Go/z-biased coupling profile of the dopamine D3 receptor

Go/z-biased coupling profile of the dopamine D3 receptor | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Go/z-biased coupling profile of the dopamine D3 receptor Lucrezia Zanetti , Luca Franchini , Shirsha Saha , Yini Liao , View ORCID Profile Brian S. Muntean , View ORCID Profile Cesare Orlandi doi: https://doi.org/10.1101/2025.08.08.668522 Lucrezia Zanetti 1 Department of Pharmacology and Physiology, University of Rochester Medical Center , Rochester, NY 14642 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Luca Franchini 1 Department of Pharmacology and Physiology, University of Rochester Medical Center , Rochester, NY 14642 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shirsha Saha 1 Department of Pharmacology and Physiology, University of Rochester Medical Center , Rochester, NY 14642 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yini Liao 2 Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University , Augusta, GA 30912 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Brian S. Muntean 2 Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University , Augusta, GA 30912 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Brian S. Muntean Cesare Orlandi 1 Department of Pharmacology and Physiology, University of Rochester Medical Center , Rochester, NY 14642 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Cesare Orlandi For correspondence: cesare_orlandi{at}urmc.rochester.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Dopamine receptors are G protein coupled receptors (GPCRs) that serve as key targets for FDA-approved drugs used to treat various neuropsychiatric disorders. Notably, ∼11% of all marketed GPCR-targeting drugs act on dopamine receptors. Five GPCRs mediate the effects of endogenous dopamine and compounds used to treat Parkinson’s disease, schizophrenia, and other conditions. However, on-target side effects associated with these medications highlight the need to analyze dopamine receptor signaling to design safer, more effective therapeutics. We characterized the G protein coupling of dopamine D2-like receptors and observed the striking inability of D3R to engage with G i proteins while effectively activating G o and G z subtypes. Applying orthogonal cell-based assays that utilize wild-type G proteins both in parental and ΔGα i/o/z cells, we conclusively established that D3R does not activate G i proteins. Further analysis of Gα i2 :Gα oA and D2R:D3R chimeras revealed that this selective inability is driven by molecular determinants located within the α5 helix of Gα i and the intracellular loop 2 (ICL2) of D3R. Guided by cryo-EM structures, we modeled the interface between these regions to better understand the structural basis of this selectivity. Finally, we treated hippocampal neurons in acute brain slices with selective agonists for D2R and D3R and observed marked differences in their ability to regulate endogenous adenylyl cyclase to produce cAMP, highlighting the neurophysiological significance of our findings. INTRODUCTION Dopamine is a critical catecholaminergic neurotransmitter that regulates a broad range of functions in both the central and the peripheral nervous systems 1 – 3 . Dopamine actions are mediated by the activation of five G protein-coupled receptors (GPCRs) that are classified into two families based on their structural and signaling properties: D1-like receptors (D1R, D5R) and D2-like receptors (D2R, D3R, D4R). D1-like receptors generally couple to heterotrimeric G s proteins that stimulate adenylyl cyclase (AC) to produce cAMP, whereas D2-like receptors predominantly couple to inhibitory G i/o/z proteins, resulting in AC inhibition and activation of additional downstream signaling pathways, including those mediated by β-arrestin recruitment 3 – 11 . A key emerging concept in signal transduction is that a single receptor can adopt multiple conformations, each selectively engaging different intracellular transducers and triggering distinct cellular responses 12 – 15 . This signaling bias includes preference not only between G protein and β-arrestin pathways but also among different G protein subtypes 16 – 19 . Dysregulation of D2-like receptor signaling has been implicated in a wide range of neurological and psychiatric disorders, including Parkinson’s disease (PD), Huntington’s disease (HD), schizophrenia (SZ), major depressive disorder (MDD), attention-deficit/hyperactivity disorder (ADHD), and substance use disorders 20 – 24 . These associations highlight the critical importance of understanding the fundamental mechanisms of dopamine receptor signaling. In particular, biased signaling offers a promising framework for the development of targeted and effective therapeutics with fewer side effects, by selectively modulating disease-relevant signaling cascades 25 . Achieving this goal requires a deeper understanding of the molecular details related to how each D2-like receptor engages with different intracellular transducers. The D2R is the most extensively studied subtype, with detailed characterization of its coupling to G i/o/z proteins 16 , including differences between its two long and short splicing isoforms 19 . In contrast, D3R and D4R, despite sharing structural and signaling features with D2R, are less understood in terms of their selective coupling to Gα protein subtypes. In particular, the Gα protein coupling profile of D3R is debated, with some studies suggesting it fails to engage Gα i subtypes and others reporting broader coupling across the G i/o/z family 26 – 29 . In this