Dual-mode intramolecular agonist-dependent mechanoactivation of the adhesion GPCR ADGRG1/GPR56

Dual-mode intramolecular agonist-dependent mechanoactivation of the adhesion GPCR ADGRG1/GPR56 | 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 Dual-mode intramolecular agonist-dependent mechanoactivation of the adhesion GPCR ADGRG1/GPR56 View ORCID Profile Chaoyu Fu , Cheng Quan , View ORCID Profile Ying Zhang , View ORCID Profile Gaojie Song , View ORCID Profile Tobias Langenhan , View ORCID Profile Jie Yan doi: https://doi.org/10.1101/2025.09.16.676693 Chaoyu Fu 1 Department of Physics, National University of Singapore , Singapore 117551 2 Mechanobiology Institute, National University of Singapore , Singapore 117411 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chaoyu Fu Cheng Quan 2 Mechanobiology Institute, National University of Singapore , Singapore 117411 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ying Zhang 3 Genome Institute of Singapore (GIS), Agency for Science, Technology and Research (A*STAR) , Singapore 138672 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ying Zhang Gaojie Song 4 School of Life Sciences, East China Normal University , Shanghai, China , 200241 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gaojie Song Tobias Langenhan 5 Rudolf Schönheimer Institute of Biochemistry, Division of General Biochemistry, Medical Faculty, Leipzig University , Leipzig, Germany , 04103 6 Institute of Biology, Faculty of Life Sciences, Leipzig University , 04103 Leipzig, Germany 7 Comprehensive Cancer Center Central Germany (CCCG) , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tobias Langenhan For correspondence: tobias.langenhan{at}gmail.com phyyj{at}nus.edu.sg Jie Yan 1 Department of Physics, National University of Singapore , Singapore 117551 2 Mechanobiology Institute, National University of Singapore , Singapore 117411 8 Centre for Bioimaging Sciences, National University of Singapore , Singapore 117557 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jie Yan For correspondence: tobias.langenhan{at}gmail.com phyyj{at}nus.edu.sg Abstract Full Text Info/History Metrics Preview PDF SUMMARY PARAGRAPH Mechanical stimuli instruct cardinal cellular decisions pertaining to their fate, proliferation, morphology, and movement 1 , 2 . How adhesion G protein-coupled receptors (aGPCRs), a large family of mechanosensors with more than 30 members in humans 3 – 5 , transduce mechanical cues into metabotropic commands, has been a matter of debate due to the lack of suitable approaches to analyze receptor activation during mechanotransduction in live cells 6 . Here we use human ADGRG1/GPR56 (G1), an aGPCR with roles in brain development 7 , skeletal muscle 8 , and platelet function 9 , to study the events during aGPCR mechanotransduction. We show that G1 dissociation, taking place at ∼18 pN (ref. 10 ), occurs at retracting fibers during cell migration over an adhesive substrate 11 , an effect that can be enhanced by cell stretching. Simultaneous live recording of G protein recruitment through total internal reflection fluorescence (TIRF) imaging and pharmacological assays during force transmission onto G1 show graded receptor responses with sub-maximal signaling before receptor dissociation, and maximal activation thereafter. Both modes require the intramolecular agonist/Stachel element 12 , 13 of the receptor. Our findings demonstrate that non-dissociative and dissociative aGPCR signaling can emerge from the same aGPCR depending on mechanical stimulus magnitude, demonstrating the capability of aGPCRs for graded receptor responses upon force detection. aGPCRs are complex cell surface molecules with extended extracellular regions (ECR) that contain receptor-specific adhesion motifs for interaction with cellular and matricellular ligands 3 . The ECRs are mounted on the archetypical heptahelical transmembrane (7TM) domain of all GPCRs, while their intracellular regions (ICR) potentially provide anchoring to the cytoskeleton 3 , 5 , 14 , 15 . After biosynthesis many aGPCRs self-cleave into N-terminal (NTF) and C-terminal fragments (CTF) and form non-covalently associated complexes at the cell surface 16 – 19 . This remarkable post-translational feature is permitted through the presence of a GPCR autoproteolysis-inducing (GAIN) domain, the hallmark domain of all aGPCRs, which is located in the ECR near the membrane 19 . Receptor self-cleavage occurs at a GPCR proteolysis site (GPS) within the GAIN domain, which divides the GAIN domain itself into a larger N-terminal and a smaller C-terminal portion, the latter of which corresponds to the most C-terminal β-strand of the GAIN domain 19 . Apart from its structural role within the GAIN domain, this element stimulates receptor activity by interacting with the 7TM domain as a tethered/intramolecular agonist (also termed Stachel). Ample pharmacological 12 , 13 , structural 20 – 23 and in vivo evidence 9 , 24 – 26 attests to this activation paradigm across the aGPCR family. Their molecular structure of aGPCRs lends itself to the activation by mechanical forces, e.g. during neuronal mechanosensation 6 , 27 , 28 , neural development 29 , myelination 30 , immune cell function 25 , 31 , 32 or the control of surfactant levels in the lung 26 . The ECRs of individual aGPCRs sample the presence of local cognate adhesive ligands outside the receptor expressing cells, which laterally anchor them in the membrane 33 , and receive mechanical forces transmitted onto the receptors by relative movement between receptor and ligand. Incoming mechanical stimuli are thought to dissociate the NTF-CTF structure of autoproteolysed aGPCRs 29 thereby exposing the Stachel agonist to allow its interaction with the 7TM domain (dissociation model) as exemplified for ADGRD1/Gpr133 34 , ADGRE2/EMR2 32 , ADGRE5/CD97 25 , 31 , ADGRF5/Gpr116 26 , G1 9 , and ADGRG6/Gpr126 30 . Other studies demonstrated that despite suppression of aGPCR self-cleavage via mutagenesis, receptor function can remain unperturbed in vitro 35 and in vivo 28 , 36 , 37 dependent on the integrity of the Stachel core sequence even though NTF-CTF separation is not feasible (non-dissociation model) 12 , 22 , 24 , 38 , 39 . This could be achieved by the conformational flexibility of the GAIN domain, which allows the Stachel to interact with the 7TM domain while still bound to the GAIN domain 40 , or by other yet unknown means. In addition, single-molecule measurements indicated that separation of cleaved GAIN domains, and thus, NTF-CTF separation of a cleaved aGPCR, requires mechanical forces above a threshold defined by the non-covalent network of hydrophobic interactions within the autoproteolyzed domains 19 . E.g. G1, ADGRL1/LPHN1, and ADGRL3/LPHN3 dissociate at physiologically relevant forces of approximately 10-20 pN (refs. 10 , 41 ) after partial unfolding of the subdomain B of the GAIN domain, which occurs around 5-10 pN for both cleavable and non-cleavable receptor versions. Collectively, these findings imply that mechanical forces can stimulate aGPCR activity prior to receptor dissociation and transduce graded force stimuli before and after receptor dissociation, respectively. How aGPCR self-cleavage, dissociation, and activation are related to each other has been hampered by the lack of approaches to apply physiologically relevant mechanical forces to aGPCRs, independently observe the impact on their constituent NTF and CTF, and to simultaneously record their metabotropic output. In order to assess the relationship between ligand-dependent mechanical stimulation of aGPCRs through physiologically relevant forces, GAIN domain autoproteolysis, receptor dissociation and intramolecular agonism, we chose the human aGPCR ADGRG1/Gpr56 (G1) as a model ( Fig. 1a ). G1 has a wide spectrum of physiological tasks in neural development 7 , immune 42 and platelet 9 functions, skeletal muscle homeostasis 8 , and relevance for the emergence and metastasis of neoplasias 43 . G1 is a minimalist aGPCR containing only pentraxin and laminin/neurexin/sex hormone-binding globulin-like (PLL) and GAIN domains in its ECR 44 ( Fig. 1a ), which interacts with multiple ligands 43 , 45 . G1 is autoproteolysed 46 , its agonistic Stachel agonist has been defined and is required for receptor activation in vitro 13 and in vivo 9 . Further, the cryo-EM structure of the active conformation of the G1-CTF has been obtained recently 21 . Finally, mechano-activated G1 couples to Gα 13 , and controls a RhoA-dependent pathway that governs cytoskeleton reorganization 9 , 13 , 43 , 46 . Download figure Open in new tab Fig. 1. Methods for Real-Time Monitoring of GAIN Domain Dissociation and aGPCR Activation (a) G1 receptor structure: PLL and GAIN domains in the ECD, a 7TM domain, and an ICD. GPS cleavage produces NTF and CTF, which remain associated. Activation requires the intramolecular Stachel agonist and can occur via ligand or mechanical stimuli. (b) G1 receptor design for ‘migration force assay’: extracellular Spy and Halo Tags enable adhesion (via SpyCatcher) and JF646 labeling, with an intracellular EGFP fusion. α-G1 targets the GAIN domain. (c) ‘Migration force assay’ setup: SpyTag-HaloTag-G1-EGFP adheres to SpyCatcher+FN-coated surfaces, visualizing receptor dissociation during cell movement. (d) G1 GPS sequence, autoproteolysis site, and signaling-impacting mutations (H381S, T383A, F385A, L388A/M389A) (left). Western blot analysis of COS-7 cells expressing G1 variants (G1 X corresponds to GPS mutations) (right). (e) Quantification of Western blot cleavage ratios for indicated G1 variants. Data (n≥5 replicates for all groups) shown in a scatter plot with bar (data points plotted as mean ± SD); analyzed via an ordinary one-way ANOVA with Tukey’s multiple comparisons test. P values indicated. RESULTS Protein design We engineered a G1 version, whose NTF was bestowed with a HaloTag N-terminal to the PLL domain. The addition of JF646, an organic dye, to the cell culture medium, leads to covalent HaloTag binding to mark G1-NTF for imaging analyses. To enable concomitant visual inspection of G1-CTF in live cells, the C-terminus of the receptor was fused to EGFP. We appended a SpyTag to the G1-NTF to accommodate faithful force transmission onto G1 when the SpyTag forms a stable covalent bond upon binding to SpyCatcher protein 47 ( Fig. 1b ). To this end, SpyCatcher was coated on a surface as an artificial ligand interface for migrating COS-7 cells expressing the final SpyTag-HaloTag-G1-EGFP protein layout ( Fig. 1c ). To test for intact auto-proteolytic activity of SpyTag-HaloTag-G1-EGFP, it was expressed in COS-7 