uptake. Transport assay was performed by incubating synaptosomes isolated from the DRN with 20 nM [\u003csup\u003e3\u003c/sup\u003eH]5–HT at 22 °C for 10 min and 5–HT accumulated in synaptosomes was measured. \u003cstrong\u003e(E) \u003c/strong\u003eSERT expression on the synaptosomal surface. Membrane proteins on the synaptosomal surface were biotinylated and the biotinylated SERT was detected by immunoblot analysis with anti–SERT antibody. \u003cstrong\u003e(F)\u003c/strong\u003e The SERT–sGC association in synaptosomes. The SERT–sGC association was assessed by co–immunoprecipitation of the protein complexes with anti–SERT antibody, followed by immunoblot analysis for measuring sGCβ1 immunoreactivity with anti–sGCβ1 antibody. \u003cstrong\u003e(G)\u003c/strong\u003e Extracellular 5–HT levels in the hippocampus. Data represent mean values ± SEM (\u003cem\u003en\u003c/em\u003e ≥ 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/f18be7153c4e28918e033036.jpg"},{"id":102297858,"identity":"bb33ffd7-f20c-4978-8d44-d3cd88d5ac13","added_by":"auto","created_at":"2026-02-10 10:29:26","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6052359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCartoon presentation of the modulation of 5–HT signaling by the IL4–mediated SERT–sGC association. \u003c/strong\u003eIn physiological conditions, SERT subcellular localization is regulated by two competitive SERT–sGC and SERT–SEC interactions, which maintain a dynamic equilibrium of SERT distribution. However, exposure to susceptibility factors breaks the equilibrium by augmenting the SERT–sGC interaction, leading to a reduced SERT cell surface distribution and a decreased 5–HT reuptake in the DRN, which, in turn, impairs 5–HT transmission by activating 5–HT\u003csub\u003e1A\u003c/sub\u003e autoreceptors from the DRN to other brain regions, such as hippocampus and cortex. On the other hand, SERT–IL4 peptide reverses the effects of the SERT–sGC interaction by dissociating SERT from sGC and subsequently by increasing SERT distribution on the cell surface in the DRN, thus resulting in an enhancement of 5–HT transmission by deactivating 5–HT\u003csub\u003e1A\u003c/sub\u003e autoreceptors.\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/30547c8f74cde5945baedfb7.jpg"},{"id":102746037,"identity":"70cfaca5-d8a8-48a2-b0df-bed8bf4ce058","added_by":"auto","created_at":"2026-02-16 08:55:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19666254,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/bab2e2d6-3e85-474a-a434-1ab2ed5abc01.pdf"},{"id":102277739,"identity":"24726ebc-fb71-4ce9-9391-c0e93fa537bd","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3715957,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/1dc3dddd32901e175a4cede6.docx"},{"id":102277746,"identity":"bc8e55b2-d8df-4317-8602-ed041430841e","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3546324,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/479b5e5d70cf703717d62523.tif"},{"id":102277749,"identity":"dadfbb92-7919-4363-9aba-a1bb66f0d2bd","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":8464632,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/dea7c895b6ad13bdb72a95ff.tif"},{"id":102277743,"identity":"0db62733-c6e7-43cd-b5b0-3471c7f21843","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2122284,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/43ca968cd8aa740ae406df17.tif"},{"id":102277745,"identity":"3b395c93-3934-4bd6-9f66-b34424213546","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2368952,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/aee48ba2d8fe1e7b5810264a.tif"},{"id":102277740,"identity":"bee482d9-4798-48b5-a9db-7756b5b40ff4","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":419084,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/89a74836045487e06fc1b881.tif"},{"id":102277741,"identity":"be8214c9-9a12-42e5-9e91-e85058cd8e51","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1184448,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS6.tif","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/5e192f8ec39833482f1bbd6f.tif"},{"id":102277747,"identity":"14c65da2-bd92-4264-bf4c-998dceda505b","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":6830388,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS7.tif","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/4dcfe26d8b99568c78b844f4.tif"},{"id":102277744,"identity":"562c31c1-e565-40cd-92c2-2de7e56d8394","added_by":"auto","created_at":"2026-02-10 06:18:21","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":11969752,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS8.tif","url":"https://assets-eu.researchsquare.com/files/rs-8689963/v1/5f70ccccb79073ab2b9f61ba.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Disrupting the physical interaction between serotonin transporter and soluble guanylate cyclase produces a fast–acting antidepressant activity","fulltext":[{"header":"Background","content":"\u003cp\u003eSerotonin (5\u0026ndash;hydroxytryptamine, 5\u0026ndash;HT) signaling plays a central role in the modulation of mood, cognition, appetite, and motor behavior. It is generally acknowledged that abnormality in 5\u0026ndash;HT signaling is a major risk in the pathophysiology of several psychiatric disorders, including major depressive disorder (MDD) (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Serotonin transporter (SERT) precisely regulates synaptic 5\u0026ndash;HT signaling through the reuptake of 5\u0026ndash;HT after its release from presynaptic neurons. Notably, agents that specifically inhibit SERT, so\u0026ndash;called selective serotonin reuptake inhibitors (SSRIs), are widely used to treat MDD and other psychiatric disorders (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, these conventional antidepressants have many limitations (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), supporting the development of fast\u0026ndash;acting and more effective agents with a novel mechanism of action.\u003c/p\u003e \u003cp\u003eMultiple signal transduction pathways have been demonstrated to modulate 5\u0026ndash;HT signaling by regulating SERT subcellular localization or catalytic function (\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Thus, understanding of these signal systems and molecular mechanisms underlying SERT regulation has recently become a major research focus (\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Evaluation of physiological significance and clarification of the specificity of key molecules involved in SERT regulation may provide insights into potential drug targets in the treatment of 5\u0026ndash;HT\u0026ndash;linked psychiatric disorders.\u003c/p\u003e \u003cp\u003eThe cGMP signal cascade is one of the most striking transduction pathways in SERT regulation because it has been indicated to be implicated in the pathophysiological states of several psychiatric disorders (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Interestingly, several components in the cGMP signal cascade, such as neuronal nitric oxide synthase (nNOS) and cGMP\u0026ndash;dependent protein kinase (PKG), were previously shown to form complexes with SERT, respectively (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Formation of these regulatory complexes was proposed to be effective locally for production of the second messengers NO and cGMP so as to rapidly activate cGMP signaling in SERT phosphorylation (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, these individual physical associations by themselves were also shown to exert specific effects on SERT activity (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). For example, enhancement of the SERT\u0026ndash;nNOS association was indicated to reduce SERT cell surface expression and subsequently to decrease 5\u0026ndash;HT uptake (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Furthermore, disruption of the SERT\u0026ndash;nNOS interaction has recently been demonstrated to produce a fast onset antidepressant activity in mice (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoluble guanylate cyclase (sGC), another indispensable component in the cGMP signal cascade, is a heterodimeric protein consisting of α and β subunits and catalyzes synthesis of the messenger cGMP upon binding of NO to a prosthetic heme group in sGCβ1 subunit (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). sGC is prevalently expressed in the somatodendritic compartment of neurons in most brain regions and plays an important role in neuroplasticity and memory formation (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In the present study, we focus on sGC to establish the molecular basis and functional consequence for the interaction between SERT and sGC as well as biological responses of the SERT\u0026ndash;sGC association to the pathophysiological factors of psychiatric disorders. Because SERT is predominantly expressed in serotoninergic neurons in the dorsal raphe nucleus (DRN) (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), its specific localization provides an opportunity to selectively manipulate the SERT\u0026ndash;sGC interaction in the DRN for modulating 5\u0026ndash;HT signaling in other brain regions, such as hippocampus that plays a major role in modulating mood (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Our results may lead to the development of a novel drug target in the treatment of 5\u0026ndash;HT\u0026ndash;linked mental disorders.