Helix-8 and Carboxyl Terminal Tail Regulation of Proteinase Activated Receptor-4 (PAR4) signalling

Helix-8 and Carboxyl Terminal Tail Regulation of Proteinase Activated Receptor-4 (PAR4) signalling | 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 Helix-8 and Carboxyl Terminal Tail Regulation of Proteinase Activated Receptor-4 (PAR4) signalling View ORCID Profile Pierre E. Thibeault , View ORCID Profile Amr A.K. Mousa , View ORCID Profile Victor M. Mirka , View ORCID Profile Rithwik Ramachandran doi: https://doi.org/10.1101/2025.02.10.637468 Pierre E. Thibeault 1 Department of Physiology and Pharmacology, University of Western Ontario , London, ON, N6A 5C1, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pierre E. Thibeault Amr A.K. Mousa 1 Department of Physiology and Pharmacology, University of Western Ontario , London, ON, N6A 5C1, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Amr A.K. Mousa Victor M. Mirka 1 Department of Physiology and Pharmacology, University of Western Ontario , London, ON, N6A 5C1, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Victor M. Mirka Rithwik Ramachandran 1 Department of Physiology and Pharmacology, University of Western Ontario , London, ON, N6A 5C1, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rithwik Ramachandran For correspondence: rramach{at}uwo.ca Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The 8th-helix (H8) and carboxyl terminal tail (CT) of GPCRs are crucial for interactions with intracellular signaling molecules and regulatory proteins. We investigated how H8 and CT residues influence signaling in PAR4, a tethered-ligand activated GPCR. Our analysis revealed that PAR4 activation by thrombin or AYPGKF-NH2 stimulates β-arrestin-1/-2 recruitment and activates multiple Gα proteins (Gαq, Gα11, Gαz, Gα15, Gα12, Gα13, Gαi1-3, GαoA/B). Mutation of the H8 sequence (R 352 AGLFQRS 359 ) significantly reduced G protein activation and β-arrestin recruitment, with residues Leu 355 -Glu 357 being particularly important. Mutations of specific lysine residues in H8 and CT also impaired signaling. Additionally, we identified a crucial TM7-H8 interaction and found that CT phosphorylation sites regulate β-arrestin recruitment. Finally, using AlphaFold3 we predicted interactions between PAR4, β-arrestins, and G proteins, revealing novel receptor regulatory sites in the intracellular loops, transmembrane domains, H8 and CT. Introduction G protein-coupled receptors (GPCRs) are the largest family of cell membrane signalling proteins expressed in eukaryotic cells 1 , 2 . These receptors convert extracellular ligand binding into intracellular signalling to mediate cellular affects. Ligand binding activates the GPCR through a series of structural reorganizations that allow for effectors, such as G proteins, to bind and become activated. In addition to the intracellular loops of GPCRs, the H8 and CT also play important roles in effector interaction. Proteinase activated receptors (PARs) are a family of four (PAR1-4) rhodopsin-like (Class A) GPCRs, which are activated by a host of proteolytic enzymes from coagulation cascade-, immune cell-, and pathogen-derived sources 3 – 5 . PARs are unique compared to other Class A GPCR counterparts in their mechanism of activation which involves proteolysis of the receptor N-terminus to reveal a novel sequence, often termed the tethered ligand, that is capable of activating the receptor through intramolecular binding 3 . Alternatively, subtype-selective PAR activation can be achieved in the absence of proteolytic cleavage, through application of synthetic tethered ligand-mimetic peptides 6 – 9 . PAR4 performs important functions in regulating thrombosis and hemostasis through its role as a platelet thrombin receptor 3 , 10 – 12 . In addition to thrombin, PAR4 is also activated by other enzymes including trypsin and neutrophil Cathepsin-G 5 . Proteolytic cleavage of PAR4 by thrombin unmasks the tethered ligand sequence, G 48 YPGQV.., which binds intramolecularly to the receptor, leading to its activation. Alternatively, tethered ligand-mimicking peptides, such as AYPGKF-NH 2 , can activate PAR4 without proteolytic cleavage 7 , 13 . PAR-mediated platelet activation downstream of thrombin release at the site of vessel injury is well-described, and as such, these receptors have been the target for anti-platelet therapeutic approaches 10 , 11 , 14 , 15 . When activated with either the thrombin cleavage-revealed tethered ligand, G 48 YPGQV 53 …, or tethered ligand-mimicking peptide, AYPGKF-NH 2 , PAR4 engages Gα q/11 , Gα 12/13 , and Gα i/o G protein subtypes as well as β-arrestins 7 , 13 , 16 , 17 , although PAR4 cupling to Gα i independent of crosstalk with purinergic receptors has been questioned in certain tissue contexts 7 , 16 , 18 – 20 . Previously, we demonstrated key differences in CT regulation of β-arrestin-1/-2 recruitment and G protein mediated signaling between peptide and proteolytic activation of the related PAR2 receptor 21 . In contrast to PAR2 and unlike many other Class A GPCRs, PAR4 lacks certain conserved residues and motifs implicated in effector interaction and signalling, such as a H8 cysteine residue and CT clusters of serine/threonine residues. Thus, the molecular mechanisms that underlie PAR4 signalling, and signal regulation are of interest – both for understanding the mechanisms of effector interactions with PAR4 as well as for the development of targeted therapeutic strategies. Further, probing differential contributions of residues to signalling and regulation downstream of either peptide activation or proteolytic cleavage provides