cells, which do not endogenously produce detectable G1 ( Fig. 1d ). We also generated mutated SpyTag-HaloTag-G1 H 381 S -EGFP, SpyTag-HaloTag-G1 T 383 A -EGFP, SpyTag-HaloTag-G1 F 385 A -EGFP, and SpyTag-HaloTag-G1 L 388 A/M389A -EGFP receptor variants with different effects on G1 self-cleavage and activation. All point mutations are located in subdomain B of the G1 GAIN domain and flank the GPS of G1 (ref. 44 ) ( Fig. 1d ). Western blot analysis of cell lysates confirmed autoproteolysis of the fusion protein by generation of a ∼95 kDa SpyTag-HaloTag-G1-NTF fragment ( Fig. 1d ), consistent with the highly N-glycosylated G1-NTF (ref. 48 ). The H381S mutation prevents GAIN domain-mediated autoproteolysis while retaining an intact agonistic Stachel sequence 19 , 44 , 49 , 50 . Mutations T383A and F385A also impair receptor self-cleavage but additionally perturb the Stachel sequence 10 , 50 , 51 . In particular, F385 is a highly conserved Stachel residue that is critical for G1 activation 13 . Finally, the L388A/M389A double mutant retains autoproteolysis but severely impairs intramolecular agonist-dependent receptor activity 38 . Western blot analysis showed largely reduced cleavage efficiencies apparent by the generation of full-length protein at ∼150 kDa compared to the SpyTag-HaloTag-G1-EGFP protein, corresponding to 18 % (H381S), 5 % (T383A), 34 % (F385A), and 95 % (L388A/M389A), respectively ( Fig. 1d-e ). We also observed reduced total protein expression due to suppression of G1 self-cleavage as reported previously ( Fig. 1d-e ) (ref. 44 ). A real-time assay to monitor migration force and ligand-dependent aGPCR dissociation We developed two mechanotransduction paradigms to investigate ligand- and mechanodependent activation of G1 signaling. In the first paradigm (henceforth termed ‘migration force assay’) we surmised that during cell migration the covalent SpyTag-SpyCatcher bond provides a stable force-bearing point. This allows force transmission onto the G1-ECR at a physiologically relevant loading rate in the pN/s range 52 , 53 and is sufficient for separation of G1’s self-cleaved GAIN domain and, thus, also dissociation of the NTF-CTF heteroprotomer structure 16 , 17 , 40 . Using magnetic tweezers, we previously determined that the isolated GAIN domain of G1 dissociates at forces between 10-20 pN (at a loading rate of 1.0 pN/s) 10 , while single-molecule atomic force microscopy (smAFM) measurements found a G1-GAIN domain separation at 100-160 pN (at loading rates of 1,000-40,000 pN/s) 54 . Both receptor dissociation thresholds are well below the SpyTag-SpyCatcher breakage force of ∼1.9 nN (ref. 47 ). COS-7 cells expressing cleavable and non-cleavable SpyTag-HaloTag-G1-EGFP proteins ( Fig. 1d ) were seeded on fibronectin (FN) to support cell migration, or an FN mixed with SpyCatcher (FN+Spy)-coated polydimethyl-siloxane (PDMS) surface for receptor adhesion ( Fig. 1c ). Based on the fluorescent labels attached at the NTF (JF646, magenta) and CTF (EGFP, green), intact non-dissociated G1 receptors showed by an overlay of both labels (for better channel recognition the color palette was inverted; thus NTF+CTF is false-colored in black). We harnessed the rear detachment phenomenon 11 of migrating cells and imaged their trailing edge upon cell movement, as dissociated NTF should remain adhered to the ligand-coated surface, where it is discernible at high contrast. Due to the labeling strategy, we could differentiate between intact aGPCR heteroprotomers in migrasomes 14 , vesicles formed by plasma membrane material upon breaking off retracting fibres during migration 55 , and dissociated G1 receptors, where CTF resident in the retracting fiber membrane is separated from the NTF adhered to the extracellular ligand surface ( Supplementary Fig. 1a ). 3 hours after seeding we commenced imaging with a spinning disk confocal microscope for 30 min at a sampling rate of 1/min to follow the fate of individual G1 receptors over this period ( Fig. 2a , black circles; SI video. 1). In each image frame series we assessed the dissociation ratio of individual receptor clusters based on their fluorescence signals. First, we found non-dissociated SpyTag-HaloTag-G1-EGFP receptors at the cell surface, with an average receptor cluster size of 0.44 ± 0.17 µm 2 ( Supplementary Fig. 2a ). During cell migration JF646-labeled NTF-only profiles were left in the migratory tracks of the cells, indicating that the receptor became dissociated during cell movement ( Fig. 2a , magenta circles; Supplementary Fig. 2b ). To quantify the time course of GAIN domain dissociation, we calculated the NTF/CTF fluorescence intensity ratio in fixed size ROIs (0.8 × 0.8 µm) over time. We found increased NTF/CTF ratios for all receptor clusters ( Fig. 2b ). Kymographic analyses of individual SpyTag-HaloTag-G1-EGFP clusters confirmed NTF-CTF separation during cell migration ( Fig. 2c ), with an average separation time of 6.2 ± 2.6 minutes ( Supplementary Fig. 2c ). Download figure Open in new tab Fig. 2. Visualizing of real-time GAIN domain dissociation during cell migration (a) Visualization of SpyTag-HaloTag-G1-WT-EGFP dissociation in COS-7 cells on FN+Spy-coated surfaces. Frames (30’; 1 frame/min) show non-dissociated G1 (NTF [magenta]-CTF [green], black circles) and dissociated G1-NTF (magenta, red circles). Arrows indicate dissociation events; blue chevrons show migration direction. Scale bar: 10 µm. (b) Representative NTF/CTF fluorescence intensity traces and (c) Kymographs of individual G1 clusters over 30 min. The cluster size is shown below the spectrogram. (d) Cleavage ratio analysis (dissociated/total G1 clusters) for SpyTag-HaloTag-G1-EGFP variants. Data (n≥5 replicates, 3 independent experiments) shown as mean ± SD scatter plots with bar; analyzed with an ordinary one-way ANOVA with Tukey’s multiple comparisons test. P values provided. (e) Immunostaining of G1-NTF (magenta) and fluorescence imaging of G1-CTF (green) in COS-7 cells. Blue chevrons indicate migration direction. Yellow arrows highlight dissociated NTFs (zoom-in). Scale bar: 20 µm; zoom-in: 10 µm. (f) Pearson’s correlation coefficient between G1-NTF and G1-CTF signals. Data (n ≥ 10 replicates, 3 independent experiments) are shown in box-and-whisker plots (all data points plotted; median: horizontal line; boxes: 25th–75th percentiles; whiskers: min and max). Data were analyzed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test.; P values provided. To control for GAIN domain cleavage-dependent receptor dissociation, which may alternatively occur through processing by other secreted or membrane-bound proteases 56 , we repeated the migration force assay with the autoproteolysis-deficient SpyTag-HaloTag-G1-EGFP (H381S, T383A, F385A) and autoproteolysis-competent SpyTag-HaloTag-G1-EGFP (L388A/M389A) versions. In line with the Western blot data ( Fig. 1d-e ), NTF-CTF separation of the uncleavable receptor mutants were reduced to 13 % (H381S), 2 % (T383A), and 45 % (F385A) compared to the self-cleaved receptor protein, while the cleavage ratio of the L388A/M389A receptor was not significantly altered ( Fig. 2d ). We also analyzed SpyTag-HaloTag-G1-EGFP GAIN domain dissociation by direct NTF and CTF fluorescence imaging at the cell surface after overnight cell migration ( Fig. 2e ). COS-7 cells expressing the different G1 receptor variants were allowed to migrate on differently coated surfaces (FN vs. FN+Spy). Non-cleaved G1 on the thin membrane tubes was indicated by a white fluorescence signal. Dissociated G1-NTF ligated to the slide surface through SpyTag-SpyCatcher bonding was indicated by a magenta fluorescence signal only ( Fig. 2e ). Pearson’s correlation between NTF and CTF indicated that GAIN domain dissociation occurred only in cells containing SpyTag-HaloTag-G1-EGFP, SpyTag-HaloTag-G1 F 385 A -EGFP, and SpyTag-HaloTag-G1 L 388 A/M389A -EGFP, which shows residual GAIN domain cleavage upon migration on the FN+Spy surface ( Fig. 2e, f ). Consistently, no dissociation was observed for receptors that contained the H381S or T383A GPS mutations, respectively ( Fig. 2e, f ). Moreover, no receptor dissociation was detected on the FN surface lacking SpyCatcher ligand with any G1 variant ( Fig. 2f , Extended Data Fig. 1 ). To rule out cell-type specific effects, the migration force assay was repeated in Human Foreskin Fibroblasts (HFF). Also in migrating HFF cells GAIN domain dissociation occurred only in SpyTag-HaloTag-G1-EGFP and SpyTag-HaloTag-G1 F385A -EGFP and required FN+Spy coating, while no dissociation was observed in H381S and T383A mutants ( Supplementary Fig. 3a-b ). Taken together, this set of observations demonstrates the suitability of the migration force assay to capture NTF-CTF dissociation dynamics of aGPCRs upon migration-dependent mechanotransduction in a quantitative manner. aGPCR dissociation requires ligand-receptor interaction, force generation through cell migration, and GAIN domain autoproteolysis. G1 dissociation occurs at retracting fibers within minutes after formation G1 dissociation primarily occurs at the tips of retracting fibers, which extend along the migratory axis of the cells’ rear edge 11 ( Fig. 2 ). In COS-7 cells, the length of these retracting fibers increased at a typical rate of 0.77 ± 0.29 µm/min ( Supplementary Fig. 2d-e ). As cells continued migrating, the retracting fibers broke, leading to the formation of migrasomes, which were left on the surface ( Supplementary Fig. 1a ). We observed that both COS-7 cells and HFF cells were capable of forming retracting fibers and migrasomes. This was further confirmed by the presence of integrin α5 and actin filaments in the retracting fibers, whereas migrasomes contained integrin α5 but lacked actin filaments ( Supplementary Fig. 1b ) (ref. 57 ). Similar to ADGRE5/CD97 (ref. ( 14 )), we found that G1 was located in both the retracting fiber and the migrasome ( Supplementary Fig. 1a-b ). During cell migration, G1 dissociated at a fiber length of 4.56 ± 2.07 µm ( Supplementary Fig. 2f ). These results demonstrate that along the direction of migration, G1-GAIN domain dissociation occurs within minutes of retraction fiber formation. Since the retraction fiber contains F-actin, we reasoned that the force required to maintain a retracting fiber tube is similar to that required to maintain a filopodium, which is in the range of several pN at pulling rates in order of µm/min (ref. 58 ). This is similar to the force range in which partial unfolding and dissociation of GAIN domains of different aGPCRs, including G1, was observed 10 , 41 . During the force exertion process by cell