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids\u003c/h2\u003e \u003cp\u003eThe lentiviral plasmid (Lenti\u0026ndash;EF\u0026ndash;1α\u0026ndash;SERT\u0026ndash;BSD) encoding C\u0026ndash;terminally Flag\u0026ndash;tagged SERT for generating stable cell line was constructed using the Mut Express II Kit (Vazyme). The plasmids for SERT, DAT, NET, sGCα1 and sGCβ1 in pcDNA3.1 were from Dr. Gary Rudnick\u0026rsquo;s Laboratory at Yale University School of Medicine. The plasmids for SERT mutants, SERT\u0026ndash;IL1, and SERT\u0026ndash;IL4 in pcDNA3.1 were generated by PCR amplification of the sequences using the Mut Express II Fast Mutagenesis Kit V2 (Vazyme) and confirmed by full\u0026ndash;length DNA sequencing. The prokaryotic expression plasmids were generated by amplifying the sequences encoding N\u0026ndash;terminally GST\u0026ndash;tagged cytoplasmic regions of SERT and inserting them into an E. coli expression vector pGEX under the control of the tac promoter using the ClonExpress II One Step Cloning Kit (Vazyme).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunocytochemistry\u003c/h3\u003e\n\u003cp\u003eImmunocytochemistry was performed according to a previous protocol (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Briefly, cells grown on circular poly\u0026ndash;D\u0026ndash;lysine\u0026ndash;coated coverslips in 12\u0026ndash;well plates were fixed with 4% paraformaldehyde at room temperature for 20 min, permeabilized by 0.1% Triton X\u0026ndash;100, and blocked with 10% goat serum, followed by application of a primary antibody overnight at 4\u0026deg;C. The primary antibodies used were rabbit anti\u0026ndash;SERT (Synaptic Systems, 1:500), mouse anti\u0026ndash;sGCβ1 (Santa Cruz, 1:50), mouse anti\u0026ndash;sGCα1 (Santa Cruz, 1:50), rabbit anti\u0026ndash;calnexin (Huabio, 1:500), and mouse anti\u0026ndash;calnexin (Huabio, 1:500). In the next day, cells were treated with a secondary antibody conjugated with either fluorophores Alexa Fluor 488 or Alexa Fluor 594 at room temperature for 1 h. Immunofluorescence images were captured using a LSM 900 confocal microscope (Zeiss) and quantified using Image J. Fluorescence intensity correlation quotient (ICQ) values were calculated as evidence for dependent staining or colocalization when the ICQ value falls between 0 and +\u0026thinsp;0.5. Independent staining or lack of colocalization was indicated when ICQ values were not different from 0 or were between 0 and \u0026minus;\u0026thinsp;0.5.\u003c/p\u003e\n\u003ch3\u003eCo–immunoprecipitation and pull–down experiments\u003c/h3\u003e\n\u003cp\u003eCo\u0026ndash;immunoprecipitation and pull\u0026ndash;down experiments were carried out as described previously (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Briefly, cell lysates in RIPA buffer (50 mM Tris\u0026ndash;HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 1% Triton X\u0026ndash;100) were centrifugated at 15,000 x g for 20 min at 4\u0026deg;C and the resulting supernatants were incubated with anti\u0026ndash;Flag agarose gel, Ni\u0026ndash;NTA beads, or antibodies such as anti\u0026ndash;SERT (Synaptic Systems, 1:1000), anti\u0026ndash;sGCα1 (Santa Cruz, 1:200) or anti\u0026ndash;sGCβ1 (Santa Cruz, 1:200), with protein A/G agarose beads, respectively, overnight at 4\u0026deg;C with gentle rotation. After washed three times with ice\u0026ndash;cold RIPA buffer, proteins bound to the beads were eluted into 100 \u0026micro;L SDS\u0026ndash;PAGE sample buffer.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 (DE3) cells were transformed with an expression plasmid for a GST\u0026ndash;tagged SERT cytoplasmic region (GST\u0026ndash;SERT\u0026ndash;X, X refers to a cytoplasmic region of SERT) and cultured in 25 mL LB broth at 37\u0026deg;C. When the OD\u003csub\u003e600\u003c/sub\u003e reached to 0.6\u0026ndash;0.8, 1 mM IPTG was added to induce expression of GST\u0026ndash;tagged SERT\u0026ndash;X and the cells were further grown at 16\u0026deg;C overnight. IL1, IL3, IL4, IL5, N86, C36, or C15, transformed with a plasmid encoding N\u0026ndash;terminally GST\u0026ndash;fused SERT\u0026ndash;IL1, IL3, IL4, IL5, N\u0026ndash;terminal region comprising 1\u0026ndash;86 residues, C\u0026ndash;terminal tail comprising 36 residues from 594\u0026ndash;630, or C\u0026ndash;terminal region comprising 15 residues from 615\u0026ndash;630, respectively. The bacterial cells were lysed in PBS buffer using a combination of lysozyme and a gentle sonication step, and the supernatants of cell lysates prepared from a centrifugation at 15,000 x g for 20 min at 4\u0026deg;C were incubated with 30 \u0026micro;L of glutathione agarose beads at 4\u0026deg;C for 4 hours. After washed three times with cold PBS, the beads were further incubated overnight at 4\u0026deg;C with 500 \u0026micro;L of cell lysates prepared from 2 x 10\u003csup\u003e6\u003c/sup\u003e HEK\u0026ndash;293 cells transiently transfected with an expression plasmid, pcDNA3.1\u0026ndash;sGCβ1 or pcDNA3.1\u0026ndash;sGCα1. After washing 4 times with PBS, proteins bound to the beads were eluted by incubating with 100 \u0026micro;L of 50 mM reduced glutathione in PBS buffer.\u003c/p\u003e\n\u003ch3\u003e[H]5–HT or APP uptake assay\u003c/h3\u003e\n\u003cp\u003e[\u003csup\u003e3\u003c/sup\u003eH]5\u0026ndash;HT (27.1 Ci/mmol, PerkinElmer) uptake assay was performed by incubating cells expressing SERT or synaptosomes isolated from the DRN of mice with 20 nM [\u003csup\u003e3\u003c/sup\u003eH]5\u0026ndash;HT in KRH buffer containing 20 mM HEPES, pH 7.4, 120 mM NaCl, 1.3 mM KCl, 2.2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1.2 mM MgSO\u003csub\u003e4\u003c/sub\u003e, and 0.1% (w/v) glucose at room temperature for 10 min, and [\u003csup\u003e3\u003c/sup\u003eH]5\u0026ndash;HT accumulation into cells or synaptosomes was measured by a MicroBeta2 microplate counter (PerkinElmer), as described previously (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). For kinetic analysis, cells expressing SERT or its mutants were incubated with 5\u0026ndash;HT at various concentrations generated by adding unlabeled 5\u0026ndash;HT to a constant concentration of [\u003csup\u003e3\u003c/sup\u003eH]5\u0026ndash;HT. Nonspecific [\u003csup\u003e3\u003c/sup\u003eH]5\u0026ndash;HT uptake was determined in the presence of 100 \u0026micro;M fluoxetine. For APP\u003csup\u003e+\u003c/sup\u003e uptake assay, cells expressing SERT, DAT, or NET were incubated with 2 \u0026micro;M APP\u003csup\u003e+\u003c/sup\u003e at room temperature for 5 min, and the extent of APP\u003csup\u003e+\u003c/sup\u003e accumulated in the cells was measured by fluorescence spectrometry with an Infinite 200 Pro microplate reader (Tecan) as described previously (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Nonspecific APP\u003csup\u003e+\u003c/sup\u003e uptake was determined in the presence of 100 \u0026micro;M fluoxetine, GBR-12909, or desipramine, respectively.\u003c/p\u003e\n\u003ch3\u003eCell surface biotinylation\u003c/h3\u003e\n\u003cp\u003eCell or synaptosome surface biotinylation was performed using a membrane\u0026ndash;impermeant biotinylation reagent sulfo\u0026ndash;NHS\u0026ndash;SS\u0026ndash;biotin, as described previously (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In brief, cells expressing SERT or synaptosomes were labeled with sulfo\u0026ndash;NHS\u0026ndash;SS\u0026ndash;biotin, and biotinylated proteins were captured by streptavidin\u0026ndash;agarose beads. After washed, biotinylated proteins were eluted into 100 \u0026micro;L SDS\u0026ndash;PAGE sample buffer. Samples were separated on a 10% SDS\u0026ndash;polyacrylamide gel and visualized by immunoblot analysis using an eBlot touch imager (eBlot).