insight into how these two different modes of activation may differentially regulate these interactions. In this study, we use Alphafold modelling and mutagenesis to determine H8 and CT residues and motifs important for thrombin or AYPGKF-NH 2 activated PAR4 interaction with signaling effectors. We find that residues in both the H8 and CT differentially impact PAR4 interaction with G proteins and β-arrestins. Together our findings provide novel insights into molecular mechanisms regulating PAR4 functions and may aid in the development of novel therapeutics targeting this receptor. Results G protein activation profile of PAR4 in response to activation with the tethered-ligand mimicking peptide, (AYPGKF-NH 2 ) or thrombin Our understanding of the G protein-coupling profile of PAR4 is largely based on second messenger signaling responses. To gain an understanding of G protein subtype selectivity and agonist-dependent differences, we performed TRUPATH assays following activation of PAR4 with the thrombin revealed tethered-ligand GYPGQV…, or the PAR4 tethered ligand-mimicking peptide AYPGKF-NH 2 to assess the G protein subtypes that are activated. TRUPATH is a BRETII assay wherein 14 Gα-Rluc8 proteins are co-transfected with untagged Gβ, and Gγ-GFP2 22 . Decrease in BRETII signal signals a change in conformation or dissociation of the βγ complex from the Gα protein – indicative of G protein activation 22 . We transfected HEK-293 or CRISPR/Cas9 PAR1-knockout (PAR1-KO) HEK-293 cells 17 with PAR4 and the various TRUPATH Gα-β-γ constructs and monitored responses to AYPGKF-NH 2 and thrombin. We observed that AYPGKF-NH 2 activation of PAR4 stimulated Gα q , Gα 11 , Gα z , Gα 15 , Gα 12 , Gα 13 , Gα i1 , Gα i2 , Gα i3 , Gα oA , Gα oB , but not Gα sS , Gα sL , or Gα Gustducin (Gα q EC 50 = 18.4 ± 6.1 µM, Gα 11 EC 50 = 5.9 ± 2.0 µM, Gα z EC 50 = 35.1 ± 7.4 µM, Gα 15 EC 50 = 2.9 ± 1.9 µM, Gα 12 EC 50 = 1.6 ± 0.5 µM, Gα 13 EC 50 = 2.8 ± 0.6 µM, Gα i1 EC 50 = 24.5 ± 5.6 µM, Gα i2 EC 50 = 24.4 ± 10.4 µM, Gα i3 EC 50 = 29.3 ± 5.4 µM, Gα oA EC 50 = 9.7 ± 2.0 µM, Gα oB EC 50 = 8.5 ± 1.5 µM, Fig. 1 ). Following activation of PAR4 with thrombin, we observed that PAR4 triggered Gα q , Gα 11 , Gα z , Gα 15 , Gα 12 , Gα 13 , Gα i1 , Gα i3 , Gα oA , Gα oB and similarly did not Gα sS , Gα sL , or Gα Gustducin . In contrast to AYPGKF-NH 2 , thrombin activation of PAR4 did not stimulate Gα i2 activity (Gα q EC 50 = 0.27 ± 0.17 Units/mL, Gα 11 EC 50 = 0.25 ± 0.01 Units/mL, Gα z EC 50 = 2.06 ± 0.54 Units/mL, Gα 15 EC 50 = 0.27 ± 0.18 Units/mL, Gα 12 EC 50 = 0.04 ± 0.01 Units/mL, Gα 13 EC 50 = 0.17 ± 0.06 Units/mL, Gα i1 EC 50 = 0.70 ± 0.31 Units/mL, Gα i3 EC 50 = 0.79 ± 0.70 Units/mL, Gα oA EC 50 = 0.55 ± 0.09 Units/mL, Gα oB EC 50 = 0.43 ± 0.17 Units/mL; Fig. 2 ). Thus, PAR4 appears to selectively activate members of the Gα q/11 , 12/13, and i/o subfamilies, but not Gα s proteins. Further, selectivity within these G protein subfamilies is evident between agonists (Apparent rank order derived from EC 50 AYPGKF-NH 2 -Gα 12 , Gα 13 , Gα 15 , Gα 11 , Gα oB , Gα oA , Gα q , Gα i2 , Gα i1 , Gα i3 , Gα z ; Apparent rank order derived from EC 50 thrombin – Gα 12 , Gα 13 , Gα 11 , Gα q , Gα 15 , Gα oB , Gα oA , Gα i1 , Gα i3 , Gα z ). Download figure Open in new tab Fig. 1 AYPGKF-NH 2 -activated PAR4 stimulates Gα q/11 , Gα 12/13 , and Gα i/o G protein-subtypes, but not Gα s family subtypes. Assays with HEK-293 cells expressing wild-type PAR-YFP receptor were conducted for each of the TRUPATH BRET pairings with stimulation of PAR4 by the tethered ligand-mimicking peptide AYPGKF-NH 2 . Curve fitting was conducted by three-parameter, non-linear regression and EC 50 values are reported for each G protein subtype. Where curve fitting could not be conducted EC 50 values are reported as “not determined” ( n.d. ). Technical replicates were collected in triplicate for each experimental replicate ( n = 3-4 ) Download figure Open in new tab Fig. 2 Thrombin-activated PAR4 stimulates Gα q/11 , Gα 12/13 , and Gα i/o G protein-subtypes, but not Gα s family subtypes. Assays with PAR1-KO-HEK-293 cells expressing wild-type PAR-YFP receptor were conducted for each of the TRUPATH BRET pairings with stimulation of PAR4 by enzymatic activation with thrombin. Curve fitting was conducted by three-parameter, non-linear regression and EC 50 values are reported for each G protein subtype. Where curve fitting could not be conducted EC 50 values are reported as “not determined” ( n.d. ). Technical replicates were collected in triplicate for each experimental replicate ( n = 3-4 ). Overall, here we established the G protein subtypes that can be activated by PAR4 in HEK-293 and PAR1-knockout HEK-293 cells and our findings are consistent with well-established Gα q/11 , Gα 12/13 , Gα i signalling reported following PAR4 activation in physiologically relevant cell types 7 , 12 , 19 , 23 . With the complete PAR4 G protein coupling profile in hand, we next turned to examining the effect of mutations to PAR H8 and CT residues on G protein and β-arrestin recruitment. Membrane expression of wild-type and mutant PAR4-YFP receptors In certain Class A GPCRs, H8 and CT mutations analogous to those discussed in the remainder of this study negatively impacted cell surface expression. For instance, Mutation of H8 lysine residues reduce membrane expression of both MCH 1 R and B 2 R 24 , 25 . Additionally, mutation of the highly-conserved NPxxYx 5,6 F motif in rhodopsin and β 2 AR significantly affect cell surface expression of these receptors 26 – 29 . Previously, we demonstrated that H8 R 352 AGLFQRS 359 deletion did not