migration, COS-7 and HFF cells expressing GAIN domain cleavage-deficient SpyTag-HaloTag-G1-EGFP mutants displayed retracting fibers and the migrasomes with similar properties and directionality to the cleavable receptor variant ( Fig. 2e , Extended Data Fig. 1 , Supplementary Fig. 3a ). G1 dissociation results in receptor activation and Gα 13 recruitment in migrating cells The non-dissociation model of aGPCR activation posits that receptor activation can occur in intact NTF-CTF receptor complexes, while dissociative signaling requires NTF release before CTF stimulation through tethered/intramolecular agonism 4 , 5 , 59 , 60 . Both are relevant in vivo . Since partial unfolding of the GAIN subdomain B of G1 occurs before GAIN domain dissociation 10 , changes in GAIN domain conformation take place in autoproteolysed and uncleaved aGPCRs 40 . Capitalizing on the migration force assay we directly tested the relationship between receptor dissociation and activation during mechanotransduction under physiological force application. For these assays we used a modified G1 version without a HaloTag (SpyTag-G1-EGFP) ( Fig. 3a ). COS-7 cells expressing SpyTag-G1-EGFP were seeded on FN+Spy-coated PDMS ( Fig. 2e ) or glass surfaces ( Supplementary Fig. 4a ). After overnight migration ( Fig. 3b ), both conditions exhibited similar GAIN domain dissociation characteristics ( Fig. 2f , Supplementary Fig. 4b ). After 3 hours of seeding immobilized receptors, assayed by total internal reflection fluorescence (TIRF) microscopy using their EGFP signal, were organized in ridge-like arrays that reflect their lateral anchorage in the membrane due to adhesion complex formation 33 ( Fig. 3c, d, SI Video. 2), indicating that G1 was covalently tethered to the SpyCatcher surface (receptor ridges are hereafter referred to as ‘Spy adhesion sites’). Besides immobilized G1 ridges, highly mobile, non-tethered G1 puncta were also visible (SI Video. 2). Download figure Open in new tab Fig. 3. Dynamic mechanical activation of G1 during cell migration (a) G1 receptor design for the ‘migration force assay’, combining G protein recruitment imaging. (b) Force-dependent GAIN domain dissociation at SpyCatcher sites activates G1, dynamically visualized via Halo-miniG13 recruitment and JF646 labeling in real-time using TIRFM. (c) TIRFM images of COS-7 cells expressing SpyTag-G1-EGFP variants (green) migrating on FN+Spy-coated glass surfaces. HaloTag-miniG13 (magenta) is recruited to WT G1 and partially to the H381S mutant but not to Stachel-defective T383A, F385A and L388A/M389A variants. A zoomed-in white box (∼10 µm, shown in (d) ) highlights the 60-minute time point. Scale bar: 10 µm. (d) Zoom-in region (top) and normalized intensity profiles (bottom) for G1 (green) and miniG13 (magenta) signal at Spy adhesion sites. MiniG13 intensity reflects net increases during the 60-minute interval. Dashed lines mark G1 signal peaks at 60 minutes. Scale bar: 3 µm. (e) Dynamic changes in normalized G1 intensity (I EGFP /I EGFP,0 , green) and miniG13 intensity (I miniG13 /I miniG13,0 , magenta) at Spy adhesion sites over 60 minutes. (f) Average I EGFP /I EGFP,0 and (g) I miniG13 /I miniG13,0 values from the final 5 minutes of imaging in (e) . Data (n ≥ 30 replicates from ≥ 6 cells/group) are shown as scatter plots with mean ± SD. Statistical analysis: one-way ANOVA with Tukey’s test (P values shown). (h) G1 activation during the force migration assay was assessed via SRE-Luciferase activity. (i) Relative luciferase activity for G1 constructs on FN, FN+TG2 (D3D4 domains) or FN+Spy surfaces during migration. Data (n ≥ 5 replicates from 3 independent experiments) are shown as scatter plots with mean ± SD. Statistical analysis: one-way ANOVA with Tukey’s test (P values shown). (j) Migration speed of COS-7 cells expressing SpyTag-G1-EGFP variants on FN or FN+Spy surfaces. Data (n ≥ 13 cells from 2 independent experiments) are shown as scatter plots with mean ± SD. Statistical analysis: one-way ANOVA with Tukey’s test (P values shown). As immobile ligated G1 in Spy adhesion sites were subjected to mechanical force transmission via cell migration, we surmised that G1 could be activated mechanically there. As G1 activation leads to engagement of Gα 13 (refs. 9 , 13 , 21 ), we dynamically assayed this step in G1 signal transduction through a fluorescent HaloTag-miniG13 probe 61 ( Fig. 3b ) under live conditions during cell migration with TIRF imaging (SI Video. 2). Mechanically activated SpyTag-G1-EGFP at Spy adhesion sites recruited HaloTag-miniG13 during the migration force assay as visualized by JF646-labeling ( Fig. 3c , SI Video. 2). This allowed us to speculate that ( i ) at Spy adhesion sites undergoing migration-dependent force transmission sufficient for G1 activation, low sub-saturation HaloTag-miniG13 levels could incur when low mechanical forces for G1 activation apply; ( ii ) HaloTag-miniG13 signals may increase until saturation, or ( iii ) due to photobleaching or receptor internalization HaloTag-miniG13 signals may decay 62 . All three outcomes were observed in our experiments ( Supplementary Fig. 5a-b , SI Video. 3). Receptor activation also occurs before G1 dissociation Cells expressing SpyTag-G1-EGFP or SpyTag-G1 H 381 S -EGFP showed recruitment of HaloTag-miniG13 to the receptors at Spy adhesion sites ( Fig. 3c,d ), consistent with residual signaling ability of this autoproteolysis-deficient G1 mutant, which contains the unfazed intramolecular Stachel agonist ( Fig. 1d ). In contrast, no HaloTag-miniG13 recruitment to mutant receptors carrying either the T383A, F385A, or L388A/M389A mutations was observed in the migration force assay ( Fig. 3c ). These results were confirmed in TIRF-ROI scans of SpyTag-G1-EGFP and JF646-HaloTag-miniG13 intensities ( Fig. 3d ). To capture the dynamic changes in SpyTag-G1-EGFP and HaloTag-miniG13 colocalization, several Spy adhesions sites from each cell were analyzed, and the average fluorescence signal intensities were normalized to the corresponding background average intensities outside Spy adhesion sites. The obtained Spy adhesion site enrichment ratio is noted as I EGFP , and the miniG13 enrichment ratio is I miniG13 , respectively, with I EGFP,0 and I miniG13,0 indicating the normalized intensities at the start of a 1 h imaging time interval, during which the dynamic changes in I EGFP /I EGFP,0 (green) and I miniG13 /I miniG13,0 (magenta) were plotted at a rate of 1 frame/min ( Fig. 3e ). Each data point represents the average of > 30 Spy adhesion sites (n = 5 sites/cell; n > 6 cells). We observed a significant increase in I miniG13 /I miniG13,0 and a decrease in I EGFP /I EGFP,0 for the wild-type GAIN domain receptor protein, indicating G1 activation followed by receptor internalization and/or out-diffusion of the dissociated G1-CTF from the Spy adhesion sites ( Fig. 3e-g , SI Video. 2). The increase in I miniG13 /I miniG13,0 , indicates that miniG13 recruitment occurs at a higher rate than receptor internalization and/or out-diffusion from the imaging site. In contrast, SpyTag-G1 H 381 S -EGFP showed only a moderate increase in I miniG13 /I miniG13,0 and near-constant I EGFP /I EGFP,0 consistent with a low activation level due to self-cleavage deficiency but intact signaling ability due to the unharmed Stachel sequence ( Fig. 3e-g , SI Video. 4). In line with these observations, the T383A mutant showed constant I miniG13 /I miniG13,0 and I EGFP /I EGFP,0 values indicating immobilization and non-dissociation of the mutant receptor protein due to its cleavage deficiency resulting in no activation ( Fig. 3e-g , SI Video. 5). Finally, SpyTag-G1 F 385 A -EGFP and SpyTag-G1 L 388 /M389A -EGFP showed no I miniG13 /I miniG13,0 changes during the migration force assay, while I EGFP /I EGFP,0 decreased moderately, in keeping with the partial cleavage inflicted by the F385A mutation and cleavage-competence of the L388A/M389A mutation, but no receptor activation due to the disabled Stachel ( Fig. 3e-g , SI Video. 6-8). We specifically assessed the requirement of adhesion site-formation or force generation through cell migration for G1 signaling and miniG13 recruitment. Cells expressing any SpyTag-G1-EGFP variant on FN-only SpyCatcher-lacking surfaces showed no I miniG13 /I miniG13,0 miniG13 recruitment ( Supplementary Fig. 6a ). Conversely, a G1-EGFP protein without the SpyTag, unable to form Spy adhesions, lacked miniG13 recruitment on FN+Spy surfaces ( Supplementary Fig. 6b ). In cells treated with the Src kinase inhibitor Dasatinib (100 nM), which significantly reduces or blocks cell migration 63 ( Supplementary Fig. 7a ), migration speed reduction nearly abolished GAIN domain dissociation ( Supplementary Fig. 7b-c, f ) and G1 activation ( Supplementary Fig. 7d-e, g ). Collectively, we conclude that activation of G1 and the recruitment of miniG13 upon force transmission requires the formation of Spy adhesion contacts and cell migration. Non-dissociative signalling reflects submaximal G1 activation To further support the force-dependent activation of G1, we used a serum response element (SRE)-luciferase reporter assay ( Fig. 3h ). Cells expressing SpyTag-G1-EGFP exhibited substantially higher SRE activity on the FN+Spy surface compared to the FN-only condition ( Fig. 3i ), indicating that migration forces activate G1. SRE activity of FN cultured cells was comparable to that of the no receptor control (empty vector, EV) on the FN surfaces ( Fig. 3i ), suggesting minimal activation of G1 in the absence of force. Furthermore, cells expressing SpyTag-G1-EGFP exhibited significantly higher SRE activity compared to the mutant G1 proteins when migrating on FN+Spy surfaces ( Fig. 3i ). Notably, the activation level of the SpyTag-G1 H 381 S -EGFP mutant on FN+Spy surfaces was lower than that of the WT receptor, but higher than that of the control (EV) on the FN+Spy surfaces, and cells expressing WT and H381S proteins on FN surfaces lacking mechanical anchoring of the receptor’s NTF to the substrate ( Fig. 3i ). This confirms again that, while the H381S mutation severely impacts autoproteolytic activity of the G1 GAIN domain ( Fig. 1d-e ), migration-dependent force exerted on SpyTag-G1 H 381 S -EGFP can partially activate the receptor. In contrast, T383A, F385A, and L388A/M389A mutants, all lacking an intact Stachel ( Fig. 1d ), did not exhibit receptor activation during cell migration ( Fig. 3i ), even though two mutants, SpyTag-G1 F 385 A -EGFP and SpyTag-G1 L 388 A/M389A -EGFP, retained considerable receptor self-cleavage activity ( Fig. 2d-f ). To investigate non-dissociative signaling triggered by mechanotransmission