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMicrodialysis\u003c/h2\u003e \u003cp\u003eMicrodialysis was performed according to a procedure described previously (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Mice were anesthetized with isoflurane and fixed on stereotaxic apparatus. A positioning needle was inserted into the ventral hippocampus (AP\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;2.9 mm, ML\u0026thinsp;=\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 mm, DV\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;3.6 mm, from bregma) (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). A probe cannula was implanted to a depth of 3.6 mm using a probe holder. Dental cement was prepared by mixing dental powder with water to fix the probe cannula. After dental cement was solidified, the holder was removed. Mice were singly housed and allowed to recover for 1 day before microdialysis.\u003c/p\u003e \u003cp\u003eOn the day of dialysis, a microdialysis probe was inserted through the guide cannula. The probe was perfused at a constant flow rate of 1.0 \u0026micro;L/min with an artificial cerebrospinal fluid containing 2.4 mM KCl, 125.9 mM NaCl, 1.1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.85 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 27.5 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 0.5 mM Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 0.5 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and 0.2 mM ascorbate, pH 7.4. After a 90\u0026ndash;min equilibration period, the baseline dialysate samples were collected for consecutive 60 min from all mice. Then, a subset of mice received an intraperitoneal injection of Tat\u0026ndash;IL4 (100 \u0026micro;g). Starting 90 min post\u0026ndash;administration, additional dialysate samples were collected for 60 min from the Tat\u0026ndash;IL4 group.\u003c/p\u003e \u003cp\u003eThe concentrations of 5\u0026ndash;HT in the dialysates were determined by high\u0026ndash;performance liquid chromatography (HPLC) with electrochemical detection (Sykam). Separation was performed on a C\u003csub\u003e18\u003c/sub\u003e reverse\u0026ndash;phase column (Thermo Scientific, 250 \u0026times; 4.6 mm) with a mobile phase consisting of 100 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.2 mM ascorbate, 0.74 mM sodium octane sulfonate, 0.027 mM EDTA, 2 mM KCl, and 10% methanol, pH 3.0, at a flow rate of 0.2 mL/min. 5\u0026ndash;HT was detected using an electrochemical detector with the electrode set at +\u0026thinsp;0.70 V. Quantification was performed according to an external standard curve (1, 5, 10, 50, and 100 ng/mL), which was linear over this range of 5\u0026ndash;HT concentrations with a correlation coefficient (R\u0026sup2;) of 0.99972.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBehavioral tests\u003c/h3\u003e\n\u003cp\u003eC57BL/6 male mice (6\u0026ndash;8 weeks old) were obtained from the Experimental Animal Center at Southern Medical University (Guangzhou, China). Mice were maintained in a standard condition with a 12\u0026ndash;hour light/12\u0026ndash;hour dark cycle and ad libitum access to diet during the experiments. Animal use and procedure were approved by the Animal Use and Care Committee of Southern Medical University. Synaptosomes were isolated and purified from the DRN regions according to the work described previously (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBehavioral tests were conducted after a 4\u0026ndash;week chronic unpredictable mild stress (CUMS) procedure (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Mice were subjected to three behavioral tests, including sucrose splash test (SST), tail suspension test (TST), and forced swim test (FST), in 3 consecutive days, according to a previous work (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). For SST, mice were placed in a cage and sprayed with a 10% sucrose solution on their backs. Grooming time was measured for 5 min during 2\u0026ndash;6 min. In TST, the tail tips of mice were fixed at approximately one\u0026ndash;third of its length, and mice were suspended with their heads facing downward. Immobility time during 2\u0026ndash;6 min was measured. In FST, mice were placed in a glass cylinder filled with water (18 cm depth, 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) to swim for 6 min. Immobility time during 2\u0026ndash;6 min was measured.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were derived from experiments replicated a minimum of three times. Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analysis was performed using one\u0026ndash;way ANOVA followed by Tukey\u0026rsquo;s post hoc tests, and statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSERT associates with sGC to form a stable complex\u003c/h2\u003e \u003cp\u003eTo analyze sGC gene products engaged in SERT regulation, we examined the expression of sGC isoforms in RBL\u0026ndash;2H3 cells, which have been used as a cell model for exploring the cGMP signaling\u0026ndash;mediated regulation of SERT (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), by using qualitative RT\u0026ndash;PCR on total RNA with oligonucleotide primers specific for all sGC subunits (sGCα1, sGCα2, sGCβ1 and sGCβ2). Compared to mice midbrain RNA, where we identified expression of sGCα1 and sGCβ1 as predominant isoforms with a smaller amount of sGCα2 estimated by an additional quantitative RT\u0026ndash;PCR (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA), RBL\u0026ndash;2H3 RNA only yielded amplification of sGCα1 and sGCβ1 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). In the present study, we, thus, focus on the predominant sGCα1/β1 heterodimeric isoforms in the model cells or mice midbrain. To examine the association between endogenous sGC with SERT, we performed co\u0026ndash;immunoprecipitation of protein complexes from 1% Triton X\u0026ndash;100 extracts from RBL\u0026ndash;2H3 cells by using antibodies specific for SERT or sGC (sGCα1 or sGCβ1). Anti\u0026ndash;sGCα1 or \u0026ndash;sGCβ1 immunoblot demonstrated the existence of sGCα1 or sGCβ1 immunoreactivity in the SERT immunoprecipitates, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Similarly, an additional anti\u0026ndash;SERT blot also indicated the presence of SERT immunoreactive bands in the sGCα1 or sGCβ1 immunoprecipitates (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). These results provided evidence that a detergent\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:-\\)\u003c/span\u003e\u003c/span\u003eresistant complex of SERT with sGCα1/β1 is formed in RBL\u0026ndash;2H3 cells.\u003c/p\u003e \u003cp\u003eFor subcellular colocalization studies of SERT and sGC, RBL\u0026ndash;2H3 cells were probed with antibodies for SERT and sGC (sGCα1 or sGCβ1) under permeabilized conditions. Double labeling experiments revealed evidence of significant colocalization of SERT with either sGCα1 or sGCβ1 throughout the cell soma and plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To assess SERT and sGC colocalization, we calculated the intensity colocalization quotient (ICQ), which represents a measure of the extent of correlation of intensity values in space for two separate fluorophores (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Both SERT/sGCα1 and SERT/sGCβ1 ICQ values fall significantly above zero (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), suggesting that a remarkable colocalization of SERT/sGC occurs in both the cell soma and plasma membrane. These results are supported by our co\u0026ndash;immunoprecipitation analyses for the subcellular association between sGC and SERT. SERT or sGCβ1 (as a representative subunit of sGC) immunoreactivity was detected in anti\u0026ndash;sGCβ1 or anti\u0026ndash;SERT immunoprecipitates of both the subcellular membrane and plasma membrane fractions, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and S1D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further examine sGC translocation to associate with SERT in the plasma membrane, we performed transient transfection of HeLa or stable HeLa\u0026ndash;SERT cells with a plasmid encoding sGCβ1, respectively. Our immunofluorescence analysis indicated that sGCβ1 