study, we conclusively defined the signaling properties of D3R and D4R, demonstrating that D3R displays a distinct and consistent inability to couple with Gα i proteins, despite robust activation of Gα o and Gα z . This biased coupling pattern was further corroborated by exploring structural elements and defining the responsible molecular determinants. Finally, we demonstrated the physiological relevance of this discovery in neurons. Offering physiologically relevant evidence that D3R selectively couples to Gα o and Gα z , we resolved previous inconsistencies and paved the way for the development of targeted D3R-based therapies. RESULTS Unique coupling profile of the dopamine D3 receptor We initially assessed the G protein coupling specificity of the dopamine D2-like receptors (D2R, D3R, and D4R) to confirm their preferential interaction with heterotrimeric G proteins of the G i/o/z subfamily, as previously reported 4 . To this goal, we transfected HEK293T cells with a representative Gα subunit from the four major G protein families, Gα i/o/z , Gα s , Gα q , and Gα 12/13 , and we applied a kinetic G protein nanoBRET assay to assess each G protein activation by D2R, D3R, and D4R in real time. Upon stimulation with either quinpirole ( Figure 1A ), a selective D2-like receptor agonist, or the non-selective endogenous agonist dopamine ( Supplementary Figure 1A), all three receptors displayed clear and exclusive activation of the Gα o subunit. No activation of Gα s , Gα q , or Gα 13 proteins was observed under any condition, confirming the well-established Gα i/o/z -selective signaling profile of D2-like receptors. To further investigate the receptor-specific coupling preferences, we assessed the ability of D2R, D3R, and D4R to activate each of the six main Gα i/o/z subunits (Gα oA , Gα oB , Gα i1 , Gα i2 , Gα i3 , and Gα z ). Applying the same G protein nanoBRET assay, we obtained concentration–response curves to quantitatively compare the coupling efficiency of the three dopamine receptors in response to both quinpirole and dopamine ( Figure 1B and Supplementary Figure 1B). As expected, D2R and D4R displayed robust activation of all six G proteins with a preference for G o over G i and G z . In contrast, D3R showed a distinct and restricted coupling profile, with no detectable activation of any Gα i subtype (Gα i1 , Gα i2 , and Gα i3 ), while retaining efficient coupling to Gα oA , Gα oB , and G αz ( Figure 1B and Supplementary Figure 1B). This allowed the calculation of EC 50 and E max values for individual G proteins, which are visualized as radar plots to facilitate comparison of both potency and efficacy ( Figure 1C ). These analyses further reinforced the functional similarity between D2R and D4R, while once again confirming the markedly different and selective profile of D3R. Taken together, these results suggest that D3R exhibits a selective signaling bias within the Gα i/o/z family, favoring Gα o/z over Gα i subtypes. Download figure Open in new tab Figure 1. D3R shows a Gα o/z -selective coupling profile. ( A ) Representative kinetic activation profiles measured using the G protein nanoBRET assay for dopamine receptors D2R, D3R, and D4R in response to application of 10 µM quinpirole applied at 10 seconds. Co-transfected Gα subunits from the four major G protein families are indicated. ( B ) Concentration-response curves for D2R, D3R, and D4R co-transfected with indicated Gα i/o/z subunits and stimulated with increasing concentrations of quinpirole. Data were normalized to the signal obtained with Gα oA and shown as mean ± SEM. N=5 independent replicates. ( C ) Radar plots summarizing pEC 50 and E max for D2R, D3R, and D4R in response to quinpirole across all six Gα i/o/z subunits. The observation that D3R fails to couple to Gα i subtypes, prompted us to investigate whether this reflects true molecular selectivity or results from suboptimal expression of either the receptor or the G protein in our system. We first tested whether varying the expression of D3R would rescue the coupling to Gα i . From this point onwards we used Gα i2 as a model to test D3R coupling. Accordingly, we transfected HEK293T cells with a fixed amount of either Gα oA or Gα i2 plasmids and co-transfected increasing amounts of the D3R plasmid. We found that D3R robustly activated Gα oA at all receptor expression levels tested, demonstrating the functionality of the assay across different transfection ratios ( Supplementary Figure 2A). In stark contrast, no activation of Gα i2 was observed with any amount of transfected D3R-encoding plasmid ( Supplementary Figure 2B). This suggests that receptor expression levels do not impact the assay ability to detect G protein activation effectively. To test whether the amount of Gα i2 was a limiting factor, we obtained concentration-response curves by treating cells transfected with a fixed amount of D3R and systematically increasing the levels of Gα oA , Gα i2 , and Gα z . As a crucial negative control, no signal was generated in cells not transfected with Gα proteins. As expected, increasing the expression of Gα oA and Gα z resulted in a concentration-dependent decrease in the maximal response (E max ) due to a shift of the equilibrium in the formation of the Gβγ-venus:GRK3-Nluc complex towards the G protein heterotrimer ( Supplementary Figure 2C). In contrast, Gα i2 failed to produce any measurable activation signal, demonstrating that the lack of coupling is an intrinsic property and not an assay artifact. Taken together, these control experiments further substantiate that the observed Gα o/z -selective coupling