between G1 and one of its native ligands, we coated the surface with the D3D4 domain of tissue transglutaminase 2 (TG2)(refs. 43 , 64 ), whose interaction with G1 regulates extracellular matrix remodeling and melanoma progression (ref. 43 ). Cells expressing SpyTag-G1-EGFP and spreading on FN surfaces co-coated with TG2 exhibited receptor activation comparable to that on FN+Spy surfaces, and significantly higher than on FN-only surfaces ( Fig. 3i ). Consistent with the results on FN+Spy surfaces, cells expressing the cleavage-deficient SpyTag-G1 H 381 S -EGFP mutant on FN+TG2 surfaces showed reduced activation compared to the wild-type receptor, but significantly higher than the EV control, indicating submaximal G1 activation ( Fig. 3i ). Furthermore, the T383A, F385A, and L388A/M389A mutants failed to activate on FN+TG2 surfaces, mirroring their loss of activation on FN+Spy surfaces ( Fig. 3i ). Notably, in cells expressing SpyTag-G1-EGFP migrating on FN+TG2 surfaces we observed residual GAIN domain cleavage, reflected by a lower Pearson’s correlation between NTF and CTF signals ( Supplementary Fig. 8a–b ) though the degree of dissociation was less pronounced than on FN+Spy surfaces. This demonstrates that cell migration dependent mechanotransmission between TG2 and G1 is sufficient for G1 dissociation and activation, even to a submaximal level as in the cleavage-deficient H381S mutant. The migration force assay enabled us to recapitulate both receptor dissociation and activation, supporting the hypothesis that force-induced receptor dissociation exposes the Stachel element leading to receptor activation. To directly test intramolecular agonism of G1 under mechanical force stimulation, we applied the G1 antagonist dihydromunduletone (DHM) 65 during cell migration on FN+Spy surfaces. While background SRE levels were sensitive to DHM already without G1 expression, increased SRE activity observed in cells expressing SpyTag-G1-EGFP was abolished upon 10 µM DHM treatment, ( Supplementary Fig. 9a ). Consistently, in the presence of DHM no miniG13 recruitment (I miniG13 /I miniG13,0 ) to SpyTag-G1-EGFP was detected during cell migration on FN+Spy surfaces ( Supplementary Fig. 9b-e ). These findings demonstrate that forces generated during cell migration expose the Stachel sequence and activate G1, which can be mitigated by DHM. We also measured the migration speed of COS-7 cells expressing the indicated G1 versions. Previous studies reported that G1 activation reduces cell migration speed of neural progenitors 66 and neurons 45 . Compared to cells spreading on FN surfaces, cells overexpressing the receptors with signaling capability (SpyTag-G1-EGFP and SpyTag-G1 F385A -EGFP) displayed significantly reduced migration speed on FN+Spy surfaces ( Fig. 3j ). In contrast, cells expressing the activation-deficient receptors (SpyTag-G1 T 383 A -EGFP, SpyTag-G1 F 385 –EGFP) showed no such difference ( Fig. 3j ). Importantly, these findings support a model in which G1 metabotropic signal activation inhibits cell migration, and that not mere adhesion forces between G1 and its ligands account for the speed reduction. In summary, these results demonstrate mechanical force activation of G1 through force-dependent GAIN domain dissociation and non-dissociative conformational GAIN domain changes. Interestingly, non-dissociative mechanical stimulation of G1 results in lower metabotropic signaling levels than stimuli that caused GAIN domain dissociation. However, both modes require an intact Stachel sequence in our assays. G1 encodes progressive increase in mechanical stimulation into increased signalling activity To extend our findings to another scenario in which cells physiologically experience forces, we mimicked force transmission during tissue deformation on G1. In this second mechanotransduction paradigm (henceforth termed ‘deformation force assay’), we seeded SpyTag-HaloTag-G1-EGFP expressing COS-7 cells on a flexible polydimethylsiloxane (PDMS) surface co-coated with FN+Spy, and stretched the elastomer by 16 % for 1 h ( Fig. 4a , Supplementary Fig. 10a ). This expansion range corresponds to 5-20 % ECM deformation observed during physiological processes and is commonly used in mechanotransduction studies 67 . We asked whether cell migration and stretch are additive mechanical stimuli that result in increased GAIN domain dissociation levels and higher levels of G1 activation. Indeed, we found that GAIN domain dissociation of G1 increased significantly when both were combined ( Fig. 4b, c ). In contrast, no GAIN domain dissociation was observed on the FN surface lacking SpyCatcher ( Fig. 4b, c ). Download figure Open in new tab Fig. 4. A graded mechanical activation mechanism of G1 (a) G1 dissociation and activation triggered by ECM stretching in the ‘deformation force assay’. (b) Immunostaining of COS-7 cells expressing SpyTag-G1-EGFP on FN or FN+Spy surfaces, under unstretched or 16 % stretched conditions. NTF (α-GAIN, magenta) and CTF (EGFP, green) are visualized. Scale bars: 20 µm (main) and 10 µm (zoom-in). (c) Correlation between NTF (α-GAIN) and CTF (EGFP) signals as measured in (b) . Pearson’s correlation coefficients were calculated. Data (n > 10 biological replicates from at least 3 independent experiments) are shown as box-and-whisker plots (all data points plotted; median: horizontal line; boxes: 25th–75th percentiles; whiskers: min and max). One-way ANOVA with Tukey’s test was performed, with P-values indicated. This dataset is also shown in Ext. Fig. 2b . (d) G1 activation in the ‘deformation force assay’ assessed by SRE-luciferase reporter assay under conditions in (b) . Empty vector (EV) served as a normalization control. Data (n > 6 replicates from 3 experiments) are shown as scatter plots with bars (mean ± SD). One-way ANOVA with Tukey’s test were performed; P-values are shown. This dataset is also shown in Ext. Fig. 2c . (e) Activation of G1 variants upon 16 % stretch. Data are presented as in (d) . This dataset is also shown in Ext. Fig. 2c . (f) Immunostaining of COS-7 cells expressing SpyTag-G1-EGFP variants on FN+Spy surfaces under 16% stretch. Cells were fixed post-seeding and stretching. Color scheme as in (b) . Yellow arrows in the zoom-in region indicate dissociated NTF. Scale bars: 20 µm (main) and 5 µm (zoom-in). Consistent with this, cells expressing G1 on FN+Spy surfaces in the deformation force assay showed a significant increase in receptor activation compared to cells without stretching ( Fig. 4d ). Additionally, cells expressing G1 exhibited higher SRE-luciferase activity on FN+Spy surfaces compared to FN-only surfaces, supporting the requirement for mechanical G1-NTF linkage to a stable ligand ( Fig. 1c , Fig. 4d , Supplementary Fig. 10b ). Remarkably, cells expressing self-cleavage deficient SpyTag-HaloTag-G1 H381S -EGFP and subjected to deformation forces also showed increased activation on FN+Spy-coated surfaces compared to FN-coated surfaces, though at a lower level than autoproteolyzed G1 ( Fig. 4e , Extended Data Fig. 2 ). This reinforces the conclusion from the previous results that non-cleavable G1 with an intact Stachel sequence can be mechanically activated, but to a lesser extent than cleavable G1. The observed deformation-dependent enhancement of G1 activation suggests that ECM stretch rapidly induces GAIN domain dissociation. To directly visualize ECM stretch-dependent GAIN domain dissociation, cells were seeded only for 1 h to reduce migration-dependent dissociation while allowing proper cell spreading 68 . This was followed by a 16 % stretch for 1 h. GAIN domain dissociation at the cell periphery occurred only in cells expressing G1 when subjected to 16 % stretch ( Fig. 4f ), indicating that deformation-induced GAIN domain dissociation of G1 can occur within 1 h. In conclusion, our results suggest a two-step mechanism of mechanical activation for G1, involving both GAIN domain deformation and dissociation. DISCUSSION Unravelling the steps of aGPCR mechanotransduction is critical for grasping the physiological functions that these complex receptor molecules govern 5 , 60 . Crucially, it is also a prerequisite for their manipulation by pharmacological means, which is still at large 69 . A fundamental limitation thus far has been the lack of methods to simultaneously assess if and how aGPCR activation is accompanied by receptor dissociation and G protein coupling under mechanical stimulation. Current approaches to dissect these relationships were restricted to a few receptor homologs, for which genetic animal models exist, and in which physiologically relevant mechanical stimuli were defined. This concerned vibration and sound stimulation to neuronal aGPCRs 24 , 27 , 28 , 70 , blood flow to receptors expressed in blood-borne cells 9 , 31 or exposed to circulation 25 , 71 , or receptors putatively activated by the inspiration-expiration cycle of the lung 26 . Nonetheless, direct observation of aGPCR-dependent metabotropic signals governed by force application in vivo is cumbersome and often technically not feasible yet. Conversely, in vitro approaches with the ability to acutely stimulate aGPCR activation and better accessibility to receptor readouts use force regimes for aGPCR mechanostimulation such as cell culture shaking 30 , whose physiological relevance is unclear, or entirely artificial means to initiate aGPCR signaling, e.g. antibody treatment 72 – 75 or protease activation of engineered receptors 34 , 38 , 76 . Here, we established experimental paradigms to address this need by applying force stimuli to the human aGPCR G1 through two physiologically relevant processes, cell migration and tissue deformation. We observed that self-cleavage and dissociation of the NTF-CTF complex of G1 were required for full receptor signaling. Self cleavage-deficient G1 mutants 13 , 19 , 44 , 50 , 51 were unable to undergo receptor dissociation, however, showed differential behaviors that allowed us to distinguish mechano-dependent receptor dissociation and signaling. Real-time recording of miniG13 recruitment to G1 and its mutants, and an SRE activation assay, during force application revealed a nuanced picture of mechanoactivation of G1. A cleavage-deficient mutant with an intact intramolecular agonist sequence (H381S) remained signaling-competent, while mutants that lacked both cleavage and its Stachel core (T383A) or with retained cleavage but without a functional intramolecular agonist (F385A, L388A/M389A) were completely signaling-defective in line with previous findings in G1 9 , 13 ,2113, 19 , 44 , 50 , 51 and other aGPCRs 20 , 24 , 38 , 39 . These results