impact appropriate cell membrane localization of PAR4 30 . Moreover, truncation studies wherein the CT of PAR4 is removed from H8 Lys 350 (ΔK350) or CT Lys 367 (ΔK350) reported appropriate cell membrane expression and agonist-dependent internalization 31 . We therefore assessed cell surface expression of PAR4 H8 and CT mutants and observed no deficits in cell membrane expression compared to the wild-type receptor ( Supplementary Figure 1 ). AYPGKF-NH 2 - and thrombin-stimulated G protein signalling is dependent on H8 R 352 AGLFQRS 359 residues Previously, we reported that the deletion of PAR4 H8 residues R 352 AGLFQRS 359 (construct designated dRS-PAR4) exhibited defects in agonist-stimulated Gα q/11 -mediated calcium signalling 30 . Here we employed a more conservative approach through alanine scanning mutations to rule out the potential for structural changes introduced by deletion contributing to the previously observed signalling defects. PAR4 activation-triggered calcium signalling was measured as a proxy for Gα q/11 activation by PAR4 since we have previously demonstrated that the Gα q/11 inhibitor YM254890 completely abolished PAR4 calcium responses 32 and calcium signaling provided a more consistent means of assessing Gα q/11 mediated signaling compared to the TRUPATH G protein activation assay ( Supplementary Figures 2 and 3 ). We find that mutation of the H8 RAGLFQRS motif to alanine (PAR4 R352-S359A -YFP) significantly decreased both AYPGKF-NH 2 - (EC 50 = n.d. , Max. = 2.5 ± 2.5% A23187, where “Max.” represents mean ± SEM of percent of A23187 calcium signal at 300 µM AYPGKF-NH 2 or 10 units/mL thrombin; Fig. 2A ) and thrombin-stimulated (EC 50 = n.d. , Max. = 6.1 ± 1.0% A23187, Fig. 3H ) calcium signalling compared to wild-type PAR4 (AYPGKF-NH 2 EC 50 = 26.7 ± 9.6 µM, Max. = 26.3 ± 2.5% A23187, Fig. 3A ; thrombin EC 50 = 0.3 ± 0.1 units/mL, Max. = 14.8 ± 1.4% A23187, Fig. 3H ). Building on our previous findings, we investigated the impact of R 352 AGLFQRS 359 to alanine mutation, on G protein activation by PAR4 ( Fig. 1 and Fig. 2 ; Gα 12/13 subtypes represented by Gα 13 TRUPATH biosensor and Gα i/o subtypes represented by Gα oB TRUPATH biosensor). Interestingly, while substantial deficits were observed in Gα q/11 -mediated calcium signalling, we did not see any significant alterations to PAR4 R352-S359A -YFP mediated Gα 13 or Gα oB activation compared to wild-type receptor when stimulated with either thrombin or AYPGKF-NH 2 (PAR4 R352-S359A -YFP Gα 13 : AYPGKF-NH 2 EC 50 = 20.3 ± 6.4 µM, Max. = -0.18 ± 0.01 net BRET, Fig. 3B ; Thrombin EC 50 = 2.5 ± 0.8 units/mL, Max. = -0.17 ± 0.02 net BRET, Fig. 3I ) (PAR4 R352-S359A -YFP Gα oB : AYPGKF-NH 2 EC 50 = 25.3 ± 14.6 µM, Max. = - 0.12 ± 0.02 net BRET, Fig. 3C ; Thrombin EC 50 = n.d. , Max. = -0.39 ± 0.27 net BRET, Fig. 3J ) (WT PAR4-YFP Gα 13 : AYPGKF-NH 2 EC 50 = 15.6 ± 2.5 µM, Max. = -0.17 ± 0.01 net BRET, Fig. 3B ; Thrombin EC 50 = 1.6 ± 0.3 units/mL, Max. = -0.14 ± 0.01 net BRET, Fig. 3I ) (WT PAR4-YFP Gα oB : AYPGKF-NH 2 EC 50 = 27.4 ± 7.6 µM, Max. = -0.16 ± 0.01 Net BRET, Fig. 3C ; Thrombin EC 50 = 5.4 ± 3.3 units/mL, Max. = -0.17 ± 0.05 Net BRET, Fig. 3J ). Download figure Open in new tab Fig. 3 H8 Lys 355 -Glu 357 residues are essential for Gα q/11 activation and β-arrestin-1/-2 recruitment to both AYPGKF-NH 2 - and thrombin-stimulated PAR4. G protein activation (A-C, H-J). Calcium signalling from wild-type PAR4-YFP and R 352 AGLFQRS 359 total or sequential mutant PAR4 receptors in response to AYPGKF-NH 2 (A) or thrombin (H) stimulation. Nonlinear regression curve fits are shown (mean ± S.E.) for three independent experiments ( n = 3 ). G protein activation (TRUPATH) was recorded in cells expressing wild-type, R 352 AGLFQRS 359 total or sequential mutant PAR4 receptors in response to either AYPGKF-NH 2 [HEK-293 cells; Gα 13 (B), Gα oB (C)] or thrombin [PAR1-KO-HEK-293 cells; Gα 13 (I), Gα oB (J)]. Nonlinear regression curve fits are shown (mean ± S.E.). Technical replicates were collected in triplicate for each experimental replicate ( n = 3-4 ). β -arrestin recruitment (D-F, K-M). Recruitment of β-arrestin-1/-2 to PAR4-YFP wild-type or R 352 AGLFQRS 359 Ala mutant PAR4 following stimulation with AYPGKF-NH 2 (D) or thrombin (K). Recruitment of β-arrestin-1 (E) or -2 (F) to wild-type and sequential R 352 AGLFQRS 359 mutant receptors in response to AYPGKF-NH 2 . Recruitment of β-arrestin-1 (L) or -2 (M) to wild-type and sequential R 352 AGLFQRS 359 mutant receptors in response to thrombin stimulation. Nonlinear regression curve fits are shown (mean ± S.E.) for three to four independent experiments with triplicate data points for each concentration and receptor collected within each experiment. ( n = 3-4) . Summary data normalized to wild-type receptor (G, N) . For ease of comparison, signalling of mutant PAR4 receptors was normalized to signalling recorded from wild-type PAR4 receptor in response to AYPGKF-NH 2 (G) or thrombin (N) at the highest concentrations tested (300 µM AYPGKF-NH 2 , 10 units/mL thrombin) for each of the G protein activation and β-arrestin recruitment assays (A-F, H-M). Significance was calculated on raw (non-normalized) values for each assay by T-test and is indicated (*p < 0.05 compared to wild-type PAR4-YFP receptor). To further delineate H8 residues involved in PAR4-mediated G protein signalling, we recorded agonist-stimulated Gα q/11 -mediated calcium signalling and Gα 13 and Gα oB TRUPATH sensor BRET in cells expressing PAR4 receptor mutants with sequential H8 mutations. Mutation of Arg 352 -Gly 354 Ala did not significantly alter AYPGKF-NH 2 or