profile is an intrinsic property of D3R. Ligand-bias does not explain the lack of Gα i coupling of D3R The concept of biased agonism proposes that ligands can stabilize distinct active receptor conformations, promoting selective engagement with specific transducers and activation of particular signaling pathways 12 . To determine whether quinpirole might act as a biased ligand by stabilizing D3R conformations incapable of coupling to Gα i proteins, we compared its activity with multiple other dopamine receptor agonists. In addition to quinpirole and the endogenous ligand dopamine, we also included rotigotine, a potent D1R, D2R, and D3R agonist, which was recently used to induce an active conformation of D3R subsequently determined by cryo-EM structure 30 , 31 , sumanirole, a reported D2R-selective agonist, and ML417, a D3R-selective agonist. Using the G protein nanoBRET assay, we generated concentration-response curves for each ligand based on their ability to activate Gα oA , Gα i2 , or Gα z via D2R ( Figure 2A ) and D3R ( Figure 2B ). Notably, none of the tested agonists induced dissociation of the G i heterotrimer in D3R-expressing cells, although all were capable of activating G oA and G z ( Figure 2B ). We further confirmed that ML417 preferentially activates D3R over D2R. While a partial activation of Gα oA via D2R was observed at high ML417 concentrations, the ligand was 63-fold more potent at D3R and showed no activation of G i2 or G z . Additionally, we found that sumanirole triggered G z heterotrimer dissociation with similar potency at both receptors but was five times more effective at activating G oA via D2R compared to D3R. These findings exclude ligand bias as the cause of the inability of D3R to activate G i -dependent signaling. Download figure Open in new tab Figure 2. :The unique D3R activation profile is not a result of biased agonism. ( A ) Concentration-response curves obtained using the G protein nanoBRET assay in wild-type HEK293 cells expressing D2R with indicated G proteins and treated with indicated agonists. Data are shown as means ± SEM. N=5 independent replicates. ( B ) Concentration-response curves for D3R-transfected cells with indicated G proteins and treated with indicated agonists. Data were normalized to the signal obtained by applying dopamine to cells expressing Gα oA and shown as mean ± SEM. N=5 independent replicates. Orthogonal GloSensor assay confirms lack of D3R-Gα i2 coupling To further validate our G protein nanoBRET data, we employed the GloSensor cAMP inhibition assay in ΔGα i/o/z cells to eliminate background interference from endogenous Gα i/o/z proteins. Consistent with the nanoBRET data, stimulation with 1 µM quinpirole led to a marked decrease in cAMP levels across all the receptor-G protein combinations, except when D3R was co-expressed with Gα i2 ( Figure 3A–C ). The absence of cAMP inhibition in this condition further confirms that D3R does not functionally couple to Gα i2 , supporting the conclusion that D3R preferentially signals through Gα o and Gα z , rather than Gα i subunits. Download figure Open in new tab Figure 3. Orthogonal assays in ΔGα i/o/z cells confirm lack of Gα i2 coupling by D3R. Percentage fold change of cAMP accumulation in response to stimulation with 1 µM quinpirole of D2R ( A ), D3R ( B ), D4R ( C ) co-transfected with G oA , G i2 , and G z in ΔGα i/o/z HEK293 cells. Fold change was calculated as the ratio of maximal amplitude over baseline. cAMP levels were measured using the GloSensor assay in HEK293 cells lacking endogenous Gα i/o/z proteins (ΔGα i/o/z cells). Data are shown as mean ± SEM. N=5 independent replicates. ****P < 0.00001. ( D-E ) ΔGα i/o/z HEK293 cells were co-transfected with either D2R or D3R, one part of a plasmid encoding for Gα oA (208 ng) and increasing amounts of Gα i2 (208 ng for each part). G protein activation was measured using the G protein nanoBRET assay following stimulation with 1 µM quinpirole ( D ), or 1 µM dopamine ( E ). Data were normalized to 100% of the signal obtained by transfecting only Gα oA (CNT). Data are shown as mean ± SEM. N=3 independent replicates. To further confirm the selective coupling profile of D3R, we performed a competition experiment in ΔGα i/o/z HEK293 cells co-transfected with either D2R or D3R, a constant amount of Gα oA , and increasing amounts of Gα i2 . This setup divides the Gβγ-Venus sensor into two distinct pools: one associated with Gα oA and the other with Gα i2 . D2R, which couples to both G proteins, served as a control. As expected, increasing Gα i2 expression did not diminish D2R-mediated signaling in response to quinpirole ( Figure 3D ) or dopamine ( Figure 3E ), as D2R can activate both Gα pools, maintaining a strong overall signal. In contrast, D3R showed a markedly different profile. Increasing Gα i2 levels led to a concentration-dependent reduction in signal for both agonists, with over 86% reduction at the highest Gα i2 dose. This inhibition reflects the strict selectivity of D3R: because D3R cannot activate Gα i2 , the excess Gα i2 sequesters Gβγ-Venus into inactive complexes, thereby limiting the Gβγ available for Gα oA -mediated signaling. These results further demonstrate that D3R does not functionally couple to Gα i2 , even in the presence of excess Gα i2 protein. Structural determinants of D3R G protein coupling selectivity Building on our previous findings, we sought to investigate the molecular determinants of the D3R-G protein coupling selectivity. We focused on the C-terminal α5 helix of the Gα subunit, a region known to be a primary interface for GPCR interaction 32 – 34 . An alignment of the Gα oA and Gα i2 C-termini revealed key differences within the final 10 amino acids ( Figure 4A ). To test the importance of this region, we engineered two chimeric proteins by replacing the last 6 or 10 amino acids of the C-terminus of Gα i2 with the corresponding sequence from Gα oA , creating Gα i2 Gα oA(6) and Gα i2 Gα oA(10) ( Figure 4A-B ). To confirm the functional integrity of these chimeras, we assessed their activation by D2R in response to quinpirole ( Figure 4C ). As anticipated, both chimeras produced a clear activation signal, confirming productive coupling to the receptor. When tested with D3R, both chimeras exhibited a partial yet significant rescue of the coupling ( Figure 4D ). Specifically, Gα i2 Gα oA(6) restored 31% of the activation signal observed with wild-type Gα oA , while Gα i2 Gα oA(10) restored 37% of the signal. These results demonstrated that the Gα C-terminus is a critical determinant of coupling specificity for D3R. However, the incomplete nature of the rescue indicates that while these residues are essential, they are not the sole factor governing the interaction, suggesting that other regions of the G protein and possibly other contributors also influence the selective recognition. Download figure Open in new tab Figure 4. The C-terminal α5 helix (H5) is important for G protein coupling to dopamine receptors, but it is not the sole driver. ( A ) Alignment of the C-terminal α helix of Gα i2 , Gα oA , and two chimeric constructs in which the last 6 or 10 residues of Gα i2 were replaced with the corresponding residues from Gα oA , respectively indicated as G i2 G oA (6) and G i2 G oA (10). Gα i2 -specific residues are highlighted in red; GαoA-specific residues in blue. ( B ) Models of chimeric G protein constructs. ( C ) Concentration– response curves measured using the G protein nanoBRET assay for D2R (left) and D3R (right) co-transfected with Gα oA , Gα i2 , G i2 G oA (6), or G i2 G oA (10). All experiments were conducted in wild-type HEK293T cells stimulated with increasing concentrations of quinpirole. Data were normalized to the signal obtained with Gα oA and shown as mean ± SEM. N=5 independent replicates. Having established the importance of the Gα C-terminus, we applied a similar approach to investigate the molecular determinants of the coupling selectivity of D3R. The intracellular loop 2 (ICL2) and ICL3 are known to be critical G protein interaction sites 35 – 38 . We therefore aligned the amino acid sequences of the ICL2 and ICL3 regions of D2R and D3R to identify differences and similarities ( Figure 5A ). Based on this analysis, we engineered six chimeric receptors by swapping ICL2 and/or ICL3 between D2R and D3R ( Figure 5B ). To confirm that these receptor chimeras retained the ability to respond to quinpirole and activate G proteins, we adopted the G protein nanoBRET and used G oA as positive control. All six chimeric receptors produced a clear BRET signal upon stimulation, confirming they were properly expressed and functional ( Figure 5C ). The magnitude of activation was largely consistent with that of the wild-type receptor, except for the D3R D2-ICL3 chimera that showed significantly reduced coupling to Gα oA ( Figure 5C ). Although further structural modeling is needed, we speculate that the reduced signaling may result from steric clashes, as the D3R D2-ICL3 chimera combines the longer ICL2 of D3R with the longer ICL3 of D2R. Nonetheless, this control confirmed that all chimeric receptors were functionally competent. We then co-transfected the wild-type and chimeric receptors with Gα i2 and analyzed their response to quinpirole ( Figure 5D ). The results revealed a striking pattern that depended exclusively on the origin of the ICL2. Every transfected receptor containing the ICL2 of D2R, such as wild-type D2R, D2R D3-ICL3 , D3R D2-ICL2 , and D3R D2-ICL2/ICL3 , showed robust coupling to Gα i2 . Conversely, every receptor containing the ICL2 of D3R, such as wild-type D3R, D3R D2-ICL3 , D2R D3-ICL2 , and D2R D3-ICL2/ICL3 , showed no detectable Gα i2 activation. These findings pinpoint ICL2 as the primary molecular switch preventing the interaction between D3R and Gα i , thereby enforcing the selective signaling profile observed for D3R throughout this study. Download figure Open in new tab Figure 5. Involvement of dopamine receptor intracellular loops in defining G protein selectivity. ( A ) Sequence alignment of intracellular loop 2 (ICL2) and intracellular loop 3 (ICL3) of human D2R and D3R, highlighting the degree of conservation across the aligned sequences: *=full conservation, .=strong conservation, :=weak conservation. ( B ) Schematic representation of chimeric receptor constructs in which ICL2 and/or ICL3 of D2R and D3R were swapped. ( C ) Concentration–response curves obtained using the G protein nanoBRET assay for Gα oA activation by wild-type and loop-swapped D2R and D3R constructs. ( D ) Corresponding concentration–response curves for Gα i2 activation. All experiments were conducted in wild-type HEK293T cells stimulated with increasing concentrations of quinpirole. Data were normalized to the signal obtained with Gα oA and shown as mean ± SEM. N=5 independent replicates. An investigation of the recently published structure of D3R bound to Gα oA -subtype of G protein (PDB:9F33) revealed that G350 is positioned in close proximity to a negatively charged cavity on D3R ( Figure 6A ). The presence of an Asp residue in this location in Gα i might lead to electrostatic repulsion, destabilizing the interaction. In contrast, in D2R bound to Gα i1 (PDB: 7JVR), the corresponding Asp residue (D350) is stabilized by a surrounding positively charged cavity ( Figure 6A ). In addition to this, T142 ICL2 on D3R is reported to get phosphorylated 39 , and the bulky phosphate group can be accommodated by a Gly residue. However, the presence of an Asp residue in this position would not only lead to electrostatic repulsion but also a steric clash. Interestingly, in the reported structures of D2R bound to either Gα oA (PDB:8TZQ) or Gα i1 (PDB:7JVR), the corresponding T144 4. 