underline the importance of Stachel-dependent signaling for mechanotransduction via G1, and also indicate that direct force transmission to the 7TM domain is not sufficient for receptor activation. Our findings that mutations of the Stachel core sequence (T383A, F385A, L388A/M389A) abolish G1 signaling are consistent with previous studies using CTF-only receptor versions, in which the Stachel carried an inactivating F385M mutation. In those experiments, a synthetic Stachel peptide lacking T383 induced significantly weaker G1 activation than an intact peptide 13 . Similarly, other studies reported that T383A severely impaired G1-dependent cancer cell proliferation 77 and CG4-induced IL-6 production of melanoma cells 78 . In contrast, a more recent study found no difference in G1-CTF activation between wild-type and T383A G1 21 . This apparent discrepancy can be reconciled by considering both affinity and effective concentration between soluble or tethered Stachel element and its orthosteric binding site in the 7TM domain. When the mutant T383A Stachel remains covalently attached to the CTF and fully exposed, its infinitely high local concentration could compensate for the reduced affinity allowing for G1 activation. In contrast, when the mutant Stachel sequence is presented as a soluble peptide or, as in our assay, where the mutant Stachel sequence remains fully or partially encrypted within the GAIN domain due to lack of NTF/CTF separation, this results in a reduction of the effective local concentration in addition to the lowered affinity of the T383A Stachel sequence resulting in impaired G1 signaling. Supporting this, a study on another aGPCR, ADGRL3/LPHN3, compared the effect of a homologous T923G mutation introduced either in a L3-CTF protein or the full-length L3 receptor. The T923G mutation impaired full-length L3 activity but had no effect on L3-CTF 38 . This observation is also corroborated by the observed G1 activation of the H381S mutant, which retains an intact Stachel sequence with higher binding affinity, despite reduction in Stachel exposure due to impaired receptor self-cleavage. Collectively, our results support a mechanotransduction model for G1, in which the receptor is capable of relaying force stimulus intensities after but also before receptor dissociation ( Fig. 5 ). The former signaling mode is also observed on other mechanosensitive molecules such as Notch receptors, which react with a binary one-and-done response based on proteolytic receptor destruction 79 and that has also been proposed for aGPCRs 5 , 13 . We documented here that G1 can respond to forces before NTF release off the CTF. This is in line with single molecule magnetic tweezer studies, which have determined that partial unfolding of the GAIN subdomain B occurs at forces of 5-10 pN, while the autoproteolyzed domain dissociates within seconds at ∼18 pN (ref. 10 ). This suggests that the forces encountered by individual G1 receptors during cell migration and tissue deformation range around these thresholds, a reasonable assumption given that retracting fibers are maintained at approximately several pN at pulling rates in the order of µm/min (ref. 58 ). Download figure Open in new tab Fig. 5. Proposed mechanisms for a dual mode of mechanical-dependent activation of G1 (a) G1 undergoes self-cleavage at the GPS by the GAIN domain. (b) Mechanical stimuli, caused by NTF binding to adhesive ligands and relative movement to the membrane-integral CTF, partially unfold the GAIN B subdomain at 5–10 pN, activating G1 via Stachel agonist interaction with the 7TM domain. (c) If the mechanical stimulation increases beyond the GAIN domain dissociation threshold (approximately 10-20 pN) 10 , the G1 NTF-CTF heteroprotomer is separated, the Stachel is completely uncapped and fully interacts with the orthosteric binding pocket in the 7TM domain, leading to maximal enhancement of G1 activity. In future projects, it will be necessary to analyze mechanotransmission of forces from the physiological ligands of G1 including collagen III (ref. 45 ) and tissue transglutaminase-2 (ref. 43 ) onto the receptor NTF using single-molecule biophysical assays to complete our picture of aGPCR mechanotransduction. Finally, how the Stachel-7TM domain interaction at low force stimulation comes about when GAIN domain and receptor heteroprotomer structure are still intact, may rely on the intrinsic structural flexibility of the GAIN domain exposing the intramolecular agonist commensurate with the impacting force stimulation 10 , 40 . Nonetheless, this process requires novel insights into the structure-function duality of aGPCRs. Our assays will aid in quantifying force-response characteristics of other aGPCRs and provide novel platforms to decipher how their activation results in the generation of intracellular signals. The approaches provided in this work also offer a unique toolkit to identify and study allosteric modulators that specifically interfere with the events that drive aGPCR mechanotransduction. MATERIALS AND METHODS Cell culture and reagents COS-7 cells and HFF cells were cultured at 37 ℃ in a 5 % CO2 incubator in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % Fetal Bovine Serum (FBS), 100IU/ml Penicillin/Streptomycin and 1 mM sodium pyruvate (Invitrogen). The cells were tested for mycoplasma contamination and found negative. Human G1 cDNA was cloned into a pcDNA3.1-EGFP vector as described previously 10 . A SpyTag and HaloTag were introduced downstream of the signal peptide sequence in the cDNA to generate the SpyTag-HaloTag-G1-WT-EGFP construct. Mutant SpyTag-HaloTag-G1-H381S/T383A/F385A/(L388A/M389A)-EGFP constructs were created by site-directed mutagenesis using Q5 Site-Directed Mutagenesis Kit (NEB). These mutant constructs were confirmed by Sanger sequencing. pGL4.33[luc2P/SRE/Hygro] were purchased from Promega. HaloTag-miniG13 was a gift from Dr. Nevin, Lambert’s laboratory from Augusta University. For experiments related to the G1 activation, we engineered the G1 constructs by removing the HaloTag to generate SpyTag-G1-WT/H381S/T383A/F385A/(L388A/M389A)-EGFP constructs. G1-EGFP was generated by removing the SpyTag from the SpyTag-G1-EGFP. COS-7 cells were seeded into a 6-well plate with 60-70 % confluence at day 0 and co-transfected with indicated G1 constructs along with either a HaloTag-miniG13 or a pGL4.33 vector using lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions on day 1. Then the transfected cells were used in the spreading or stretching experiment protocol. A Spy-EGFP construct without the G1 gene was used as an empty vector (EV). Dasatinib (BMS-354825) (100nM) (Selleckchem) and DHM (10µM) (Molport) were used in this study. Protein expression and purification pET28a(+) plasmids containing Avi-SpyCacher or Avi-TG2 D3D4 domain were transformed in E.coli BL21(DE3) cells with biotin protein ligase (BirA) and purified using a His-tag affinity column following the previous protocol 80 . Colonies transformed with the respective plasmids were first grown overnight in 10 mL LB medium containing 100 μg/mL ampicillin at 37 °C. The overnight cultures were then inoculated into 1 L of fresh LB medium with ampicillin and incubated at 37 °C for 4–6 hours until the optical density at 600 nm (OD600) reached ∼0.6. Protein expression was induced by adding 0.4 mM IPTG and 0.5 mM biotin, followed by overnight incubation at 20 °C. Biotinylation was catalyzed by BirA, enabling site-specific conjugation to the AviTag. Bacterial cells were harvested by centrifugation at 6000 g and pellets were resuspended in lysis buffer (50 mM Tris, 300 mM NaCl, pH 7.4) containing 1 mM PMSF, and lysed using a French press. Lysates were centrifuged at 40,000 g for 30 minutes, and the supernatant was incubated with Co²⁺-NTA resin (Thermo Scientific) for 1 hour. After washing with buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 7.4), proteins were eluted using buffer containing 200 mM imidazole. The eluted proteins were further purified by size-exclusion chromatography (Superdex 200, ÄKTA Pure, GE Healthcare, MA). Protein-containing fractions were analyzed by SDS-PAGE, dialyzed into PBS, aliquoted with 10% (v/v) glycerol, flash-frozen in liquid nitrogen, and stored at −80 °C. Protein concentration was determined by BCA assays (Thermo Scientific). Cell migration and stretching The transfected single COS-7 or HFF cells were seeded on the glass bottom dishes (ibidi), or spin-coated with a layer of rigid PDMS (2MPa) as described previously 81 , and coated with Fibronectin (Roche) (10 µg/ml), Fibronectin together with TG2 D3D4 domain (10 µg/ml), or Fibronectin together with SpyCatcher (10 µg/ml). The cells were incubated with a Janelia Fluor -646 (JF646) HaloTag Ligands (Promega) (1:3000 dilution) for 1 hour before cell spreading. The process of cell migration was performed on W1 Spinning disc confocal microscopy for visualizing the real-time GAIN domain dissociation and on widefield microscopy EZ (Olympus IX81) for calculating the migration speed. The cell migration was also performed on the total internal reflection fluorescence microscopy (TIRFM), with those cells seeded on the glass bottom dishes without the PDMS coating. The real-time GAIN domain dissociation and dynamic recruitment of miniG13 during cell migration were monitored with a TIRFM. Imaging was done on a Nikon TIRF system with 488 and 640 laser lines. For cell stretching, transfected COS-7 cells were seeded on the cell stretching system (STREX) coated with Fibronectin or Fibronectin together with SpyCatcher. Cells were then stretched to 16% strain for 1h and were fixed for immunofluorescence. Immunofluorescence After undergoing cell spreading or stretching, COS-7 or HFF cells were fixed with a pre-warmed solution of 4 % paraformaldehyde in PBS at 37°C for 15 minutes. This was followed by permeabilization with 0.2 % Triton X-100 in TBS for 30 minutes at room temperature to allow for antibody penetration. To minimize non-specific binding, the samples were then treated with a blocking solution of 1% Bovine Serum Albumin (BSA) in TBS for 1 hour. The samples were then incubated with primary antibodies (Anti-G1 Antibody (G-6) N-terminal (Santa Cruz, sc-390192), diluted 1:200, integrin α5 (BD Pharmingen, Purified NA/LE (CD49e), 555614), diluted 1:200) overnight at 4°C, followed by incubation with secondary antibodies and Alexa Fluo Plus 405 Palloindin (Invitrogen) for 1 hour at room temperature. The co-localization of G1-EGFP with the NTF of G1 was analyzed by calculating Pearson’s correlation coefficient between the two channels. Western blot Confluent cells were lysed in a