immunoreactivity was observed intracellularly, with a pattern similar to that seen for the endoplasmic reticulum (ER) marker calnexin in the host cells lacking SERT expression, consistent with the fact that sGC is a cytosolic protein (Fig. S2A). By contrast, a strong sGCβ1 immunoreactivity was detected in the plasma membrane, while little overlap with calnexin occurred in sGCβ1\u0026ndash;transfected HeLa\u0026ndash;SERT cells, indicating sGC translocation to the plasma membrane (Fig. S2B). In addition, SERT immunoreactivity was mainly observed in the plasma membrane of the cells co\u0026ndash;expressing SERT and sGCβ1 (Fig. S2C), thereby exhibiting extensive overlap with the immunoreactivity of sGCβ1 in confocal images (Fig. S2D). These results support the proposal that the mammalian cells maintain a stable association of sGC with SERT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIL4 in SERT is responsible for the association with sGC\u003c/h2\u003e \u003cp\u003eTo map structural region of SERT responsible for the interaction with sGC, we constructed several prokaryotic expression plasmids that produce various N\u0026ndash;terminally GST\u0026ndash;tagged cytoplasmic regions of SERT in \u003cem\u003eEscherichia coli\u003c/em\u003e cells (except for IL2 because it is too short, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and performed pull\u0026ndash;down experiments using glutathione affinity beads (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Glutathione Sepharose\u0026ndash;bound recombinant GST\u0026ndash;fused SERT fragments were incubated with total lysates of HEK\u0026ndash;293 cells transiently transfected with a plasmid encoding sGCα1 or sGCβ1, followed by elution of GST fusion proteins from the beads by reduced glutathione displacement. Immunoblot analysis using sGC antibodies showed that specific sGCα1 or β1 immunoreactivity was only observed in eluates from the glutathione beads bound with GST\u0026ndash;tagged internal loop 4 (IL4) of SERT (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and S3), indicating that sGC associates with the affinity\u0026ndash;purified SERT\u0026ndash;IL4 fragment.\u003c/p\u003e \u003cp\u003eTo confirm the role of IL4 motif in the SERT\u0026ndash;sGC association in mammalian expression systems, we performed anti\u0026ndash;Flag pull\u0026ndash;down experiments with HeLa\u0026ndash;SERT (C\u0026ndash;terminally Flag\u0026ndash;tagged) stable cells transiently transfected with plasmids encoding sGCβ1 or sGCβ1\u0026thinsp;+\u0026thinsp;IL4, respectively. In comparison to a low immunoreactivity of endogenous sGCβ1 detected with the cells without exogenous sGCβ1 expression, overexpression of sGCβ1 by transient transfection significantly increased sGCβ1 immunoreactive intensity in elutes from anti\u0026ndash;Flag agarose beads (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). On the other hand, co\u0026ndash;expression of the SERT\u0026ndash;IL4 fragment effectively reduced sGCβ1 immunoreactivity, suggesting that the IL4 fragment competitively displaced SERT from the SERT\u0026ndash;sGC complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe SERT\u0026ndash;sGC association decreases SERT transport activity and cell surface expression by interfering with SERT trafficking\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo examine functional consequences of the SERT\u0026ndash;sGC association, we carried out transport assay with HeLa\u0026ndash;SERT stable cells transiently transfected with plasmids encoding sGCβ1, sGCβ1\u0026thinsp;+\u0026thinsp;SERT\u0026ndash;IL1, or sGCβ1\u0026thinsp;+\u0026thinsp;SERT\u0026ndash;IL4. Compared to the control cells, exogenous expression of sGCβ1 significantly decreased SERT ability to transport 5\u0026ndash;HT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Co\u0026ndash;expression of SERT\u0026ndash;IL4 but not SERT\u0026ndash;IL1 fragment effectively reversed sGCβ1\u0026ndash;induced reduction in SERT activity, indicating that SERT\u0026ndash;IL4 exerts a specific effect on SERT transport activity. In addition, we investigated SERT\u0026ndash;IL4\u0026rsquo;s influence on the cell surface expression of SERT. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, sGCβ1 overexpression had little effect on total SERT expression but significantly reduced SERT expression on the cell surface. Co\u0026ndash;expression of SERT\u0026ndash;IL4, however, effectively reversed sGCβ1\u0026ndash;induced decrease in SERT cell surface expression.\u003c/p\u003e \u003cp\u003eThe SEC23/SEC24C complex as an essential component of the coat protein complex II has been demonstrated to play a critical role in SERT trafficking by binding with SERT (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). To examine the effect of the SERT\u0026ndash;sGC association on the interaction of SERT with SEC23/SEC24C, we performed transient co\u0026ndash;transfection of HeLa\u0026ndash;SERT stable cells to express SEC24C at a constant amount and sGCβ1 with a gradually increasing level. As sGCβ1 expression was increased, SERT activity to uptake 5\u0026ndash;HT was decreased, consistent with the proposal that augmentation of the SERT\u0026ndash;sGC association reduces SERT cell surface expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Strikingly, a sGCβ1 expression level\u0026ndash;dependent decrease in SERT-Flag immunoreactivity was observed in a His-tagged SEC24C protein complex bound to Ni-NTA beads, suggesting that the SERT\u0026ndash;sGCβ1 association inhibits the interaction of SERT with SEC24C. Hence, we assume that enhancement of the SERT\u0026ndash;sGC association leads to an impairment of SERT trafficking by decreasing the binding of SEC24C to SERT, thereby reducing SERT expression on the cell surface.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eThe unique SERT\u0026ndash;sGC association\u003c/h2\u003e \u003cp\u003eSERT belongs to the monoamine transporter family, which also includes transporters for dopamine (DAT) and norepinephrine (NET). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA shows an amino acid sequence alignment of the IL4s of human monoamine transporters, in which the residues are highly conserved between DAT and NET but less with SERT. Interestingly, there is a potent α\u0026ndash;helix\u0026ndash;breaking residue, Pro455, in SERT\u0026ndash;IL4 but not in either DAT or NET. The high\u0026ndash;resolution structures show that the IL4 is packed as a full or partial α\u0026ndash;helix in NET or DAT (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). By comparison, a pronounced kink that leads to IL4 unwinding is observed in the SERT structures (47) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), suggesting the presence of a unique folding in SERT\u0026ndash;IL4. To learn if sGC also associates with DAT\u0026ndash; or NET\u0026ndash;IL4, we performed pull\u0026ndash;down experiments by using N\u0026ndash;terminally GST\u0026ndash;tagged DAT\u0026ndash; or NET\u0026ndash;IL4 produced in prokaryotic expression systems. Unlike SERT\u0026ndash;IL4, neither DAT\u0026ndash; nor NET\u0026ndash;IL4 was able to associate with sGCβ1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Additionally, transport assay with a fluorescent substrate APP\u003csup\u003e+\u003c/sup\u003e for all three monoamine transporters showed that sGCβ1 co\u0026ndash;expression remarkably reduced APP\u003csup\u003e+\u003c/sup\u003e uptake by SERT but not by DAT or NET (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), suggesting that functional regulation by the sGC\u0026ndash;IL4 association is specific for SERT.\u003c/p\u003e \u003cp\u003eTo further investigate the effects of SERT\u0026ndash;IL4 residues on the SERT\u0026ndash;sGC interaction, we performed alanine\u0026ndash;scanning mutagenesis (except for Ala459), one at a time, and assessed the association between sGCβ1 and individual N\u0026ndash;terminally GST\u0026ndash;tagged SERT\u0026ndash;IL4 alanine mutants by glutathione affinity pull down experiments. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, compared to SERT\u0026ndash;IL4 WT, six of seven mutants, F454A, P455A, H456A, W458A, K460A, and R461A, exerted significant changes in their ability to interact with sGCβ1; of those, one mutant, P455A, showed a\u0026thinsp;\u0026gt;\u0026thinsp;3\u0026ndash;fold increase in its interaction with sGCβ1, whereas other five mutants exhibited a remarkably reduced interaction with sGCβ1, suggesting that the entire SERT\u0026ndash;IL4 region plays a critical role in the SERT\u0026ndash;sGC association. We then mutated two residues critical for maintaining the sGC\u0026ndash;SERT\u0026ndash;IL4 association, His456 or Trp458 by alanine, one at a time, in the C\u0026ndash;terminally Flag\u0026ndash;tagged full\u0026ndash;length SERT and examined the effects of these substitutions on the SERT\u0026ndash;sGC association and SERT cell surface expression and transport activity, respectively. Anti\u0026ndash;Flag agarose pull\u0026ndash;down experiments demonstrated that replacement of His456 or Trp458 with alanine decreased the SERT\u0026ndash;sGC association (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), consistent with those observed with GST\u0026ndash;tagged SERT\u0026ndash;IL4 mutants produced in our prokaryotic expression systems. Furthermore, although the alanine mutants, H456A and W458A, showed slight decreases in their cell surface expression (~\u0026thinsp;87% of SERT WT for H456A and ~\u0026thinsp;72% for W458A, respectively), possibly due to the impacts of these mutations themselves, co\u0026ndash;expression of sGCβ1 significantly reduced the cell surface expression of SERT WT but had little effect on these mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Correspondingly, transport assay indicated that sGCβ1 co\u0026ndash;expression significantly decreased transport activity of SERT WT but not H456A or W458A mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Our kinetic analysis also showed that sGCβ1 co\u0026ndash;expression decreased \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e of 5\u0026ndash;HT transport with a similar \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e for the substrate in WT but had little effect on kinetic parameters in these alanine mutants (Figs. S4A, 4B, 4C, 4D). These results suggest that the SERT\u0026ndash;sGC association modulates SERT activity by alternating its subcellular distribution not catalytic function. Furthermore, HeLa cells were transiently co\u0026ndash;transfected with equal amounts of cDNAs encoding sGCβ1, His-tagged SEC24C, and SERT or its mutants and then used for examining the effects of the two mutations, H456A and W458A, on the interaction between SERT and SEC24C. The cells were lysed and the resulting lysates were then incubated with Ni\u0026ndash;NTA beads. Immunoblot analysis indicated a remarkably increased SERT immunoreactivity in the imidazole elutes with H456A or W458A mutant, compared to that with SERT WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), consistent with our previous observation that sGCβ1 competes with SEC24C to interact with SERT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThe SERT\u0026ndash;sGC interaction is responsive to susceptibility factors in both RBL\u0026ndash;2H3 cells and mice\u003c/h2\u003e \u003cp\u003eInflammation and oxidative stress have been demonstrated to be the primary risk factors in the pathology of depression and other psychiatric illnesses (\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). To learn if the SERT\u0026ndash;sGC association is altered by exposure to these susceptibility factors, we examined the SERT\u0026ndash;sGC association under the treatment with a potent activator of inflammation, lipopolysaccharides (LPS), pro\u0026ndash;inflammatory cytokines, tumor necrosis factor alpha (TNF\u0026ndash;α) and interleukin\u0026ndash;1 beta (IL\u0026ndash;1β), or an oxidative agent, hydrogen peroxide, in RBL\u0026ndash;2H3 cells. Exposure to these agents in culture medium led to an increase in 5\u0026ndash;HT uptake by SERT, compared to that with the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The enhancement of SERT transport activity is apparently due to an increased SERT expression on the cell surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Importantly, our co\u0026ndash;immunoprecipitation experiments indicated that exposure to these agents impairs the SERT\u0026ndash;sGC interaction in RBL\u0026ndash;2H3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThen, we asked if the SERT\u0026ndash;sGC association influences sGC activity or cGMP\u0026ndash;mediated phosphorylation of SERT. To this end, we assessed sGC activity in response to various treatments in the absence or presence of a sGC stimulator, S\u0026ndash;nitroso\u0026ndash;N\u0026ndash;acetyl penicillamine (SNAP), which breaks down spontaneously to produce NO in an aqueous medium, by measuring cGMP production in RBL\u0026ndash;2H3 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, SNAP stimulated sGC activity to produce significantly increased cGMP, compared to those detected in the absence of SNAP. However, exposure to various agents had little effect on cGMP production either in the absence or presence of SNAP, although these treatments have been indicated to reduce the SERT\u0026ndash;sGC interaction in RBL\u0026ndash;2H3 cells. Additionally, we evaluated cGMP\u0026ndash;mediated phosphorylation of SERT in response to these treatments in RBL\u0026ndash;2H3 cells. Compared to the control, no statistical difference in phosphorylation level of SERT\u0026ndash;Thr276 was detected under any treatment tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), supporting that the SERT\u0026ndash;sGC association does not alter sGC activity. Moreover, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, our results also demonstrated that incubation of RBL\u0026ndash;2H3 cells with 5\u0026ndash;HT at various concentrations had little effect on cGMP production, indicating that 5\u0026ndash;HT transport does not affect sGC activity, a different action from the SERT\u0026ndash;nNOS association on sGC activity (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we examined the SERT\u0026ndash;sGC association in response to stressful events in mice by using a CUMS procedure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Exposure to CUMS for 4 weeks led to depression\u0026ndash;like behaviors including a decrease in the grooming time in SST and increases in the immobility time in both TST and FST in our animal models (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). We then isolated synaptosomes from the DRN of mice and measured 5\u0026ndash;HT uptake by somatodendritic SERT in the serotonergic neurons. Compared to the control, CUMS treatment resulted in an impaired 5\u0026ndash;HT uptake, reflecting an increased extracellular level of 5\u0026ndash;HT in the DRN (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Immunoblot analysis for the biotinylated SERT indicated that CUMS exposure caused a significant reduction in SERT distribution on the synaptosomal surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Co\u0026ndash;immunoprecipitation experiments further revealed that the CUMS procedure led to a stronger immunoreactivity of sGCβ1 than that detected with the control group in anti\u0026ndash;SERT immunoprecipitates, indicating that the SERT\u0026ndash;sGC interaction in the DRN is augmented by the CUMS exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDisrupting the SERT\u0026ndash;sGC association in the DRN produces a fast\u0026ndash;acting antidepressant\u0026ndash;like activity in mice\u003c/h2\u003e \u003cp\u003eWe synthesized a N\u0026ndash;terminally Tat\u0026ndash;fused peptide that links the HIV Tat protein transduction domain to SERT\u0026ndash;IL4 (Tat\u0026ndash;IL4, YGRKKRRQRRRGGGFPHVWAKR), allowing Tat peptide to deliver the SERT\u0026ndash;IL4 into cells, and then examined its effects on 5\u0026ndash;HT uptake by incubating HeLa\u0026ndash;SERT stable cells transiently transfected with a plasmid encoding sGCβ1 with Tat\u0026ndash;IL4. Our results showed that Tat\u0026ndash;IL4 treatment significantly reversed sGC\u0026ndash;induced decrease in 5\u0026ndash;HT uptake, indicating Tat\u0026ndash;IL4 effectively dissociated SERT from sGC (Fig. S5). To learn behavioral and functional consequences of Tat\u0026ndash;IL4 disruption of the SERT\u0026ndash;sGC association, mice were administered by intraperitoneal injection (i.p.) of Tat\u0026ndash;IL4 after exposure to a 4\u0026ndash;week CUMS procedure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At 2 hours after administration, Tat\u0026ndash;IL4 peptide labelled with Cy7.5 fluorophore was detected in the midbrain of mice by \u003cem\u003ein vivo\u003c/em\u003e imaging, indicating Tat\u0026ndash;IL4 crossed the blood\u0026ndash;brain barrier into