34 exhibits a differential rotamer and points away from the Gα subunit ( Figure 6B ). Superimposing the structures reveals that A345, R349, and Y354 occupy a similar position in both D2R and D3R ( Figure 6C ) as well as the corresponding residues in Gα i1 in D3R-bound Gα i1 ( Figure 6C ) . While Y354 could potentially facilitate additional interactions with the surrounding positively charged pocket as compared to F354, the electron cloud over the benzene ring in both helps stabilize the G proteins in their corresponding receptor cavities. Download figure Open in new tab Figure 6. Structural comparison of the G protein binding cavity of D2R and D3R. ( A ) Surface representation of the charge distribution in the pocket occupied by Gα oA in D3R (top) and Gα i1 in D2R (bottom). ( B ) T142 ICL2 faces towards the ⍺5-helix in D2R, while T144 4. 34 in D3R exhibits a different rotamer, facing away from the ⍺5-helix. ( C ) Superimposition of the G⍺ subunit in the various structures reveals the orientation of the differing residues. (D3R-Gα oA : PDB – 9F33, D3R – cornflower blue, Gα oA – rosy brown; D2R-Gα oA : PDB – 8TZQ, D2R – pale violet red, Gα oA – gray; D2R-Gα i1 : PDB – 7JVR, D2R – brick red, Gα i1 – peach puff). The unique coupling profiles of D2R and D3R produce distinct responses in hippocampal CA1 neurons To examine the unexpected coupling profile of D3R in a physiological context, we focused on cAMP signaling in CA1 hippocampal neurons that richly express D2R and D3R 40 . We crossed the cAMP Encoded Reporter ( CAMPER ) mice, which conditionally express the T EPac VV FRET-based cAMP reporter 41 , with the CaMKIIα-Cre strain to achieve expression in the hippocampus 42 . Indeed, 2-photon imaging in acute brain slices from CaMKIIα-Cre:CAMPER +/- revealed robust biosensor expression in the CA1 region ( Figure 7A ). We then used bath application of agonists at a low concentration (100 nM) to target D2R (sumanirole) or D3R (ML417) activation ( Figure 7B ). The Gα s -coupled β-adrenergic receptor was stimulated with isoproterenol (100 nM) as a control ( Figure 7B ). Sumanirole induced robust inhibition of cAMP, which is consistent with D2R-mediated Gα i activation ( Figure 7C ). On the other hand, ML417 application paradoxically led to an increase in cAMP production ( Figure 7C ). As a control, the recording buffer alone exerted no change in cAMP dynamics. Subsequent application of isoproterenol increased cAMP in both the buffer and ML417 experiments, however the effect of isoproterenol was significantly greater after ML417 priming ( Figure 7C ). Pre-treatment with sumanirole completely blocked the effect of isoproterenol ( Figure 7C ). These data suggest sumanirole strongly inhibits AC activity via D2R-Gα i and may display slow off-rate kinetics as cAMP inhibition persisted even in the presence of isoproterenol. The ability of ML417 to enhance cAMP supports the notion that D3R-Gα o leads to release of Gβγ to conditionally sensitize AC activity 43 . This likely explains the sensitized cAMP response to isoproterenol following ML417 ( Figure 7D ). Collectively, these results capture how the exquisite coupling profiles of D2R and D3R contribute toward neuronal signal transduction. Download figure Open in new tab Figure 7. Agonism of D2R, but not D3R, inhibits cAMP signaling in CA1 hippocampal neurons. (A) Schematic of approach to visualize dorsal hippocampal CA1 neuronal cAMP dynamics in acute brain slices from CaMKIIα-Cre:CAMPER +/- adult mice. Scale bar represents 100 microns. (B) Average trace of cAMP responses to agonist stimulation with ML417 500 (100 nM; n=35 neurons/6 mice), Sumanirole maleate (100 nM; n=38 neurons/6 mice), or control buffer (n=35 neurons/6 mice). In the same experiment slices were then immediately stimulated with Isoproterenol (Iso; 100 nM). ( C ) left) Maximum agonist induced cAMP amplitude calculated as peak agonist response minus basal cAMP level. One-way ANOVA was performed with Dunnett’s multiple comparisons test to buffer control. F=99.23, R 2 =0.6331, F (DFn, DFd)=1.365 (2, 115). right) Maximum Iso induced cAMP amplitude calculated as peak iso response minus peak agonist response. One-way ANOVA was performed with Dunnett’s multiple comparisons test to buffer control. F=239.4, R 2 =0.8063, F (DFn, DFd)=7.619 (2, 115). Data shown as mean ± SEM where each dot represents an individual neuron. Exact P values are depicted on the graphs. ( D ) Schematic illustration of D2R and D3R signal decoding in dorsal hippocampal CA1 neurons. DISCUSSION Our study conclusively demonstrated that D3R is incapable of productive coupling with Gα protein subtypes Gα i1 , Gα i2 , and Gα i3 , while it effectively couples to and activates Gα o and Gα z . Although previous studies have partially addressed the G protein coupling selectivity of D3R, they produced inconsistent findings across different assay platforms, likely because of using modified systems that did not employ wild-type G proteins 26 – 28 , 31 . In detail, results from Lane and colleagues suggested a preferential coupling of D3R with Gα oA , minimal or absent