RIPA buffer (Sigma) and a protease inhibitor (Roche) for 20 minutes at 4 °C. The amount of proteins extracted was determined using the BCA assay (Bio-Rad). The extracted proteins were separated by 4-20% SDS polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (Bio-Rad) using an electro-transfer process for 7 minutes (Trans-Blot Turbo system). The membranes were blocked with a solution of 5 % Bovine Serum Albumin (BSA) in TBS 0.05 % Tween 20 to prevent non-specific binding. The membranes were then incubated with primary antibodies (Anti-G1 Antibody (G-6) N-terminal [Santa Cruz, sc-390192], diluted 1:1000) overnight at 4 °C, followed by an incubation with secondary HRP antibodies (diluted 1:2000) for 1 hour at room temperature. The results were visualized using the ChemiDoc chemiluminescence detection system (Bio-Rad) and quantified using Image Lab. GAPDH was used as a reference protein to control for variations in protein loading. SRE-Luciferase Assay The co-transfected COS-7 cells were subjected to 16 hours of serum starvation. These cells were then seeded on glass bottom dishes that were coated with either fibronectin, Fibronectin together with TG2 D3D4 domain, or Fibronectin together with SpyCatcher to promote cell spreading. After 3 hours of cell spreading, the cells were lysed and analyzed using the Dual-Glo luciferase assay system (E2920, Promega), according to the manufacturer’s protocol. For the stretched cell experiment, co-transfected COS-7 cells were seeded on a cell stretching system (STREX) that was coated with either FN or FN+Spy and subjected to 16 hours of serum starvation. The cells were then stretched at 16 % strain for 1 hour, followed by 2 hours of recovery, and lysed and analyzed using the Dual-Glo luciferase assay system. The luminescence readings were taken using a Glomax Discover plate reader (Promega). The firefly luciferase data was normalized to the Renilla luciferase signal and expressed as the fold increase over the signal obtained from cells that were transfected with Spy-EGFP as an EV underwent the same spreading or stretching. Statistical analyses To ensure accuracy and reproducibility, measurements were performed at least three times in triplicate. Prism (GraphPad Software) was used for data analysis and graph plotting. The data was visualized using Adobe Illustrator. The illustrations were drawn with the help of Biorender. To determine statistical significance, an ANOVA test and an unpaired Welch’s t-test were conducted. The results were considered statistically significant if P is less than 0.05. DATA AVAILABILITY The raw Western blot data are available in figshare at DOI: xxxx. All other data are available upon request to the corresponding authors. AUTHOR CONTRIBUTIONS C.F., T.L., and J.Y. designed research C.F., and C.Q., performed the experiments C.F., C.Q, and Y.Z. analyzed the data T.L., and J.Y. co-supervised the research C.F., G.S., T.L., and J.Y. wrote the paper COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material. Correspondence and requests for materials Correspondence and requests for materials should be addressed to Yan Jie and Tobias Langenhan. EXTENDED FIGURE LEGENDS Download figure Open in new tab Extended Data Figure 1. G1 dissociation during cell migration is not occur on FN surfaces Immunostaining of G1-NTF (α-GAIN, magenta) and fluorescence imaging of G1-CTF (EGFP, green) in COS-7 cells expressing the indicated SpyTag-HaloTag-G1-EGFP versions and spreading on FN-coated surfaces. Blue chevrons indicate migration direction. Scale bar: 20 µm; zoom-in: 10 µm. Download figure Open in new tab Extended Data Figure 2. G1 dissociation and activation with ‘deformation force assays’ (a) Immunostaining of COS-7 cells expressing SpyTag-G1-EGFP variants on FN or FN+Spy surfaces, either unstretched or stretched to 16 %. NTF (α-GAIN, magenta) and CTF (EGFP, green) are visualized. Scale bars: 20 µm (main) and 10 µm (zoom). (b) Correlation between NTF (α-GAIN) and CTF (EGFP) signals from (a) , quantified with Pearson’s correlation coefficient. Data from >10 biological replicates (3+ independent experiments) are shown as box-and-whisker plots (all data points plotted; median: horizontal line; boxes: 25th–75th percentiles; whiskers: min and max). Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons test. P-values indicated. For reference, the WT dataset from Fig. 4c is displayed in this extended dataset. (c) G1 activation under deformation forces assessed via SRE-luciferase reporter assay for conditions in (a) . For reference, the full EV and WT datasets from Fig. 4d and the 16 % data for all receptor versions in Fig. 4e are displayed in this extended dataset. EV served as a control. Data (n > 6 replicates, 3 experiments) are presented as scatter plots with bars (mean ± SD). Statistical analysis: one-way ANOVA with Tukey’s test. P-values indicated. SUPPLEMENTARY MATERIAL Download figure Open in new tab Supplementary Figure 1. Formation of retracting fibers, migrasomes and occurrence of G1 dissociation during cell migration (a) Immunostaining of COS-7 and HFF cells expressing SpyTag-HaloTag-G1-EGFP on FN+Spy surfaces, showing the cell body, retracting fibers, migrasomes, and NTF/CTF dissociation. Scale bar: 20 µm. (b) Immunostaining of COS-7 and HFF cells expressing EV or SpyTag-G1-EGFP on FN surfaces, highlighting NTF (α-GAIN), CTF (EGFP), integrin α5, and actin (Phalloidin). Scale bars: 30 µm (overview) and 10 µm (zoom-in). Download figure Open in new tab Supplementary Figure 2. G1 dissociation occurs at retracting fibers within minutes after formation (a) Cluster size quantification of SpyTag-HaloTag-G1-EGFP. (b) Video frames showing NTF-CTF dissociation of SpyTag-HaloTag-G1-EGFP clusters over 1’ intervals. Blue chevrons indicate cell migration direction. Scale bars: 2.5 µm. (c) Estimated NTF-CTF separation time during cell migration on FN+Spy surfaces (violin plot). (d) Representative traces of retracting fiber length increase over time before breakage. (e) Local retraction speed and (f) retracting fiber length during cell migration on FN+Spy surfaces (violin plots). Download figure Open in new tab Supplementary Figure 3. G1 dissociation of HFF cells with ‘migration force assays’ (a) Immunostaining of HFF cells expressing the indicated G1 variants on FN+Spy or FN-coated surfaces, visualizing G1-NTF (α-GAIN) and G1-CTF (EGFP). Blue chevrons mark the direction of cell migration. Scale bars: 20 µm; zoom-in: 10 µm. (b) Pearson’s correlation coefficient between G1-NTF and G1-CTF signals in HFF cells. Data (n ≥ 8 biological replicates from ≥ 2 independent experiments per group) are shown as box-and-whisker plots (median: horizontal line; boxes: 25th–75th percentiles; whiskers: min–max values). Ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed; p-values are indicated. Download figure Open in new tab Supplementary Figure 4. G1 dissociation of COS-7 cells with ‘migration force assays’ on the glass surfaces (a) Immunostaining of COS-7 cells expressing the indicated G1 variants on FN+Spy coated glass surfaces, visualizing G1-NTF (α-GAIN) and G1-CTF (EGFP). Scale bars: 20 µm; zoom-in: 10 µm. (b) Pearson’s correlation coefficient between G1-NTF and G1-CTF signals in COS-7 cells. Data (n ≥ 10 biological replicates from ≥ 2 independent experiments per group) are shown as box-and-whisker plots (median: horizontal line; boxes: 25th–75th percentiles; whiskers: min–max values). Ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed; p-values are indicated. Download figure Open in new tab Supplementary Figure 5. Saturation and decay of HaloTag-miniG13 signals during long term recording (a) TIRFM images of cells expressing SpyTag-G1-EGFP (green) migrating on FN+Spy surfaces at indicated time points. HaloTag-miniG13 (magenta) is recruited to surface-expressed receptors in WT cells, peaking around 80 minutes and returning to baseline by 160 minutes. Scale bar: 10 µm. (b) Dynamic changes in I miniG13 /I miniG13,0 (magenta) at Spy adhesion sites over the 160-minute recording period. Download figure Open in new tab Supplementary Figure 6. Requirement of adhesion site-formation or force generation through cell migration for G1 signaling and miniG13 recruitment (a) TIRFM images of cells expressing SpyTag-G1-EGFP variants (green) migrating on FN surfaces at indicated time points, showing no HaloTag-miniG13 (magenta) recruitment over 60 minutes. Scale bar: 10 µm. Dynamic changes in I miniG13 /I miniG13,0 (magenta) are shown; n ≥ 5 cells per group. (b) TIRFM images of G1-EGFP-WT-expressing cells (green) migrating on FN or FN+Spy surfaces at indicated time points, with no HaloTag-miniG13 (magenta) recruitment over 60 minutes. Scale bar: 10 µm. Dynamic changes in I miniG13 /I miniG13,0 (magenta) are shown; n ≥ 5 cells per group. Download figure Open in new tab Supplementary Figure 7. Cell migration is required for G1 dissociation, signaling and miniG13 recruitment (a) Migration speed of COS-7 cells expressing SpyTag-HaloTag-G1-EGFP on FN+Spy surfaces with Dasatinib (100nM). Data (n ≥ 30 cells from 2 independent experiments) are shown as scatter plots with mean ± SD. Statistical analysis: Welch’s t-test (P values shown). (b) Immunostaining of COS-7 cells expressing SpyTag-HaloTag-G1-EGFP on FN+Spy surfaces with Dasatinib (100nM), visualizing G1-NTF (α-GAIN) and G1-CTF (EGFP). Scale bars: 20 µm; zoom-in: 10 µm. (c) Pearson’s correlation coefficient between G1-NTF and G1-CTF signals in COS-7 cells. Data (n ≥ 15 biological replicates from ≥ 2 independent experiments per group) are shown as box-and-whisker plots (median: horizontal line; boxes: 25th–75th percentiles; whiskers: min–max values). Welch’s t-test was performed; p-values are indicated. (d) TIRFM images of COS-7 cells expressing SpyTag-G1-EGFP (green) migrating on FN+Spy surfaces with Dasatinib (100 nM). No HaloTag-miniG13 (magenta) recruitment over 60 minutes. Scale bar: 10 µm. (e) Dynamic changes in normalized G1 intensity (I EGFP /I EGFP,0 , green) and miniG13 intensity (I miniG13 /I miniG13,0 , magenta) at Spy adhesion sites over 60 minutes with Dasatinib (100 nM). (f) Average I EGFP /I EGFP,0 and (g) I miniG13 /I miniG13,0 values from the final 5 minutes of imaging in (e) . Data (n ≥ 30 replicates from ≥ 6 cells/group) are shown as scatter plots with mean ± SD. Statistical analysis: Welch’s t-test (P values shown). This experiment was conducted in parallel with the drug treatment shown in Suppl. Fig. 9d-e using joint WT controls; therefore the same WT datasets are displayed here. Download figure Open in new tab Supplementary Figure 8. G1 dissociation of COS-7 cells with ‘migration force assays’ on TG2 surfaces. (a) Immunostaining of COS-7 cells expressing the indicated G1 variants on FN+Spy, FN+TG2(D3D4 domains), and FN coated glass surfaces, visualizing G1-NTF (α-GAIN) and G1-CTF (EGFP). Scale bars: 20 µm; zoom-in: 10 µm. (b) Pearson’s correlation coefficient between G1-NTF and G1-CTF signals in COS-7 cells. Data (n ≥ 20 biological replicates from ≥ 2 independent experiments per group) are shown as box-and-whisker plots (median: horizontal line; boxes: 25th–75th percentiles; whiskers: min–max values). Ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed; p-values are indicated. Download figure Open in new tab Supplementary Figure 9. Force generated during cell migration expose the Stachel sequence and activate G1, which can be mitigated by DHM (a) Relative luciferase activity for G1 constructs with 10µM DHM treatment on FN+Spy surfaces during migration. Data (n ≥ 3 replicates from 3 independent experiments) are shown as scatter plots with mean ± SD. Statistical analysis: one-way ANOVA with Tukey’s test (P values shown). (b) TIRFM images of COS-7 cells expressing SpyTag-G1-EGFP (green) migrating on FN+Spy surfaces with DHM (10 µM). No HaloTag-miniG13 (magenta) recruitment over 60 minutes. Scale bar: 10 µm. (c) Dynamic changes in normalized G1 intensity (I EGFP /I EGFP,0 , green) and miniG13 intensity (I miniG13 /I miniG13,0 , magenta) at Spy adhesion sites over 60 minutes with 10 µM DHM treatment. (d) Average I EGFP /I EGFP,0 and (e) I miniG13 /I miniG13,0 values from the final 5 minutes of imaging in (c) . 10 µM DHM treatment does not affect the G1 dissociation but reduces miniG13 recruitment. Data (n ≥ 30 replicates from ≥ 6 cells/group) are shown as scatter plots with mean ± SD. Statistical analysis: Welch’s t-test (P values shown). This experiment was conducted in parallel with the drug treatment shown in Suppl. Fig. 7f-g using joint WT controls; therefore the same WT datasets are displayed here. Download figure Open in new tab Supplementary Figure 10: Schematic of the stretching chamber and the protocol for SRE measurement for the ‘deformation force assay’ (a) Schematic of the stretching chamber for the ‘deformation force assay’. COS-7 cells with the indicated constructs were seeded on FN- or FN+Spy-coated chambers. Substrates were either left unstretched (top) or stretched by 16% (bottom). (b) Protocol for SRE measurement following the ‘deformation force assay’. View this table: View inline View popup Download powerpoint Supplementary Table 1 Supp. Table S1 . Primers used in this study. Supp. Video S1. Migration of COS-7 with respective SpyTag-HaloTag-G1-EGFP constructs on FN+Spy surface, recorded at 1 frame/min. Scale bar, 10 µm. Download figure Open in new tab Supp. Video S2. Migration of COS-7 with SpyTag-G1-EGFP and HaloTag-miniG13 constructs on FN+Spy surface, recorded at 2 frame/min. 1 hour duration. Scale bar, 10 µm. Download figure Open in new tab Supp. Video S3. Migration of COS-7 with SpyTag-G1-EGFP and HaloTag-miniG13 constructs on FN+Spy surface, recorded at 2 frame/min. 3 hour duration. Scale bar, 10 µm. Download figure Open in new tab Supp. Video S4. Migration of COS-7 with SpyTag-G1 H381S -EGFP and HaloTag-miniG13 constructs on FN+Spy surface, recorded at 2 frame/min. 1 hour duration. Scale bar, 10 µm. Download figure Open in new tab Supp. Video S5. Migration of COS-7 with SpyTag-G1 T383A -EGFP and HaloTag-miniG13 constructs on FN+Spy surface, recorded at 2 frame/min. 1 hour duration. Scale bar, 10 µm. Download figure Open in new tab Supp. Video S6. Migration of COS-7 with SpyTag-G1 F385A -EGFP and HaloTag-miniG13 constructs on FN+Spy surface, recorded at 2 frame/min. 1 hour duration. Scale bar, 10 µm. Download figure Open in new tab Supp. Video S7. Migration of COS-7 with SpyTag-G1 L388A/M389A -EGFP and HaloTag-miniG13 constructs on FN+Spy surface, recorded at 2 frame/min. 1 hour duration. Scale bar, 10 µm. Download figure Open in new tab Supp. Video S8. Zoom-in region for G1 (green) and miniG13 (magenta) signal at Spy adhesion sites. MiniG13 intensity reflects net increases during the 60-minute interval, recorded at 2 frame/min. 1 hour duration. Scale bar: 3 µm. Download figure Open in new tab ACKNOWLEDGEMENTS This work was supported through grants of the Deutsche Forschungsgemeinschaft (CRC-TRR166/C03) to T.L., Singapore Ministry of Education Academic Research Fund Tier 3 (MOE Grant No: MOET32021-0003) and Tier 2 (MOE Grant Nos: MOE-T2EP50123-0008), National Research Foundation (NRF) Singapore, Mechanobiology Institute under its MID-SIZED GRANT (MSG) (NRF-MSG-2023-0001) to J.Y. We thank Dr. Nevin, Lambert (Augusta University) for sharing miniG plasmids and Dr. Xianhua Piao (University of California San Francisco) for sharing the G1 plasmids, and MBI wet lab and imaging core facilities. Funder Information Declared National Research Foundation (NRF) Singapore , NRF-MSG-2023-0001 REFERENCES 1. ↵ Bukoreshtliev , N. V. , Haase , K. & Pelling , A. E . Mechanical cues in cellular signalling and communication . Cell Tissue Res . 352 , 77 – 94 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 2. ↵ Belly , H. D. , Paluch , E. K. & Chalut , K. J . Interplay between mechanics and signalling in regulating cell fate . Nat. Rev. Mol. Cell Biol . 23 , 465 – 480 ( 2022 ). OpenUrl CrossRef PubMed 3. ↵ Hamann , J. et al. International Union of Basic and Clinical Pharmacology . XCIV. Adhesion G Protein–Coupled Receptors. Pharmacol. Rev . 67 , 338 – 367 ( 2015 ). OpenUrl PubMed 4. ↵ Purcell , R. H. & Hall , R. A . Adhesion G Protein–Coupled Receptors as Drug Targets . Annu. Rev. Pharmacol. Toxicol . 58 , 1 – 21 ( 2018 ). OpenUrl PubMed 5. ↵ Vizurraga , A. , Adhikari , R. , Yeung , J. , Yu , M. & Tall , G. G . Mechanisms of adhesion G protein– coupled receptor activation . J. Biol. Chem . 295 , 14065 – 14083 ( 2020 ). OpenUrl Abstract / FREE Full Text 6. ↵ Scholz , N. , Monk , K. R. , Kittel , R. J. & Langenhan , T . Adhesion G Protein-coupled Receptors, Molecular, Physiological and Pharmacological Principles in Health and Disease . Handb. Exp. Pharmacol . 234 , 221 – 247 ( 2016 ). OpenUrl CrossRef PubMed 7. ↵ Piao , X. et al. G Protein-Coupled Receptor-Dependent Development of Human Frontal Cortex . Science 303 , 2033 – 2036 ( 2004 ). OpenUrl Abstract / FREE Full Text 8. ↵ White , J. P. et al. G protein-coupled receptor 56 regulates mechanical overload-induced muscle hypertrophy . Proc. Natl. Acad. Sci . 111 , 15756 – 15761 ( 2014 ). OpenUrl Abstract / FREE Full Text 9. ↵ Yeung , J. et al. GPR56/ADGRG1 is a platelet collagen-responsive GPCR and hemostatic sensor of shear force . Proc. Natl. Acad. Sci . 117 , 28275 – 28286 ( 2020 ). OpenUrl Abstract / FREE Full Text 10. ↵ Fu , C. et al. Unveiling Mechanical Activation: GAIN Domain Unfolding and Dissociation in Adhesion GPCRs . Nano Lett . 23 , 9179 – 9186 ( 2023 ). OpenUrl CrossRef PubMed 11. ↵ Kirfel , G. , Rigort , A. , Borm , B. & Herzog , V . Cell migration: mechanisms of rear detachment and the formation of migration tracks . Eur. J. Cell Biol . 83 , 717 – 724 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 12. ↵ Liebscher , I. et al. A Tethered Agonist within the Ectodomain Activates the Adhesion G Protein-Coupled Receptors GPR126 and GPR133 . Cell Rep . 9 , 2018 – 2026 ( 2014 ). OpenUrl CrossRef PubMed 13. ↵ Stoveken , H. M. , Hajduczok , A. G. , Xu , L. & Tall , G. G . Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist . Proc. Natl. Acad. Sci . 112 , 6194 – 6199 ( 2015 ). OpenUrl Abstract / FREE Full Text 14. ↵ Hilbig , D. et al. Mechano-Dependent Phosphorylation of the PDZ-Binding Motif of CD97/ADGRE5 Modulates Cellular Detachment . Cell Rep . 24 , 1986 – 1995 ( 2018 ). OpenUrl CrossRef PubMed 15. ↵ Cruz-Ortega , J. S. & Boucard , A. A . Actin cytoskeleton remodeling defines a distinct cellular function for adhesion G protein-coupled receptors ADGRL/latrophilins 1, 2 and 3 . Biol. Open 8 , bio039826 ( 2019 ). 16. ↵ Gray , J. X ., et al. CD97 is a processed, seven-transmembrane, heterodimeric receptor associated with inflammation . J. Immunol. (Baltim., Md : 1950) 157 , 5438 – 47 ( 1996 ). OpenUrl 17. ↵ Krasnoperov , V. G. et al. α-Latrotoxin Stimulates Exocytosis by the Interaction with a Neuron al G-Protein-Coupled Receptor . Neuron 18 , 925 – 937 ( 1997 ). OpenUrl CrossRef PubMed Web of Science 18. Lin , H.-H. et al. Autocatalytic Cleavage of the EMR2 Receptor Occurs at a Conserved G Protein-coupled Receptor Proteolytic Site Motif* . J Biol Chem 279 , 31823 – 31832 ( 2004 ). OpenUrl Abstract / FREE Full Text 19. ↵ Araç , D. et al. A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis . EMBO J . 31 , 1364 – 1378 ( 2012 ). OpenUrl Abstract / FREE Full Text 20. ↵ Ping , Y.-Q. et al. Structural basis for the tethered peptide activation of adhesion GPCRs . Nature 604 , 763 – 770 ( 2022 ). OpenUrl CrossRef PubMed 21. ↵ Barros-Álvarez , X. et al. The tethered peptide activation mechanism of adhesion GPCRs . Nature 604 , 757 – 762 ( 2022 ). OpenUrl CrossRef PubMed 22. ↵ Qu , X. et al. Structural basis of tethered agonism of the adhesion GPCRs ADGRD1 and ADGRF1 . Nature 604 , 779 – 785 ( 2022 ). OpenUrl CrossRef PubMed 23. ↵ Xiao , P. et al. Tethered peptide activation mechanism of the adhesion GPCRs ADGRG2 and ADGRG4 . Nature 604 , 771 – 778 ( 2022 ). OpenUrl CrossRef PubMed 24. ↵ Scholz , N. et al. Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons . eLife 6 , e28360 ( 2017 ). OpenUrl CrossRef PubMed 25. ↵ Liu , D. et al. CD 9 7 promotes spleen dendritic cell homeostasis through the mechanosensing of red blood cells . Science 375 , eabi5965 ( 2022 ). 26. ↵ Bridges , J. P. et al. Regulation of pulmonary surfactant by the adhesion GPCR GPR 11 6/ADGRF5 requires a tethered agonist-mediated activation mechanism . eLife 11 , e69061 ( 2022 ). OpenUrl PubMed 27. ↵ Scholz , N. et al. The Adhesion GPCR Latrophilin/CIRL Shapes Mechanosensation . Cell Rep . 11 , 866 – 874 ( 2015 ). OpenUrl CrossRef PubMed 28. ↵ Dannhäuser , S. et al. Antinociceptive modulation by the adhesion GPCR CIRL promotes mechanosensory signal discrimination . eLife 9 , e56738 ( 2020 ). OpenUrl PubMed 29. ↵ Scholz , N. et al. Molecular sensing of mechano- and ligand-dependent adhesion GPCR dissociation . Nature 615 , 945 – 953 ( 2023 ). OpenUrl CrossRef PubMed 30. ↵ Petersen , S. C. et al. The Adhesion GPCR GPR126 Has Distinct, Domain-Dependent Functions in Schwann Cell Development Mediated by Interaction with Laminin-211 . Neuron 85 , 755 – 769 ( 2015 ). OpenUrl CrossRef PubMed 31. ↵ Karpus , O. N. et al. Shear Stress–Dependent Downregulation of the Adhesion-G Protein–Coupled Receptor CD97 on Circulating Leukocytes upon Contact with Its Ligand CD55 . J. Immunol . 