thrombin-stimulated calcium signalling (AYPGKF-NH 2 EC 50 = 15.6 ± 5.0 µM, Max. = 19.7 ± 1.5% A23187, Fig. 3A ; thrombin EC 50 = 0.4 ± 0.2 units/mL, Max. = 14.4 ± 1.2% A23187, Fig. 3H ) compared to PAR4-YFP. Further, we observed no difference in Gα oB signalling with either agonist (AYPGKF-NH 2 EC 50 = 29.6 ± 8.1 µM, Max. = -0.13 ± 0.01 net BRET, Fig. 3C ; thrombin EC 50 = 13.2 ± 13.0 units/mL, Max. = -0.22 ± 0.14 net BRET, Fig. 3J ] and only observed a modest decrease in maximal signalling with AYPGKF-NH 2 -stimulated Gα 13 activation [AYPGKF-NH 2 EC 50 = 15.1 ± 3.5 µM, Max. = - 0.13 ± 0.01 net BRET (p < 0.001), Fig. 3C ; thrombin EC 50 = 2.1 ± 0.6 units/mL, Max. = -0.13 ± 0.01 net BRET, Fig. 3J ]. Similarly, there was no significant difference in Gα q/11 -mediated calcium signalling with Arg 358 -Ser 359 Ala mutation in response to either AYPGKF-NH 2 (EC 50 = n.d. , Max. = 25.8 ± 0.6% A23187, Fig. 3A ) or thrombin (EC 50 = n.d. , Max. = 13.9 ± 0.5% A23187, Fig. 3H ). We observed increased Gα 13 maximal signalling from PAR4 R358-S359A -YFP [AYPGKF-NH 2 EC 50 = 14.1 ± 3.1 µM, Max. = -0.20 ± 0.01 net BRET (p < 0.05), Fig. 3B ; Thrombin EC 50 = 2.5 ± 0.8 units/mL, Max. = -0.18 ± 0.02 net BRET (p = 0.04), Fig. 3I ]; however, when comparing the net BRET values at the highest concentrations tested, there were no significant differences between the PAR4 R358-S359A mutant and wild-type receptor [Net BRET at AYPGKF-NH 2 (300 µM) PAR4 R358-S359A -YFP -0.19 ± 0.02 (p = 0.27), Fig. 3G ; net BRET at thrombin (10 units/mL) PAR4 R358-S359A -YFP -0.15 ± 0.01 (p = 0.11), Fig. 3N ]. Similarly, we observed no significant impact of Arg 358 -Ser 359 Ala mutation on Gα oB (AYPGKF-NH 2 - EC 50 = 37.4 ± 11.9 µM, Max. = -0.14 ± 0.01 net BRET, Fig. 3C ; Thrombin EC 50 = n.d. , Max. = n.d. , net BRET at 10 units/mL = -0.08 ± 0.03 (p = 0.42), Fig. 3J & 3N] signalling compared to wild-type receptor. Interestingly, mutation of Leu 355 -Gln 357 to alanine significantly decreased thrombin stimulated calcium signalling (EC 50 = n.d. , Max. = 7.4 ± 1.9% A23187, Fig. 3A ) (EC 50 = n.d. , Max. = 2.9 ± 2.8% A23187, Fig. 3H ) but, did not impact AYPGKF-NH 2 Gα 13 (EC 50 = 20.2 ± 4.1 µM, Max. = -0.18 ± 0.01 net BRET, Fig. 3B ) or Gα oB (EC 50 = n.d. , Max. = n.d. , net BRET at 300 µM = -0.11 ± 0.02, Fig. 3C ) activation. We observed that the Leu 355 -Gln 357 Ala mutation did modestly increase thrombin-stimulated Gα 13 maximal net BRET [EC 50 = 2.5 ± 0.5 units/mL, Max. = -0.22 ± 0.02 net BRET (p < 0.001), Fig. 3I ] but had no impact on Gα oB activation (EC 50 = 17.0 ± 16.0 units/mL, Max. = -0.28 ± 0.18 net BRET, Fig. 3J ). Overall, we conclude that the loss of calcium signalling, previously reported with the H8 R 352 AGLFQRS 359 motif deletion 30 or alanine substitution in the current study, is not due to truncation or shortening of the CT, rather, the deficits in calcium signalling appear to be mediated through loss of effector interactions with this site. The H8 Leu 355 -Gln 357 residues specifically are involved in agonist-stimulated Gα q/11 coupled calcium signalling downstream of activated PAR4. Further, these residues are important for both synthetic peptide and thrombin revealed tethered ligand mediated signaling from PAR4. The modest impact of Leu 355 -Gln 357 Ala mutation on Gα 13 and Gα oB also suggest that these residues may be involved in a G protein subtype specific interaction of Gα q/11 with PAR4. AYPGKF-NH 2 and thrombin stimulated PAR4 β-arrestin-1 and -2 recruitment involves H8 Leu 355 -Gln 357 Previously, we reported that in addition to defects in agonist-stimulated Gα q/11 coupled calcium signalling, deletion of the H8 R 352 AGLFQRS 359 motif (dRS-PAR4) decreased β-arrestin recruitment to PAR4 30 . As with calcium signalling, we sought to determine the role of H8 mutations on β-arrestin-1/-2 recruitment in response to either peptide- or enzyme-mediated activation of PAR4. As previously demonstrated, the concentration effect curve for β-arrestin-1/-2 recruitment to PAR4 does not saturate in response to either peptide or thrombin activation, therefore the data presented throughout for β-arrestin recruitment are the net BRET ratio at the highest concentration tested for each agonist for comparison between receptor mutants (AYPGKF-NH 2 300 µM, thrombin 10 units/mL). In response to AYPGKF-NH 2 -stimulation, we observed β-arrestin-1 (Max. = 0.22 ± 0.01 net BRET; where “Max.” represents mean ± SEM of net BRET at 300 µM AYPGKF-NH 2 ) and β-arrestin-2 (Max. = 0.29 ± 0.01 net BRET) recruitment consistent with previous findings ( Fig. 3D-F ) 13 , 30 . Thrombin-stimulated recruitment of β-arrestin-1 (Max. = 0.18 ± 0.01 net BRET; where “Max.” represents mean ± SEM of net BRET at 10 units/mL thrombin) and β-arrestin-2 (Max. = 0.21 ± 0.01 net BRET) was also recorded ( Fig. 3K-M ) and as previously observed, recruitment of β-arrestin-1/-2 in response to thrombin exhibits decreased maximal recruitment compared to peptide activation of the receptor 17 , 30 . Mutation of the entire H8 R 352 AGLFQRS 35 sequence to alanine (PAR4 R352-S359A -YFP) mirrored the findings of our previous results using a truncation mutant providing confidence that the residues, and not the deletion, were responsible for the effects previously observed. β-arrestin- 1/-2 recruitment to PAR4 R352-S359A -YFP was significantly decreased in response to both AYPGKF- NH 2 [β-Arr-1 Max. 0.10 ± 0.01 net BRET (p < 0.0001); β-Arr-2 Max. 0.11 ± 0.00 net BRET (p < 0.0001), Fig. 3D ] and thrombin [β-Arr-1 Max. 0.08 ± 0.01 net BRET (p < 0.0001); β-Arr-2 Max. 0.08 ± 0.00 net BRET (p < 0.0001), Fig. 3K ] stimulation indicating