activation of Gα i1-3 subtypes, while coupling to Gα z was not assessed 26 . Their conclusions were supported by two sets of experiments. In the first, they engineered the tetracycline-inducible expression of PTX-resistant Gα subunits in HEK293 cells constitutively expressing D2R or D3R. Cells were treated with 10 µM dopamine, and receptor activity was assessed with a [ 35 S]GTPγS binding assay. While their data suggested D3R does not couple to Gα i2 , the induction of Gα i2 expression also elevated basal activity in unstimulated cells, complicating the interpretation of the results. Additionally, the engineered G proteins carried a point mutation in the α5 helix, a region critical for receptor interaction, raising concerns that the observed lack of Gα i coupling could have been the reflection of using an artificial system. Using an alternative approach, they generated fusion proteins of dopamine receptors and PTX-insensitive G proteins to gain control over expression stoichiometry. However, elevated basal activity of D3R compared to D2R, combined with α5 helix modifications, again left unresolved the question of true Gα i coupling. An independent group later applied miniG protein recruitment and G-CASE to profile the G protein coupling landscape of all five dopamine receptors 27 . Using a NanoBiT complementation assay based on miniG proteins, they detected miniG o recruitment by D2R, D3R, and D4R, while miniG i1 was only recruited by D2R. However, application of the G-CASE assay resulted in discrepant observations of broader coupling, with D2R, D3R, and D4R all appearing to engage each member of the G i/o/z family. Both assays have inherent limitations. MiniG proteins are truncated Gα subunits optimized for stable GPCR binding, but they lack the GTPase domain, which is a critical determinant of G protein coupling selectivity, thereby increasing promiscuity and reducing physiological relevance 44 . Similarly, the G-CASE assay does not employ wild-type G proteins, and Gα tagging positions may affect sensor performance, especially when the Gα:Gβγ interface undergoes subtle rearrangements rather than full dissociation 27 . Recent cryo-EM studies have resolved D3R-Gi complexes; however, such static structures can misrepresent physiological coupling, especially when stabilized using nanobodies, detergent micelles, or mutated receptor constructs 28 , 31 . Notably, signaling studies associated with these structures were obtained using the TRUPATH system, which shares similar design and limitations of the G-CASE assay, or with a NanoBiT recruitment performed in insect cells, which lack the mammalian post-translational modifications that could be crucial for authentic receptor behavior. In contrast, our study employed wild-type Gα proteins expressed in both parental and ΔGα i/o/z HEK293 cells in two complementary functional assays. This experimental design, combined with the inclusion of multiple positive and negative controls, minimized artifacts associated with engineered or truncated Gα proteins and enabled a more physiologically relevant evaluation of receptor coupling selectivity. Our findings confirmed that D2R and D4R couple broadly to all Gα i/o/z subtypes, while D3R exhibits exclusive coupling selectivity for Gα oA , Gα oB , and Gα z , with no detectable engagement with Gα i1 , Gα i2 , and Gα i3 under any tested condition. Employing both G protein and receptor chimeras, we explored the molecular basis underlying this coupling selectivity. We confirmed the importance of the interface between the C-terminal α5 helix of the Gα proteins and the ICL2 of D3R in defining the biased coupling of D3R toward Gα o , while we showed that the ICL3 does not contribute to this selectivity. This is in conflict with previous findings that introducing 12 amino acids from the C-terminal segment of the ICL3 of D2R into the corresponding region of D3R partially restored Gα i coupling 26 . Such discrepancies may arise from engineered features that bypass natural coupling constraints, highlighting the need for further molecular investigations. Models of the receptor-G protein interaction, based on available cryo-EM structures 28 , 31 , suggest that steric clashes and electrostatic repulsion may be responsible for the inability of D3R to engage Gα i proteins. In particular, our analysis points at the involvement of D350 in Gα i2 and T142 ICL2 in D3R, especially if phosphorylated by GRK2 as previously reported 39 . The role of these residues will require further investigations. The Gα o/z -biased coupling of D3R has significant physiological implications. Although Gα subunits within the G i/o/z family share high sequence similarity, they can engage distinct effectors and mediate non-redundant functions, as shown by the divergent phenotypes observed in Gα o , Gα i , and Gα z knockout models and by recent studies characterizing unique intracellular effectors 43 , 45 – 50 . The restricted coupling profile of D3R suggests it may activate a distinct subset of signaling pathways limited to Gα o/z and Gβγ-mediated responses, or it may bias signaling by excluding Gα i subtypes, thereby enhancing the activation of alternative effectors, such as GIRK channels or Gβγ-sensitive adenylyl cyclases. As a promising target for treating substance use and other neuropsychiatric disorders 11 , 51 , 52 , D3R offers an additional layer of selectivity by modulating specific signaling pathways within defined cellular or subcellular contexts. Such enhanced selectivity holds the promise for the development of precisely targeted therapeutic strategies with reduced side effects. In