190 , 3740 – 3748 ( 2013 ). OpenUrl Abstract / FREE Full Text 32. ↵ Boyden , S. E. et al. Vibratory Urticaria Associated with a Missense Variant in ADGRE2 . N. Engl. J. Med . 374 , 656 – 663 ( 2016 ). OpenUrl CrossRef PubMed 33. ↵ Streit , M. et al. Optimized genetic code expansion technology for time-dependent induction of adhesion GPCR-ligand engagement . Protein Sci . 32 , e4614 ( 2023 ). OpenUrl PubMed 34. ↵ Frenster , J. D. et al. Functional impact of intramolecular cleavage and dissociation of adhesion G protein–coupled receptor GPR133 (ADGRD1) on canonical signaling . J. Biol. Chem . 296 , 100798 ( 2021 ). 35. ↵ Bohnekamp , J. & Schöneberg , T . Cell Adhesion Receptor GPR133 Couples to Gs Protein* . J. Biol. Chem . 286 , 41912 – 41916 ( 2011 ). OpenUrl Abstract / FREE Full Text 36. ↵ Prömel , S. et al. The GPS Motif Is a Molecular Switch for Bimodal Activities of Adhesion Class G Protein-Coupled Receptors . Cell Rep . 2 , 321 – 331 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 37. ↵ Sando , R. , Jiang , X. & Südhof , T. C . Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins . Science 363 , eaav7969 ( 2019 ). 38. ↵ Perry-Hauser , N. A. , VanDyck , M. W. , Lee , K. H. , Shi , L. & Javitch , J. A . Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3 . J. Biol. Chem . 298 , 102594 ( 2022 ). 39. ↵ Kordon , S. P. et al. Isoform- and ligand-specific modulation of the adhesion GPCR ADGRL3/Latrophilin3 by a synthetic binder . Nat. Commun . 14 , 635 ( 2023 ). 40. ↵ Beliu , G. et al. Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain . Mol. Cell 81 , 905 – 921 .e5 ( 2021 ). OpenUrl CrossRef PubMed 41. ↵ Zhong , B. L. et al. Piconewton Forces Mediate GAIN Domain Dissociation of the Latrophilin-3 Adhesion GPCR . Nano Lett . 23 , 9187 – 9194 ( 2023 ). OpenUrl CrossRef PubMed 42. ↵ Chang , G.-W. et al. The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells . Cell Rep . 15 , 1757 – 1770 ( 2016 ). OpenUrl CrossRef PubMed 43. ↵ Xu , L. , Begum , S. , Hearn , J. D. & Hynes , R. O . GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis . Proc. Natl. Acad. Sci . 103 , 9023 – 9028 ( 2006 ). OpenUrl Abstract / FREE Full Text 44. ↵ Salzman , G. S. et al. Structural Basis for Regulation of GPR56/ADGRG1 by Its Alternatively Spliced Extracellular Domains . Neuron 91 , 1292 – 1304 ( 2016 ). OpenUrl CrossRef PubMed 45. ↵ Luo , R. et al. G protein-coupled receptor 56 and collagen III, a receptor-ligand pair, regulates cortical development and lamination . Proc. Natl. Acad. Sci . 108 , 12925 – 12930 ( 2011 ). OpenUrl Abstract / FREE Full Text 46. ↵ Paavola , K. J. , Stephenson , J. R. , Ritter , S. L. , Alter , S. P. & Hall , R. A . The N Terminus of the Adhesion G Protein-coupled Receptor GPR56 Controls Receptor Signaling Activity* . J. Biol. Chem . 286 , 28914 – 28921 ( 2011 ). OpenUrl Abstract / FREE Full Text 47. ↵ Zakeri , B ., et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin . Proc. Natl. Acad. Sci . 109 , E690 – E697 ( 2012 ). OpenUrl Abstract / FREE Full Text 48. ↵ Jin , Z. et al. Disease-associated mutations affect GPR56 protein trafficking and cell surface expression . Hum. Mol. Genet . 16 , 1972 – 1985 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 49. ↵ Zhu , B. et al. GAIN domain–mediated cleavage is required for activation of G protein–coupled receptor 56 (GPR56) by its natural ligands and a small-molecule agonist . J. Biol. Chem . 294 , 19246 – 19254 ( 2019 ). OpenUrl Abstract / FREE Full Text 50. ↵ Salzman , G. S. et al. Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region . Proc. Natl. Acad. Sci . 114 , 10095 – 10100 ( 2017 ). OpenUrl Abstract / FREE Full Text 51. ↵ Kishore , A. , Purcell , R. H. , Nassiri-Toosi , Z. & Hall , R. A . Stalk-dependent and Stalk-independent Signaling by the Adhesion G Protein-coupled Receptors GPR56 (ADGRG1) and BAI1 (ADGRB1)* . J. Biol. Chem . 291 , 3385 – 3394 ( 2016 ). OpenUrl Abstract / FREE Full Text 52. ↵ Hu , Y. et al. DNA-based ForceChrono probes for deciphering single-molecule force dynamics in living cells . Cell 187 , 3445 – 3459 .e15 ( 2024 ). OpenUrl CrossRef PubMed 53. ↵ Jo , M. H. et al. Determination of single-molecule loading rate during mechanotransduction in cell adhesion . Science 383 , 1374 – 1379 ( 2024 ). OpenUrl CrossRef PubMed 54. ↵ Dumas , L . et al. Uncovering and engineering the mechanical properties of the adhesion GPCR ADGRG1 GAIN domain . bioRxiv 2023.04.05.535724 ( 2023 ) doi: 10.1101/2023.04.05.535724 . OpenUrl Abstract / FREE Full Text 55. ↵ Ma , L. et al. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration . Cell Res . 25 , 24 – 38 ( 2015 ). OpenUrl CrossRef PubMed 56. ↵ Nieberler , M. , Kittel , R. J. , Petrenko , A. G. , Lin , H.-H. & Langenhan , T . Adhesion G Protein-coupled Receptors, Molecular, Physiological and Pharmacological Principles in Health and Disease . Handb. Exp. Pharmacol . 234 , 83 – 109 ( 2016 ). OpenUrl CrossRef PubMed 57. ↵ Wu , D. et al. Pairing of integrins with ECM proteins determines migrasome formation . Cell Res . 27 , 1397 – 1400 ( 2017 ). OpenUrl CrossRef PubMed 58. ↵ Alieva , N. O. et al. Myosin IIA and formin dependent mechanosensitivity of filopodia adhesion . Nat. Commun . 10 , 3593 ( 2019 ). OpenUrl CrossRef PubMed 59. ↵ Liebscher , I. & Schöneberg , T . Adhesion G Protein-coupled Receptors, Molecular, Physiological and Pharmacological Principles in Health and Disease . Handb. Exp. Pharmacol . 234 , 111 – 125 ( 2016 ). OpenUrl CrossRef PubMed 60. ↵ Langenhan , T . Adhesion G protein–coupled receptors—Candidate metabotropic mechanosensors and novel drug targets . Basic Clin. Pharmacol. Toxicol . 126 , 5 – 16 ( 2019 ). OpenUrl 61. ↵ Wan , Q. et al. Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells . J. Biol. Chem . 293 , 7466 – 7473 ( 2018 ). OpenUrl Abstract / FREE Full Text 62. ↵ Manchanda , Y. et al. Engineered mini-G proteins block the internalization of cognate GPCRs and disrupt downstream intracellular signaling . Sci. Signal . 17 , eabq7038 ( 2024 ). 63. ↵ Johnson , F. M. , Saigal , B. , Talpaz , M. & Donato , N. J . Dasatinib (BMS-354825) Tyrosine Kinase Inhibitor Suppresses Invasion and Induces Cell Cycle Arrest and Apoptosis of Head and Neck Squamous Cell Carcinoma and Non–Small Cell Lung Cancer Cells . Clin. Cancer Res . 11 , 6924 – 6932 ( 2005 ). OpenUrl Abstract / FREE Full Text 64. ↵ Salzman , G. S. , Zhang , S. , Fernandez , C. G. , Araç , D. & Koide , S . Specific and direct modulation of the interaction between adhesion GPCR GPR56/ADGRG1 and tissue transglutaminase 2 using synthetic ligands . Sci. Rep . 10 , 16912 ( 2020 ). 65. ↵ Stoveken , H. M. et al. Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein– Coupled Receptor Antagonist . Mol. Pharmacol . 90 , 214 – 224 ( 2016 ). OpenUrl Abstract / FREE Full Text 66. ↵ Iguchi , T. et al. Orphan G Protein-coupled Receptor GPR56 Regulates Neural Progenitor Cell Migration via a Gα12/13 and Rho Pathway . J. Biol. Chem . 283 , 14469 – 14478 ( 2008 ). OpenUrl Abstract / FREE Full Text 67. ↵ Jufri , N. F. , Mohamedali , A. , Avolio , A. & Baker , M. S . Mechanical stretch: physiological and pathological implications for human vascular endothelial cells. Vasc . Cell 7 , 8 ( 2015 ). 68. ↵ Döbereiner , H.-G. , Dubin-Thaler , B. , Giannone , G. , Xenias , H. S. & Sheetz , M. P . Dynamic Phase Transitions in Cell Spreading . Phys. Rev. Lett . 93 , 108105 ( 2004 ). 69. ↵ Bassilana , F. , Nash , M. & Ludwig , M.-G . Adhesion G protein-coupled receptors: opportunities for drug discovery . Nat. Rev. Drug Discov . 18 , 869 – 884 ( 2019 ). OpenUrl CrossRef PubMed 70. ↵ Bormann , A. et al. Intron retention of an adhesion GPCR generates 1TM isoforms required for 7TM-GPCR function . Cell Rep . 44 , 115078 ( 2025 ). 71. ↵ Tanaka , K. et al. Latrophilin-2 mediates fluid shear stress mechanotransduction at endothelial junctions . EMBO J . 43 , 3175 – 3191 ( 2024 ). OpenUrl CrossRef PubMed 72. ↵ Volynski , K. E. et al. Latrophilin fragments behave as independent proteins that associate and signal on binding of LTXN4C . EMBO J . 23 , 4423 – 4433 ( 2004 ). OpenUrl Abstract / FREE Full Text 73. Huang , Y.-S. et al. Activation of Myeloid Cell-Specific Adhesion Class G Protein-Coupled Receptor EMR2 via Ligation-Induced Translocation and Interaction of Receptor Subunits in Lipid Raft Microdomains . Mol. Cell. Biol . 32 , 1408 – 1420 ( 2012 ). OpenUrl Abstract / FREE Full Text 74. Stephan , G. , Frenster , J. D. , Liebscher , I. & Placantonakis , D. G . Activation of the adhesion G protein–coupled receptor GPR133 by antibodies targeting its N-terminus . J. Biol. Chem . 298 , 101949 ( 2022 ). 75. ↵ Mitgau , J. et al. The N Terminus of Adhesion G Protein–Coupled Receptor GPR126/ADGRG6 as Allosteric Force Integrator . Front. Cell Dev. Biol . 10 , 873278 ( 2022 ). 76. ↵ Mathiasen , S. et al. G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3 . Nat. Chem. Biol . 16 , 1343 – 1350 ( 2020 ). OpenUrl CrossRef PubMed 77. ↵ Sasaki , S. et al. Crucial contribution of GPR56/ADGRG1, expressed by breast cancer cells, to bone metastasis formation . Cancer Sci . 112 , 4883 – 4893 ( 2021 ). OpenUrl CrossRef PubMed 78. ↵ Chiang , N.-Y. et al. GPR56/ADGRG1 Activation Promotes Melanoma Cell Migration via NTF Dissociation and CTF-Mediated Gα12/13/RhoA Signaling . J. Investig. Dermatol . 137 , 727 – 736 ( 2017 ). OpenUrl PubMed 79. ↵ Rodriguez , F. S. , Sanlidag , S. & Sahlgren , C . Mechanical regulation of the Notch signaling pathway . Curr. Opin. Cell Biol . 85 , 102244 ( 2023 ). 80. ↵ Huang , W. et al. Mechanical Stabilization of a Bacterial Adhesion Complex . J. Am. Chem. Soc . 144 , 16808 – 16818 ( 2022 ). OpenUrl CrossRef PubMed 81. ↵ Prager-Khoutorsky , M. et al. Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing . Nat. Cell Biol . 13 , 1457 – 1465 ( 2011 ). OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted September 17, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Share Dual-mode intramolecular agonist-dependent mechanoactivation of the adhesion GPCR ADGRG1/GPR56 Chaoyu Fu , Cheng Quan , Ying Zhang , Gaojie Song , Tobias Langenhan , Jie Yan bioRxiv 2025.09.16.676693; doi: https://doi.org/10.1101/2025.09.16.676693 Share This Article: Copy Citation Tools Dual-mode intramolecular agonist-dependent mechanoactivation of the adhesion GPCR ADGRG1/GPR56 Chaoyu Fu , Cheng Quan , Ying Zhang , Gaojie Song , Tobias Langenhan , Jie Yan bioRxiv 2025.09.16.676693; doi: https://doi.org/10.1101/2025.09.16.676693 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41937) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15156) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)