that these H8 residues are indeed important for β-arrestin-1/-2 recruitment to PAR4. To determine whether the entire H8 R 352 AGLFQRS 359 sequence, or a subset of these residues, is participating in agonist-stimulated recruitment of β-arrestins, we utilized the sequential H8 mutant receptors and recorded recruitment. The first of these mutations, Arg 352 -Gly 354 Ala, significantly increased recruitment of β-arrestin-1 [Max. = 0.24 ± 0.01 net BRET (p < 0.05)] and -2 [Max. = 0.29 ± 0.00 (p < 0.05)] compared to wild-type PAR4-YFP in response to AYPGKF-NH 2 stimulation ( Fig. 3E & 3F). Interestingly, we observed differential agonist-dependent recruitment of β-arrestins to thrombin activated PAR4 R352-G354A -YFP. Thrombin-stimulated recruitment of β-arrestin-1 to PAR4 R352-G354A -YFP [Max. = 0.15 ± 0.01 net BRET (p = 0.69), Fig. 3L ] was not significantly different than wild-type receptor, however, the recruitment of β-arrestin-2 was significantly reduced [Max. = 0.16 ± 0.00 net BRET (p = 0.01), Fig. 3M ]. Therefore, these data may highlight differences in mechanisms underlying β-arrestin-1and 2 recruitment to PAR4. Leu 355 -Gln 357 to alanine mutation (PAR4 L355-Q357A -YFP) resulted in decreased β-arrestin-1/-2 recruitment to PAR4 in response to both agonists studied. AYPGKF-NH 2 -stimulated recruitment of β-arrestin-1 [Max. = 0.10 ± 0.00 net BRET (p < 0.0001), Fig. 3E ) and -2 [Max. = 0.10 ± 0.00 net BRET (p < 0.0001), Fig. 3F ) to PAR4 L355-Q357A -YFP was significantly reduced compared to wild-type receptor. Further, thrombin-stimulated recruitment of β-arrestin-1 [Max. = 0.10 ± 0.01 net BRET (p < 0.0001), Fig. 3L ) and -2 [Max. = 0.08 ± 0.00 net BRET (p < 0.0001), Fig. 3M ) was also significantly reduced compared to thrombin-activated PAR4. Given that both peptide- and thrombin-stimulated recruitment of β-arrestins was affected by Leu 355 -Gln 357 Ala mutation, we determine a role for these residues in β-arrestin recruitment to PAR4, independent of agonist. Finally, we evaluated the effect of alanine mutation Arg 358 and Ser 359 (PAR4 R358-S359A -YFP). We observed that AYPGKF-NH 2 -stimulated recruitment of both β-arrestin-1 [Max. = 0.17 ± 0.00 net BRET (p < 0.05), Fig. 3E ] and -2 [Max. = 0.23 ± 0.01 net BRET (p < 0.05), Fig. 3F ) was significantly reduced compared to wild-type receptor. Interestingly, as observed with Arg 352 -Gly 354 Ala mutation, thrombin-stimulated β-arrestin-1 recruitment was unaffected with Arg 358 -Ser 359 Ala mutation [Max. = 0.13 ± 0.01 net BRET (p = 0.06), Fig. 3L ); however, β-arrestin-2 recruitment was significantly reduced [Max. = 0.15 ± 0.00 net BRET (p < 0.05), Fig. 3M ). These data highlight an important role for H8 R 352 AGLFQRS 359 residues in the recruitment of β-arrestins to PAR4. Sequential mutation revealed a role for Leu 355 -Gln 357 residues in agonist-stimulated β-arrestin recruitment, regardless of which PAR4-agonist was applied. Thus, these residues may be a key regulatory site for this interaction as the phenotype is observed in response to either agonist tested. Interestingly, the data also reveal key agonist-dependent differences in which residues are important for β-arrestin recruitment. Peptide-stimulated β-arrestin-1/-2 recruitment to PAR4 was altered by all of the H8 mutations studied, with Leu 355 -Gln 357 Ala mutation more deleterious than the other mutations studied (p < 0.05). Interestingly, thrombin-stimulated recruitment of β-arrestin-1/-2 was also significantly decreased compared to wild-type receptor, like the effect observed with peptide stimulation of this mutant. All mutations studied were significantly deleterious to β-arrestin-2 recruitment downstream of thrombin-activation of the receptor; however, only Leu 355 -Gln 357 Ala mutation altered thrombin-stimulated recruitment of β-arrestin-1 in comparison to wild-type receptor. Thus, these results indicate both an agonist-dependent, and β-arrestin-subtype dependent role for H8 residues in recruitment to activated PAR4. AYPGKF-NH 2 or thrombin stimulated PAR4 activation of Gα q/11 and Gα oB signalling requires H8 Lys 350 H8 and C-terminal tail lysine residues have been shown for many Class A GPCRs to be involved in interactions with both G proteins and β-arrestins, as well as changes in the activation state of GPCRs 33 – 35 . Specifically, lysine residues located in the H8, which are adjacent to the canonical NPxxYX 5,6 F motif, have been implicated to be a binding site for various G protein subtypes including Gα t , Gα i , and Gα s 24 , 25 , 35 , 36 . The PAR4 H8 and C-terminal region contains two lysine residues, Lys 350 (8.53, Ballesteros-Weinstein numbering) and Lys 367 , which are distal to the PAR4 DPxxYX 5,6 F motif. Given the emerging role for H8 lysine residues in GPCR-G protein interaction, we investigated G protein activation in PAR4 receptors with C-terminal tail lysine mutations. Gα q/11 -mediated calcium signalling in HEK-293 cells expressing PAR4-YFP was recorded in response to AYPGKF-NH 2 - (EC 50 = 35.9 ± 12.8 µM, Max. = 26.1 ± 2.6% A23187, Fig. 4A ) or thrombin-stimulation (EC 50 = 1.3 ± 0.5 units/mL, Max. = 16.2 ± 1.7% A23187, Fig. 4H ). In comparison, cells expressing PAR4 with H8 lysine, Lys 350 Ala mutation (PAR4 K350A -YFP) had significantly decreased calcium signalling in response to both AYPGKF-NH 2 (EC 50 = n.d. , Max. at 300 µM = 8.5 ± 1.8% A23187; Fig. 4A ) and thrombin (EC 50 = n.d. , Signal at 300 µM = 4.3 ± 0.7% A23187, Fig. 4H ). Download figure Open in new tab Fig. 4 H8 Lys 350 residue is essential for Gα q/11 and Gα oB activation and β-arrestin-1/-2 recruitment to both AYPGKF-NH 2 - and thrombin-stimulated