the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Animal behavioral tests showed that a single injection of Tat\u0026ndash;IL4 significantly attenuated CUMS\u0026ndash;induced behavioral abnormalities in SST, FST and TST 2 hours after administration, exerting rapid onset antidepressant\u0026ndash;like effects in our animal models (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The DRN synaptosomes were then subjected to various biochemical analyses. Administration of Tat\u0026ndash;IL4 remarkably reversed CUMS\u0026ndash;induced alterations of 5\u0026ndash;HT uptake, SERT distribution, and the SERT\u0026ndash;sGC association (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Furthermore, we measured the extracellular 5\u0026ndash;HT levels in the hippocampus by microdialysis combined with HPLC. As expected, exposure to CUMS resulted in markedly reduced hippocampal 5\u0026ndash;HT concentrations. By contrast, Tat\u0026ndash;IL4 rapidly elevated 5\u0026ndash;HT to the levels detected in control mice 2 hours after injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). We also examined the effects of administration of a control peptide Tat\u0026ndash;8A (YGRKKRRQRRRGGGAAAAAAAA) on animal behaviors. Compared to CUMS group, a single injection of Tat\u0026ndash;8A had little effect on CUMS\u0026ndash;induced depression\u0026ndash;like behaviors (Fig. S6). Thus, we propose that disruption of the SERT\u0026ndash;sGC association by Tat\u0026ndash;IL4 led to an increased 5\u0026ndash;HT uptake by SERT in the DRN and an increased 5\u0026ndash;HT release from the presynaptic never terminals in the hippocampus, thereby exerting a fast\u0026ndash;acting antidepressant activity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we identified a stable protein\u0026ndash;protein complex formed by the physical interaction between SERT and sGC and revealed the molecular mechanism by which SERT is under a regulatory control mediated by the SERT\u0026ndash;sGC complex. Enhancement of the SERT\u0026ndash;sGC interaction reduced SERT trafficking, thereby decreasing SERT cell surface expression and 5\u0026ndash;HT uptake, and \u003cem\u003evice versa\u003c/em\u003e. Additionally, our results indicated that alteration of the SERT\u0026ndash;sGC interaction has little effect on sGC activity, exerting a unilateral modulation of SERT. Furthermore, we demonstrated that a unique structural folding of the internal loop IL4 renders a specificity for SERT to associate with sGC.\u003c/p\u003e \u003cp\u003eMost importantly, the SERT\u0026ndash;sGC association was shown to be responsive to a variety of susceptibility factors in the pathophysiology of 5\u0026ndash;HT\u0026ndash;related psychiatric disorders. Thus, we propose a possible scenario for SERT regulation by the SERT\u0026ndash;sGC association (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Under physiological conditions, SERT is present in a dynamic equilibrium between the association with sGC and dissociation from sGC, which determines SERT distribution on the cell surface. Alteration of the SERT\u0026ndash;sGC interaction affects the equilibrium, which, in turn, changes SERT subcellular localization and its ability to uptake 5\u0026ndash;HT. The finding of this phenomenon not only promotes our understanding of the pathophysiology of several 5\u0026ndash;HT\u0026ndash;linked mental diseases but also provides an opportunity to develop novel therapeutic agents targeting SERT regulation mediated by the SERT\u0026ndash;sGC association. Indeed, exposure to CUMS increases the SERT\u0026ndash;sGC interaction and then reduces SERT cell surface expression and synaptic 5\u0026ndash;HT uptake in the DRN, thereby causing a decreased 5\u0026ndash;HT level in the hippocampus and behavioral abnormalities in our models. At 2-hour after administration, however, SERT\u0026ndash;IL4 peptide rapidly reverses CUMS\u0026ndash;induced enhancement of the SERT\u0026ndash;sGC interaction and normalizes 5\u0026ndash;HT neurotransmission from the DRN to hippocampus, exerting a fast onset antidepressant activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe conventional SSRI antidepressants inhibit SERT in both the serotoninergic neurons in the DRN and presynaptic never terminals in other brain regions, leading to a non\u0026ndash;selective activation of both 5\u0026ndash;HT\u003csub\u003e1A\u003c/sub\u003e autoreceptors in the DRN and postsynaptic 5\u0026ndash;HT\u003csub\u003e1A\u003c/sub\u003e heteroreceptors in the hippocampus and cortex (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Activation of the DRN 5\u0026ndash;HT\u003csub\u003e1A\u003c/sub\u003e autoreceptors has been demonstrated to suppress 5\u0026ndash;HT release and firing rate of the serotoninergic neurons by its negative feedback mechanism (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Thus, an additional desensitization of 5\u0026ndash;HT\u003csub\u003e1A\u003c/sub\u003e autoreceptors is required for the antidepressant effects of SSRIs (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). The desensitization process has been proposed to be achieved by sustained activation of 5\u0026ndash;HT\u003csub\u003e1A\u003c/sub\u003e autoreceptors with chronic exposure to SSRIs, thereby leading to the delayed therapeutic effects and other limitations of the SERT inhibitors (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). On the other hand, specific suppression of 5\u0026ndash;HT\u003csub\u003e1A\u003c/sub\u003e autoreceptors by reducing synaptic 5\u0026ndash;HT level in the DRN can rapidly stimulate the serotonergic neurons activity and 5\u0026ndash;HT release into the hippocampus and cortex without the requirement of 5\u0026ndash;HT\u003csub\u003e1A\u003c/sub\u003e autoreceptors desensitization and then produce fast onset antidepressant effects (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). SERT is predominantly expressed in the serotonergic neurons in the DRN (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), thus allowing us to selectively control 5-HT\u003csub\u003e1A\u003c/sub\u003e autoreceptors by modulating SERT subcellular localization in the DRN with minimal effects on 5\u0026ndash;HT\u003csub\u003e1A\u003c/sub\u003e heteroreceptors in other brain regions. Hence, we propose that agents such as the SERT\u0026ndash;IL4 peptide could specifically decrease synaptic 5\u0026ndash;HT level in the DRN by disrupting the SERT\u0026ndash;sGC interaction and subsequently deactivate 5\u0026ndash;HT\u003csub\u003e1A\u003c/sub\u003e autoreceptors, thereby exerting fast\u0026ndash;acting antidepressant effects without 5\u0026ndash;HT\u003csub\u003e1A\u003c/sub\u003e autoreceptors desensitization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe SERT\u0026ndash;IL4 motif connecting transmembrane (TM) α\u0026ndash;helices 8 and 9 is fully exposed to the cytosolic medium (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e), providing an accessibility to sGC for the SERT\u0026ndash;sGC interaction. In addition, this internal loop adopts an extended conformation adjacent to an intercellular gate residue Asp\u0026ndash;452, which was proposed to control SERT conformational transitions by forming or breaking a salt\u0026ndash;bridge with N\u0026ndash;terminal Arg\u0026ndash;79 (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Comparison of SERT structures in several conformations such as outward\u0026ndash;open, occluded, and inward\u0026ndash;open conformational states showed that the structural rearrangements in the conformational transitions largely involve a hinge\u0026ndash;like movement of the unwound TM1a, which leads to a significant shift of the adjacent N\u0026ndash;terminal tail containing Arg\u0026ndash;79. By contrast, TMs 8 and 9 in a scaffold region are among several α\u0026ndash;helices with minimal structural rearrangements during the conformational transitions (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e) (Fig. S7), consistent with the proposal that the IL4 motif is conformationally insensitive and flexible. Our kinetic analysis showed that augmentation of the SERT\u0026ndash;sGC interaction decreases 5\u0026ndash;HT transport velocity but with a similar affinity for the substrate to that observed in the basal state, indicating that the IL4\u0026ndash;mediated association has a profound impact on the SERT subcellular distribution rather than its catalytic function. In addition, the C\u0026ndash;terminal residues of SERT, such as Arg\u0026ndash;607, Ile\u0026ndash;608, and Lys\u0026ndash;610, have been demonstrated to participate in the binding of the SEC23/SEC24C complex, which is critical to form vesicles for transporting proteins from the ER to plasma membrane (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Our docking indicated that the interaction between sGC and SERT\u0026ndash;IL4 forms a spatial barrier that prevents the C\u0026ndash;terminal residues from binding with SEC23/SEC24C (Fig. S8), consistent with our experimental results in showing the effect of sGC on the SERT\u0026ndash;SEC24C interaction. Hence, we propose that augmentation of the SERT\u0026ndash;sGC interaction impairs SERT trafficking to the plasma membrane, thereby resulting in a reduced SERT cell surface distribution.