this context, receptor-selective agonists are valuable both as research tools and potential treatments, offering improved outcomes compared to non-selective compounds due to differences in both receptor distribution and signaling 53 . Sumanirole, a D2R-selective agonist with 200-fold higher affinity for D2R over D3R 54 , exemplifies this approach by selectively initiating D2R-mediated effects in animal models, including prolactin release, regulation of striatal acetylcholine levels, and mediating autoreceptor-driven inhibition of nigrostriatal firing 54 . Similarly, pramipexole, a drug initially developed as a D2R agonist and later found to have high potency and selectivity for D3R 55 , has a demonstrated ability to delay L-DOPA-induced dyskinesia in the treatment of Parkinson’s disease 55 – 57 , suggesting that its clinical benefits may be partly due to D3R unique signaling properties. Although evidence for D3R-specific physiological effects is still emerging, the recent development of ML417 as a highly selective D3R agonist represents a major step forward in understanding D3R function and therapeutic potential 58 . ML417 has already been shown to possess neuroprotective properties, along with promising safety and pharmacokinetic profiles that would make it a strong lead compound for further drug development. Overall, D3R represents a unique case among GPCRs as no other GPCR with a primary coupling to G i/o/z heterotrimeric proteins has been reported to be incapable of activating a subset of the family members. These insights not only clarify previous inconsistencies in the literature but also open new avenues for selective D3R pharmacology. METHODS Ethics statement Procedures involving mice strictly followed NIH guidelines and were approved by the Institutional Animal Care and Use Committee at Augusta University. Mice were housed at consistent temperature with unlimited access to food and water under a constant 12-hour light/dark cycle. The following previously described strains were utilized: i.) cAMP E ncoded R eporter ( CAMPER ) (C57BL/6-Gt(ROSA)26Sortm1(CAG-ECFP*/Rapgef3/Venus*)Kama/J) (RRID:IMSR_JAX:032205) 41 , ii.) CaMKIIα-Cre (B6.Cg-Tg(Camk2a-cre)T29-1Stl/J) (RRID:IMSR_JAX:005359) 42 . The two strains were crossed to obtain experimental CaMKIIα-Cre:CAMPER +/- mice. Both sexes were used and animal identification was verified by automated genotyping PCR (Transnetyx). DNA plasmids and chemicals The plasmids encoding human codon-optimized DRD2, DRD3, and DRD4 were subcloned from the PRESTO-Tango library (Addgene; #66269, #66270, and #66271), a generous gift from Dr. Bryan Roth (UNC, Chapel Hill, NC). Gβ1-venus 156–239 and Gγ2-venus 1-1 55 were generous gifts from Dr. Nevin A. Lambert (Augusta University, Augusta, GA). Gα proteins and masGRK3CT-Nluc constructs were generous gifts from Dr. Kirill A. Martemyanov (UF Scripps Biomedical Research, Jupiter, FL). pGloSensor™-22F was purchased from Promega. Dopamine hydrochloride (#3548), quinpirole hydrochloride (#1061), and forskolin (#1099) were purchased from Tocris, while rotigotine (#HY-75502), sumanirole maleate (HY-70081A), and ML417 (HY-136390) were purchased from MedChemExpress. All chemicals were resuspended according to manufacturers’ instructions, aliquoted, and stored at -20°C until use. Cell cultures and transfections HEK293T/17 cells were purchased from ATCC, while Gα i/o/z -deficient HEK293A cells (ΔGα i/o/z ), a generous gift from Dr. Asuka Inoue (Tohoku University, Japan), were previously generated using three rounds of CRISPR–Cas9 mutagenesis and validated 59 . Cells were cultured in DMEM (Gibco, 10567-014) supplemented with 10% FBS (Biowest, S1520), non-essential amino acids (Gibco, 11140-050), 100 units/ml penicillin and 100 µg/ml streptomycin (Gibco, 15140-122), and 250 µg/ml amphotericin B (ThermoFisher, 15290-018) at 37°C and 5% CO 2 . For transfection, two million cells were seeded in each well of 6-well plates in 1.5 mL medium per well without antibiotics and containing 10% dialyzed FBS (Biowest, S181D) for 4 hours and then transfected with a mixture containing a 1:3 ratio of DNA plasmid (2.5 µg) and polyethylenimine (PEI; 7.5 µl) (VWR, AAA43896) in a final volume of 500 µL per well. The following amount of each plasmid was transfected, unless otherwise indicated: 0.21 µg GPCR (D2R, D3R, or D4R), 0.83 µg Gα protein (Gα oA , Gα oB , Gα i1 , Gα i2 , Gα i3 , Gα z, Gα s , Gα q , Gα 12 ), 0.21 µg Gβ1-venus 156–239 , 0.21 µg Gγ2-venus 1-1 55 , and 0.013 µg masGRK3CT-Nluc. An empty vector, pcDNA3.1, was used to normalize the ratio of transfected plasmids. Transiently transfected cells were incubated for 18-22 hours before being tested. G protein nanoBRET assay The day after transfection, cells were briefly washed with PBS, resuspended in BRET buffer (PBS supplemented with 0.5 mM MgCl 2 and 0.1% glucose), collected in 1.5 ml tubes, and centrifuged for 5 minutes at 500 x g. Pelleted cells were resuspended in 300 µl of BRET buffer, and 25 µl of cells were plated in 96-well white microplates (Greiner Bio-One). The Nluc substrate Hikarazine-103 was purchased from Synthelis and used according to the manufacturer’s instructions. BRET measurements were obtained using a POLARstar Omega microplate reader (BMG Labtech). All measurements were performed at room temperature, and the BRET signal was determined by calculating the ratio of the light emitted by Gβ1γ2-venus (collected using the emission filter 535/30) to the light emitted by masGRK3CT-Nluc (475/30). In kinetics assays, the baseline value (basal BRET ratio) was averaged from recordings of the five seconds before agonist injection. In concentration-response experiments, 25 µl of cells per well were plated and mixed with the Nluc substrate. Initial readings were performed to establish basal BRET ratio, and then agonists were added. The BRET signal was recorded for 60 seconds. ΔBRET ratios were obtained by subtracting the basal BRET ratio from the maximal amplitude measured. cAMP inhibition assay 1 million ΔGα i cells were seeded in each well of 6-well plates. 