PAR4, but not Gα 13 activation. G protein activation (A-C, H-J). Calcium signalling from wild-type PAR4-YFP, PAR4 K350A -YFP, PAR4 K367A -YFP, and PAR4 K350A, K367A -YFP receptors in response to AYPGKF-NH 2 (A) or thrombin (H) stimulation. Nonlinear regression curve fits are shown (mean ± S.E.) for three independent experiments ( n = 3-4 ). G protein activation (TRUPATH) was recorded in cells expressing wild-type or mutant PAR4-YFP receptors in response to either AYPGKF-NH 2 [HEK- 293 cells; Gα 13 (B), Gα oB (C)] or thrombin [PAR1-KO-HEK-293 cells; Gα 13 (I), Gα oB (J)]. Nonlinear regression curve fits are shown (mean ± S.E.). Technical replicates were collected in triplicate for each experimental replicate ( n = 3-4 ). β -arrestin recruitment (D-F, K-M). Recruitment of β-arrestin-1/-2 to PAR4-YFP wild-type or Lys 350 Ala mutant PAR4-YFP following stimulation with AYPGKF-NH 2 (D) or thrombin (K). Recruitment of β-arrestin-1/-2 to PAR4- YFP wild-type or Lys 367 Ala mutant PAR4-YFP following stimulation with AYPGKF-NH 2 (E) or thrombin (L). Recruitment of β-arrestin-1/-2 to PAR4-YFP wild-type or Lys 350 Ala/Lys 367 Ala double mutant PAR4-YFP following stimulation with AYPGKF-NH 2 (F) or thrombin (M). Nonlinear regression curve fits are shown (mean ± S.E.) with triplicate data points for each concentration and receptor collected within each experiment. ( n = 3) . Summary data normalized to wild-type receptor (G, N) . For ease of comparison, signalling of mutant PAR4 receptors was normalized to signalling recorded from wild-type PAR4 receptor in response to AYPGKF-NH 2 (G) or thrombin (N) at the highest concentrations tested (300 µM AYPGKF-NH 2 , 10 units/mL thrombin) for each of the G protein activation and β-arrestin recruitment assays (A-F, H-M). Significance was calculated on raw (non-normalized) values for each assay by T-test and is indicated (*p < 0.05 compared to wild-type, † p < 0.05 compared to PAR4 K367A -YFP). Additionally, we observed that Lys 350 Ala mutation decreased both AYPGKF-NH 2 -stimulated Gα 13 [EC 50 = 41.3 ± 9.2 µM (p < 0.05), Max. = -0.15 ± 0.01 net BRET (p = 0.12), net BRET at 300 µM = -0.13 ± 0.01 (p < 0.05), Fig. 4B ] and Gα oB [EC 50 = n.d. , Max. = n.d. , net BRET at 300 µM = -0.10 ± 0.01 (p < 0.05), Fig. 4C ] activation compared to wild-type receptor [Gα 13 - EC 50 = 15.6 ± 1.5 µM, Max. = -0.17 ± 0.01 net BRET, Fig. 4B ; Gα oB - EC 50 = 27.4 ± 7.6 µM, Max. = -0.16 ± 0.01 net BRET, Fig. 4C ]. In contrast, Lys 350 Ala mutation significantly impacted thrombin-stimulated Gα oB signalling [EC 50 = n.d. , Max. = n.d. , net BRET at 10 units/mL = -0.05 ± 0.02 (p < 0.05), Fig. 4J ] but not Gα 13 signalling [EC 50 = 3.6 ± 2.1 units/mL (p = 0.13), Max. = - 0.12 ± 0.03 net BRET (p = 0.50), net BRET at 10 units/mL = -0.09 ± 0.01 (p = 0.07), Fig. 4I ] compared to wild-type receptor [Gα 13 - EC 50 = 1.6 ± 0.3 units/mL, Max. = -0.14 ± 0.01 net BRET, Fig. 4I ; Gα oB - EC 50 = 5.4 ± 3.3 units/mL, Max. = -0.17 ± 0.05 net BRET, Fig. 4J ]. Mutation of the distal C-terminal lysine residue (PAR4 K367A -YFP) also decreased calcium signalling following receptor stimulation with either AYPGKF-NH 2 [EC 50 = n.d. , Signal at 300 µM = 10.6 ± 4.4% A23187 (p < 0.05); Fig. 4 .A] or thrombin [EC 50 = 0.5 ± 0.3 units/mL, Max. = 10.8 ± 1.9% A23187 (p < 0.05); Fig. 4H ]. In contrast to the decreased signalling observed with Lys 350 Ala mutation, Lys 367 Ala mutation did not significantly alter either AYPGKF-NH 2 - or thrombin-stimulated Gα 13 [AYPGKF-NH 2 - EC 50 = 18.3 ± 5.4 µM (p = 0.67), Max. = -0.15 ± 0.01 net BRET (p = 0.25), Fig. 4B ; Thrombin - EC 50 = 2.2 ± 0.6 units/mL (p = 0.29), Max. = -0.14 ± 0.01 net BRET (p = 0.66), Fig. 4I ] or Gα oB [AYPGKF-NH 2 - EC 50 = 75.7 ± 26.4 µM (p < 0.05), Max. = -0.17 ± 0.02 net BRET (p = 0.65), Fig. 4C ; Thrombin - EC 50 = 6.8 ± 6.5 units/mL (p = 0.82), Max. = -0.16 ± 0.08 net BRET (p = 0.87), Fig. 4J ] activation. Combination of these lysine mutations (PAR4 K350A, K367A -YFP) also significantly reduced AYPGKF-NH 2 -stimulated calcium signalling [EC 50 = n.d. , Max. = n.d. , Signal at 300 µM = 4.0 ± 1.7% A23187 (p < 0.05), Fig. 4A ) but not thrombin-stimulated signalling compared to wild-type [EC 50 = 1.8 ± 2.0 units/mL (p = 0.79), Max. = 9.1 ± 2.8% A23187 (p = 0.22), Signal at 300 µM = 8.2 ± 3.0 (p = 0.08), Fig. 4H ]. We observed that Gα 13 was able to reach equivalent signalling by the highest concentrations of agonist tested however revealed a distinct rightward shift in the concentration of agonist required to reach EC 50 with both AYPGKF-NH 2 [EC 50 = 32.8 ± 8.8 µM (p < 0.05), Max. = -0.15 ± 0.01 net BRET (p = 0.20), Fig. 4C ] and thrombin [EC 50 = 6.0 ± 3.7 µM (p < 0.05), Max. = -0.16 ± 0.04 net BRET (p = 0.63), Fig. 4I ]. In contrast, Gα oB activation was significantly diminished compared to wild-type receptor with AYPGKF-NH 2 stimulation [EC 50 = n.d. , Max. = n.d. , net BRET at 300 µM = -0.07 ± 0.02 (p < 0.05), Fig. 4C ] but not thrombin activation of PAR4 [EC 50 = n.d. , Max. = n.d. , BRET at 10 units/mL = -0.08 ± 0.01 (p = 0.14), Fig. 4J ]. Together, these data implicate a previously unreported role of H8 Lys 350 in PAR4 Gα q/11 -, Gα 13 -, and Gα oB -mediated signalling downstream of PAR4 activation with either agonist peptide-mediated or thrombin cleavage-mediated activation of calcium signalling. Whether the residue is involved directly in G protein effector binding or a structural interaction with other active state receptor motifs, such as the NPxxYx 4,5 F motif or through receptor post-translational modifications (e.g. ubiquitination), remains to be explored. Additionally, the role of