\u003c/p\u003e \u003cp\u003esGC is a heterodimeric hemoprotein generally activated by NO binding (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e) and each subunit contains four domains, heme NO/oxygen (H\u0026ndash;NOX) binding domain, Per\u0026ndash;Arnt\u0026ndash;Sim (PAS) domain, coiled\u0026ndash;coil (CC) domain and catalytic (CAT) domain (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Our molecular docking indicated that the SERT\u0026ndash;sGC interaction mainly occurs in the interface between the SERT\u0026ndash;IL4 and sGCα1 pseudo H\u0026ndash;NOX domain with sGCβ1 PAS domain in a sGC heterodimer (Fig. S8). The heme molecule only binds to the sGCβ1 H\u0026ndash;NOX domain but not to the sGCα1 pseudo H\u0026ndash;NOX domain (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). In addition, the PAS domain in either sGCα1 or sGCβ1 is believed to act as an anchor to stabilize each subunit in sGC activation (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). Our results showed that the SERT\u0026ndash;sGC interaction does not alter sGC activity. Thus, it is reasonable to assume that the SERT\u0026ndash;sGC interaction does not interfere NO binding in the sGCβ1 and activation of the C\u0026ndash;terminal cyclase domains in both subunits to synthesize cGMP from GTP.\u003c/p\u003e \u003cp\u003eWith the addition of sGC, it seems increasingly likely that all of the components of cGMP signal cascade are associated with SERT (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). However, functional consequences of these individual associations are not same. While the SERT\u0026ndash;nNOS association impaired 5\u0026ndash;HT uptake by reducing SERT cell surface expression, SERT was also shown to reciprocally affect nNOS enzymatic activity, which, in turn, modulated NO signaling in the central never system (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). An agent that disrupts the SERT\u0026ndash;nNOS association has recently been indicated to produce fast onset antidepressant\u0026ndash;like effects by enhancing 5\u0026ndash;HT signaling in forebrain circuits (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), the reciprocal modulation between SERT and nNOS, however, increases the concerns about its off\u0026ndash;target effects due to an altered nNOS activity. It was previously proposed that an allosteric conformational change of nNOS caused by 5\u0026ndash;HT transport in the SERT\u0026ndash;nNOS complex could increase its affinity for calmodulin, thereby elevating nNOS catalytic activity (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). By contrast, the SERT\u0026ndash;PKG association was proposed to have two effects on SERT, alteration of SERT localization and stimulation of SERT phosphorylation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Alteration of SERT distribution was demonstrated to be independent of PKG activity, because a similar effect on SERT subcellular localization was found with catalytically inactive mutants of PKG (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Unlike nNOS and sGC, enhancement of the SERT\u0026ndash;PKG interaction was shown to increase SERT cell surface expression (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In addition, PKG\u0026ndash;mediated phosphorylation of SERT was indicated to elevate 5\u0026ndash;HT uptake, which, in turn, further stimulated SERT phosphorylation (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Subsequent studies showed that phosphorylation and activation of SERT were blocked by a mutation of Thr276 near the cytoplasmic end of TM5 (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e), consistent with phosphorylation of SERT on Thr276. However, the physiological significance and specificity of the SERT\u0026ndash;PKG association have not been determined yet.\u003c/p\u003e \u003cp\u003eThe protein\u0026ndash;protein interactions (PPIs) play critical roles in life processes. Abnormal PPIs have been demonstrated to be associated with many human diseases, supporting the development of drug targeting therapeutics for the PPIs (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). Many protein\u0026ndash;protein interaction modulators such as venetoclax, sarilumab, maraviroc, adagrasib, siltuximab, and sotorasib have been approved for clinical treatment of various diseases (\u003cspan additionalcitationids=\"CR71 CR72 CR73 CR74\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). Although the Tat\u0026ndash;IL4 peptide was shown to produce a rapid\u0026ndash;onset antidepressant activity in our depressive models, it might suffer from several intrinsic drawbacks as a peptide molecule, including limited stability, poor bioavailability, and low selectivity. Development of small molecule or peptidomimetic agents targeting the SERT\u0026ndash;sGC interaction is undoubtedly a future strategy to address these limitations. Nevertheless, understanding the molecular mechanism in the functional modulation of a physiologically important membrane transporter by the interaction with its regulatory proteins could have broader implications for the development of new therapeutics targeting its regulation rather than the transporter itself.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe conventional SSRI antidepressants are plagued by their significant limitations caused by a global elevation of 5\u0026ndash;HT level in the entire brain. By comparison, regional regulation by PPIs can achieve precise, rapid, and fine\u0026ndash;tuning modulation of SERT function, while avoiding dysregulation of the essential neurological functions in other brain regions. The present study identified a physical interaction between SERT and its regulatory protein sGC. The SERT-sGC association specifically regulated SERT capability to uptake 5\u0026ndash;HT by modulating its membrane trafficking. The SERT\u0026ndash;sGC interaction was shown to be mediated by SERT\u0026ndash;IL4 motif and exerted a unilateral modulation of SERT but not sGC. Most strikingly, dissociating SERT from sGC by the SERT\u0026ndash;IL4 peptide produced a rapid-onset antidepressant activity. These findings open up a promising way for future antidepressant development. Targeting the PPI interface rather than the transporter itself represents a paradigm shift from the conventional pharmacological approaches to a novel strategy for the precise control of the serotonergic system.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e5-Hydroxytryptamine\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e5-HT\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecGMP\u0026ndash;dependent protein kinase\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePKG\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eChronic unpredictable mild stress\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCUMS\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDopamine transporter\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDAT\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDorsal raphe nucleus\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDRN\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEndoplasmic reticulum\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eER\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eForced swim test\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFST\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHeme NO/oxygen\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eH\u0026ndash;NOX\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHigh\u0026ndash;performance liquid chromatography\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHPLC\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIntensity correlation quotient\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eICQ\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMajor depressive disorder\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMDD\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNeuronal nitric oxide synthase\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enNOS\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNorepinephrine transporter\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNET\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSelective serotonin reuptake inhibitor\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSSRI\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSerotonin transporter\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSERT\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eS\u003c/em\u003e\u0026ndash;Nitroso\u0026ndash;\u003cem\u003eN\u003c/em\u003e\u0026ndash;acetyl penicillamine\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSNAP\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSoluble guanylyl cyclase\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esGC\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSucrose splash test\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSST\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTail suspension test\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTST\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e Animal use was approved by the Animal Use and Care Committee of Southern Medical University (approved code: SMUL2021177, approved date: 12 December 2021).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eAuthors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthor information\u003c/h2\u003e \u003cp\u003eSchool of Life Sciences, Guangzhou University 230 Outer Ring West Road Higher Education Mega Center, Panyu District, Guangzhou, Guangdong 510006, China\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFootnotes\u003c/strong\u003e \u003cp\u003ePublisher's Note\u003c/p\u003e \u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNational Natural Science Foundation of China 32371304 and 32071233 (Y\u0026ndash;WZ).\u003c/p\u003e \u003cp\u003eGuangdong Basic and Applied Research Foundation 2021A1515012067 (Y\u0026ndash;WZ).\u003c/p\u003e \u003cp\u003eInnovative Research Program for Guangzhou University Postgraduate Students JCCX2024\u0026ndash;017 (XH).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: XH, Y\u0026ndash;WZBench experiments: XH, CL, JLMolecular docking: XZ, YXVisualization: XZ, YXSupervision: Y\u0026ndash;WZWriting\u0026mdash;original draft: XH, Y\u0026ndash;WZWriting\u0026mdash;review \u0026amp; editing: CL, Y\u0026ndash;WZResources, project administration and funding acquisition: Y\u0026ndash;WZ, XH\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank all members in Zhang\u0026rsquo;s Lab for their suggestions in preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data are available in the main text and the supplementary materials. Any remaining raw data will be available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOwens MJ, Nemeroff CB. Role of serotonin in the pathophysiology of depression: focus on the serotonin transporter. Clin Chem. 1994;40:288\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlivier B. Serotonin: a never-ending story. Eur J Pharmacol. 2015;753:2\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElias Eriksson MJ. Serotonin in Psychiatric Pathophysiology The Biological Basis of Psychiatric Treatment. 1990;3:66\u0026ndash;119.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaswani M, Linda FK, Ramesh S. Role of selective serotonin reuptake inhibitors in psychiatric disorders: a comprehensive review. 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Mol Psychiatry. 2003;8:933\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelorme R, Betancur C, Wagner M, Krebs MO, Gorwood P, Pearl P, et al. Support for the association between the rare functional variant I425V of the serotonin transporter gene and susceptibility to obsessive compulsive disorder. Mol Psychiatry. 2005;10:1059\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang YW, Gesmonde J, Ramamoorthy S, Rudnick G. Serotonin transporter phosphorylation by cGMP-dependent protein kinase is altered by a mutation associated with obsessive compulsive disorder. J Neurosci. 2007;27:10878\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChanrion B, Mannoury la Cour C, Bertaso F, Lerner-Natoli M, Freissmuth M, Millan MJ, et al. Physical interaction between the serotonin transporter and neuronal nitric oxide synthase underlies reciprocal modulation of their activity. 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Bioinformatics. 2014;30:1771\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Serotonin, Serotonin transporter, Soluble guanylate cyclase, Regional regulation, Antidepressants, Rapid-onset antidepressants","lastPublishedDoi":"10.21203/rs.3.rs-8689963/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8689963/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMajor depressive disorder (MDD) is a refractory neurological disorder often linked to dysregulated 5\u0026ndash;hydroxytryptamine (5\u0026ndash;HT) neurotransmission. Conventional selective serotonin reuptake inhibitors (SSRIs) face criticism due to their delayed therapeutic onsets and severe adverse effects. The present study aims to illustrate the molecular mechanism by which serotonin transporter (SERT) is regulated by its physical interaction with soluble guanylate cyclase (sGC) for exploring a new therapeutic target with a novel mechanism of action.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eRBL-2H3 cells and synaptosomes isolated from the dorsal raphe nucleus (DRN) were employed to investigate the SERT\u0026ndash;sGC association under physiological conditions or in response to various treatments by using the molecular and cellular approaches. The functional consequences of the SERT\u0026ndash;sGC interaction were assessed by biotinylation of membrane proteins and 5\u0026ndash;HT uptake assay. GST-tagged intracellular fragments were produced to map the structural motif of SERT responsible for the interaction with sGC and the forth internal loop (IL4)\u0026ndash;mediated SERT\u0026ndash;sGC association was further confirmed by mutagenesis. Finally, a peptide to dissociate SERT from sGC was synthesized and its antidepressant activity was evaluated with chronic unpredictable mild stress (CUMS) mouse models.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA stable SERT\u0026ndash;sGC complex was identified. Augmentation of the SERT\u0026ndash;sGC interaction decreased SERT cell surface expression by interfering with its trafficking, thereby reducing 5\u0026ndash;HT uptake. The specific SERT\u0026ndash;sGC association was mediated by the IL4 motif with a unique structural folding in SERT and exerted a unilateral modulation of SERT without affecting sGC. Notably, the SERT\u0026ndash;sGC interaction was altered in response to treatment of a variety of susceptibility factors in both cells and animal models. Two hours after administration, SERT\u0026ndash;IL4 peptide reversed CUMS\u0026ndash;induced enhancement of the SERT\u0026ndash;sGC interaction and normalized 5\u0026ndash;HT neurotransmission from the DRN to hippocampus, exerting a fast-onset antidepressant activity.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study revealed the mechanism by which SERT is under the regulatory control by the SERT\u0026ndash;sGC association and provided insights into a novel target toward the SERT\u0026ndash;sGC interaction in developing rapid\u0026ndash;onset agents in the treatment of MDD.\u003c/p\u003e","manuscriptTitle":"Disrupting the physical interaction between serotonin transporter and soluble guanylate cyclase produces a fast–acting antidepressant activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-10 06:18:15","doi":"10.21203/rs.3.rs-8689963/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"26223127-023e-4b80-bd6f-c8b051505fcc","owner":[],"postedDate":"February 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-12T02:39:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-10 06:18:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8689963","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8689963","identity":"rs-8689963","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}