4 hours later, cells were transfected with plasmids for mammalian expression of individual dopamine receptors (D2R, D3R, or D4R), pGloSensor™-22F cAMP plasmid (Promega), and individual Gα proteins Gα oA , Gα i2 , or Gα z . 18-22 hours after transfection, cells were collected in PBS, centrifuged at 500 x g for 5 minutes at room temperature, and the supernatant was discarded. The pelleted cells were resuspended in 300 μl of BRET buffer (PBS supplemented with 0.5 mM MgCl 2 and 0.1% glucose), and 40 μl was plated into each well of a white 96-well plate, followed by the addition of 10 μl of D-Luciferin potassium salt substrate (GoldBio; #LUCK-100; final concentration 600 µg/ml). After incubation at 37°C for 1h, the plate was placed in a BMG Omega microplate reader and maintained at 28°C until a stable baseline value was recorded. Cells were then treated with 1 μM quinpirole for 30 seconds before adding 0.5 μM forskolin and recording luminescence for 25 minutes at 72-second intervals. A fold change (FC) was calculated for each sample by dividing the maximum relative light units (RLU) obtained after forskolin application by the average baseline RLU. For the quinpirole concentration-response curve, the FC for samples treated with the vehicle was averaged and considered 100% of forskolin-induced cAMP levels. The FC of all other samples was then normalized to the vehicle control FC average and expressed as a percentage of cAMP inhibition. 2-photon FRET cAMP imaging Acute brain slices were prepared from adult mice (2-4 months of age) as similarly described 60 , 61 . Animals were isoflurane anesthetized followed by decapitation and rapid extraction of the brain, which was subsequently agarose-mounted on a vibratome (Precisionary VF-310-0Z) in ice-cold oxygenated buffer consisting of (in mM): KCl (2.5), NMDG (93), glucose (25), HEPES (20), sodium ascorbate (5), sodium pyruvate (3), thiourea (2), NaH 2 PO 4 (1.2), CaCl 2 (0.5), MgCl 2 (10), NaHCO 3 (30). 300-micron thick coronal slices containing the hippocampus were sectioned and incubated for 1 hour at 34°C in an oxygenated recovery buffer consisting of (in mM): NaCl (126), KCl (2.5), CaCl 2 (2), MgCl 2 (2), NaHCO 3 (18), NaH 2 PO 4 (1.2), glucose (10). Slices were then maintained at ambient temperature oxygenated recording buffer consisting of (in mM): NaCl (125), KCl (2.5), CaCl 2 (2), MgCl 2 (2), NaH 2 PO 4 (1.25), NaHCO 3 (25), glucose (25). Individual brain sections were then constantly perfused in recording buffer at approximately 2 ml per minute in a recording chamber (Warner Instruments) for FRET imaging on a Zeiss 780 multiphoton confocal microscope (20X W Plan-Apochromat objective). Excitation of the cAMP FRET donor (mTurquoise2) was achieved by tuning a Ti:Sapphire laser (Coherent Chameleon Vision S) to 850 nm. Individual photomultiplier tubes were utilized to simultaneously capture emission from the FRET donor (455-509 nm) and FRET acceptor (Venus; 526-571 nm) at 2.5 second intervals. ML417 (MedChemExpress HY-136390), Sumanirole maleate (MedChemExpress HY-70081A), and isoproterenol hydrochloride (Iso; TCI America I0260) were bath applied as indicated in the text. FRET values were calculated using standard ImageJ tools from the raw fluorophore intensity at the neuronal cell body. Non-responsive cells were excluded based on a previously established cutoff criterion (two multiplied by the standard deviation of the baseline prior to drug treatment) 62 , 63 . Statistical analysis Statistical analysis was performed using GraphPad Prism version 10 software. Concentration-response curves were fitted to a sigmoidal four parameter logistic function (variable slope analysis) to quantify agonist potencies (pEC 50 ), maximal responses (E max ). Concentration-response curves were fitted and Hill slope values for each agonist were close to 1. At least three independent biological replicates were used for each experiment. Data Availability Statement All processed data that support the findings of this study are available within the paper and its supplemental data. Any additional information required to reanalyze the data reported in this paper is available from the corresponding author upon request. Acknowledgments This work was supported by NIH awards R01 DC022104 to C.O and R01 NS129554 to B.S.M. We thank Dr. Asuka Inoue at Tohoku University for the CRISPR-knockout ΔGα i/o/z HEK293 cell line under a Materials Transfer Agreement. The CAMPER mouse strain was a gift from Dr. Kirill Martemyanov. The Cell Imaging Core facility at Augusta University was utilized for 2P imaging. Funder Information Declared National Institute on Deafness and Other Communication Disorders , DC022104 National Institute of Neurological Disorders and Stroke, https://ror.org/01s5ya894 , NS129554 REFERENCES ↵ Klein , M. O. et al. Dopamine: Functions, Signaling, and Association with Neurological Diseases . Cell Mol Neurobiol 39 , 31 – 59 ( 2019 ). doi: 10.1007/s10571-018-0632-3 OpenUrl CrossRef PubMed Missale , C. , Nash , S. R. , Robinson , S. W. , Jaber , M. & Caron , M. G . Dopamine receptors: from structure to function . 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