the more distal Lys 367 in peptide-activated PAR4 may provide some additional insight into key differences in effector signalling between peptide-activated and thrombin-activated PAR4 given that the effect of mutation was significantly deleterious to peptide agonist-stimulated, but not thrombin-stimulated, activation of Gα q/11 and Gα oB ( Fig. 4G , 4N). Further, these data may reveal G protein subtype-specific roles for Lys 367 as Gα 13 was unaffected with Lys 367 Ala mutation in response to either agonist. Importance of Lys 350 in AYPGKF-NH 2 - and thrombin-stimulated β-arrestin-1 and -2 recruitment revealed by PAR4 CT lysine mutants Lysine residues located in the H8, adjacent to the canonical NPxxYX 5,6 F motif, have also been implicated to have a role in interactions with the β-arrestin finger-loop, assisting the β-arrestin proteins in recognizing an active conformation of the receptor. To determine if these C-terminal tail lysine residues play a role in β-arrestin recruitment to activated PAR4, we recorded β-arrestin recruitment to PAR4 K350A -YFP and PAR4 K367A-YFP . Lys 350 Ala significantly reduced agonist-stimulated β-arrestin recruitment to PAR4 in response to AYPGKF-NH 2 stimulation [PAR4 K350A -YFP, β-Arr-1 Max. = 0.13 ± 0.00 net BRET (p < 0.0001), β-Arr-2 Max. = 0.16 ± 0.01 net BRET (p < 0.0001)] compared to wild-type PAR4-YFP (β-Arr-1 Max. = 0.24 ± 0.00 net BRET, β-Arr-2 Max. = 0.29 ± 0.01 net BRET, Fig. 4D ). Additionally, thrombin-stimulated β-arrestin recruitment was also significantly decreased to PAR4 K350A -YFP [β-Arr-1 Max. = 0.12 ± 0.00 net BRET (p < 0.0001), β-Arr-2 Max. = 0.12 ± 0.00 net BRET (p < 0.0001)] compared to wild-type receptor (β-Arr-1 Max. = 0.18 ± 0.01 net BRET, β-Arr-2 Max. = 0.20 ± 0.00 net BRET, Fig. 4K ). As discussed above, the CT of PAR4 contains one additional, non-H8 lysine residue, Lys 367 . Lys 367 Ala mutation had no effect on agonist-stimulated β-arrestin recruitment to the receptor compared to wild-type receptor in response to either AYPGKF-NH 2 [β-Arr-1 Max. = 0.22 ± 0.01 net BRET (p = 0.85), β-Arr-2 Max. = 0.32 ± 0.01 net BRET (p = 0.10), Fig. 4E ] or thrombin [β-Arr-1 Max. = 0.19 ± 0.01 net BRET (p = 0.60), β-Arr-2 Max. = 0.26 ± 0.02 net BRET (0.07), Fig. 4L ] compared to wild-type receptor (AYPGKF-NH 2 β-Arr-1 Max. = 0.22 ± 0.01 net BRET, β-Arr-2 Max. = 0.29 ± 0.01 net BRET, Fig. 4E ; thrombin β-Arr-1 Max. = 0.19 ± 0.01 net BRET, β-Arr-2 Max. = 0.23 ± 0.01 net BRET, Fig. 4L ). As expected given the effects of Lys 350 Ala mutation on β-arrestin recruitment, combined mutation of both lysine residues (PAR4 K350A, K367A -YFP) significantly decreased in response to AYPGKF-NH 2 [β-Arr-1 Max. = 0.12 ± 0.00 net BRET (p < 0.0001), β-Arr-2 Max. = 0.17 ± 0.01 net BRET (p < 0.0001), Fig. 4F ] or thrombin [β-Arr-1 Max. = 0.12 ± 0.01 net BRET (p < 0.0001), β-Arr-2 Max. = 0.16 ± 0.01 net BRET (p < 0.0001), Fig. 4M ]. Interestingly, in response to peptide agonism of the double mutant receptor, β-arrestin-1 recruitment was significantly decreased when compared to Lys 350 Ala mutation alone (p < 0.05); however, β-arrestin-2 recruitment was not significantly different than single mutation of the H8 Lys 350 (p = 0.07). Oppositely, thrombin-stimulated β-arrestin-1 recruitment did not show a significantly greater reduction than with the single Lys 350 Ala mutation alone (p = 0.95), however, β-arrestin-2 recruitment was statistically recovered compared to Lys 350 Ala mutation alone (p < 0.0001). Thus, the H8 Lys 350 residue appears to have a role in agonist-stimulated β-arrestin recruitment. Additionally, there may be a role for the distal Lys 367 in agonist-dependent or subtype specific differential recruitment of β-arrestins. DPxxYx 6 F motif residues, Tyr 340 and Phe 347 , are necessary for appropriate activation of Gα q/11 , Gα 13 , and Gα oB following AYPGKF-NH 2 or thrombin stimulation of PAR4 H8 and C-terminal tail residues are involved in regulating both interactions with β-arrestins and G proteins and changes in the activation state of many Class A GPCRs 33 – 35 . Many Class A GPCRs possess the canonical NPxxYX 5,6 F motif spanning TM7 and H8, including residues Asn 7.49 , Pro 7.50 , Tyr 7.53 , and H8 Phe 8.50 . This motif is thought to be involved in stabilizing the inactive state of Class A GPCRs. Additionally, mutational studies have demonstrated deficits in G protein activation and signalling when Tyr 7.53 and Phe 8.50 are mutated to alanine 26 , 37 . In PAR4, the NPxxYx 5,6 F motif is D 7.49 PFIY 7.53 YYVSAEF 8.50 . To determine if there is similar role for Tyr 7.53 and Phe 8.50 in PAR4-mediated activation of G proteins, single amino acid mutations of Tyr 340 (PAR4 Y340A -YFP) and Phe 347 (PAR4 F347A -YFP) to alanine were generated. Gα q/11 -mediated calcium signalling in HEK-293 cells expressing PAR4-YFP was recorded in response to AYPGKF-NH 2 - (EC 50 = 19.4 ± 8.4 µM, Max. = 20.6 ± 1.8% A23187; Fig. 5A ) or thrombin-stimulation (EC 50 = 0.4 ± 0.2 units/mL, Max. = 19.4 ± 2.6% A23187; Fig. 5G ). Cells expressing PAR4 with mutation of TM7, Tyr 340 Ala (PAR4 Y340A -YFP) had significantly decreased calcium signalling in response to both AYPGKF-NH 2 (EC 50 = n.d. , Max. = n.d. , Signal at 300 µM= 0.9 ± 2.4% A23187 (p < 0.05), Fig. 5A ] and thrombin [EC 50 = n.d. , Max. = n.d. , Signal at 10 units/mL = 2.8 ± 0.5% A23187; Fig. 5G ). Download figure Open in new tab Fig. 5 TM7 and H8 DPxxYx 6 F motif residues Tyr 340 and Phe 347 are essential for G protein activation and β-arrestin-1/-2 recruitment to both AYPGKF-NH 2 - and thrombin-stimulated PAR4, but not Phe 347 in Gα 13 activation. G protein activation (A-C, H-J). Calcium signalling from wild-type PAR4-YFP, PAR4 Y340A -YFP, and PAR4 F347A -YFP receptors in response to AYPGKF-NH 2 (A) or thrombin (G) stimulation. Nonlinear regression curve fits are shown (mean ± S.E.) for three independent experiments ( n = 3 ). G protein activation (TRUPATH) was recorded in cells expressing wild-type or mutant PAR4-YFP receptors in response to either AYPGKF-NH 2 [HEK-293 cells; Gα 13 (B), Gα oB (C)] or thrombin [PAR1-KO-HEK-293 cells; Gα 13 (H), Gα oB (I)]. Nonlinear regression curve fits are shown (mean ± S.E.). Technical replicates were collected in triplicate for each experimental replicate ( n = 3-4 ). β -arrestin recruitment (D-E, J-K). Recruitment of β-arrestins to wild-type or mutant PAR4-YFP receptors following stimulation with AYPGKF-NH 2 [β-Arr-1 (D), β-Arr-2 (E)] or thrombin [β-Arr-1 (J), β-Arr-2 (K)]. Nonlinear regression curve fits are shown (mean ± S.E.) with triplicate data points for each concentration and receptor collected within each experiment. ( n = 3) . Summary data normalized to wild-type receptor (F, L) . For ease of comparison, signalling of mutant PAR4 receptors was normalized to signalling recorded from wild-type PAR4 receptor in response to AYPGKF-NH 2 (F) or thrombin (L) at the highest concentrations tested (300 µM AYPGKF-NH 2 , 10 units/mL thrombin) for each of the G protein activation and β-arrestin recruitment assays (A-E, G K). Significance was calculated on raw (non-normalized) values for each assay by T-test and is indicated (*p < 0.05 compared to wild-type PAR4-YFP receptor). To determine whether the loss of function observed with PAR4 Y340A -YFP is due to the loss of interaction with the H8 Phe 347 residue, as would be expected as part of a traditional NPxxY 6 F motif, we generated a concomitant Phe 347 Ala mutant receptor and recorded calcium signalling. Like the deficits observed with Tyr 340 Ala mutation, PAR4 F347A -YFP mutation significantly abrogated calcium signalling downstream of PAR4 activation with both AYPGKF-NH 2 (EC 50 = n.d. , Signal at 300 µM = 5.1 ± 2.4% A23187; Fig. 5A ) and thrombin (EC 50 = n.d. , Max. = 4.1 ± 0.3% A23187; Fig. 5G ) stimulation. Thus, TM7 Tyr 340 appears to perform an integral role in the DPxxYx 6 F motif in PAR4 Gα q/11 -mediated calcium signalling through association with H8 Phe 347 . To determine if the loss of Tyr 340 or Phe 347 impacted activation of other G proteins, as would be expected with a more global mechanism of PAR4 activation (as observed with many other Class A GPCRs), we recorded Gα 13 and Gα oB activation in response to either AYPGKF-NH 2 or thrombin. Mutation of Tyr 340 significantly abrogated both Gα 13 and Gα oB activation in response to either AYPGKF-NH 2 [Gα 13 - EC 50 = n.d. , Max. = n.d. , net BRET at 300 µM = 0.03 ± 0.02 (p < 0.0001), Fig. 5B ; Gα oB - EC 50 = n.d. , Max. = n.d. , net BRET at 300 µM = -0.03 ± 0.2 (p < 0.05), Fig. 5C ] or thrombin [Gα 13 - EC 50 = n.d. , Max. = n.d. , net BRET at 10 units/mL = -0.05 ± 0.02 (p < 0.05), Fig. 5H ; Gα oB - EC 50 = n.d. , Max. = n.d. , net BRET at 10 units/mL = -0.03 ± 0.1 (p < 0.05), Fig. 5I ]. Interestingly, curve fitting of Gα 13 activation in response to agonist was not possible for PAR4 F347A -YFP mutation however, the net BRET achieved at the highest concentrations tested were equivalent to those achieved with wild-type receptor [AYPGKF-NH 2 - EC 50 = n.d. , Max. = n.d. , net BRET at 300 µM = -0.18 ± 0.01 (p = 0.32), Fig. 5B ; Thrombin - EC 50 = n.d. , Max. = n.d. , net BRET at 10 units/mL = -0.11 ± 0.01 (p = 0.21), Fig. 5H ]. In contrast to Gα 13 , Gα oB activation was significantly diminished in response to both agonists [AYPGKF-NH 2 - EC 50 = n.d. , Max. = n.d. , net BRET at 300 µM = -0.08 ± 0.02 (p < 0.05), Fig. 5C ; Thrombin - EC 50 = n.d. , Max. = n.d. , net BRET at 10 units/mL = -0.06 ± 0.01 (p < 0.05), Fig. 5I ]. Together, these data demonstrate a role for the DPxxYx 6 F motif in PAR4 activation of G protein signalling. Notably, both mutations impacted G protein activation; however, both Gα q/11 and Gα oB activation was all but completely abolished compared to wild-type receptor. In the present study we did not determine whether the decreased signalling is due to a lack of receptor-effector interaction or whether the receptor is in an active state upon loss of the Tyr 340 /Phe 347 contact. We hypothesize that the latter is a likely mechanism given the evidence with many other Class A GPCRs in this contact mediating an inactive state of the receptor; thus, loss of this inactivating contact may place the receptor in a perpetually active state in which case further agonist-induced change in signalling are not seen. Further studies using PAR4 antagonists could determine if these mutations lead to constitutive activation of PAR4. The role of DPxxYx 6 F motif residues, Tyr 340 and Phe 347 , in β-arrestin-1/-2 recruitment to activated PAR4 To determine whether the PAR4 NPxxYx 6 F motif contributes to β-arrestin-1/-2 recruitment we monitored β-arrestin recruitment to PAR4 receptors with Tyr 7.53 (Tyr 340 Ala) and Phe 8.50 (Phe 347 Ala) mutations. Tyr 340 Ala mutation significantly reduced agonist-stimulated β-arrestin recruitment to PAR4 in response to AYPGKF-NH 2 stimulation [β-Arr-1 Max. = 0.09 ± 0.00 net BRET (p < 0.0001), β-Arr-2 Max. = 0.10 ± 0.00 net BRET (p < 0.0001)] compared to wild-type PAR4-YFP β-Arr-1 Max. = 0.19 ± 0.01 net BRET, β-Arr-2 Max. = 0.25 ± 0.01 net BRET, Fig. 5D & E). Interestingly, Phe 347 Ala mutation yielded almost superimposable deficits in peptide-stimulated β-arrestin recruitment, when compared to Tyr 340 Ala mutation [Phe 347 Ala, β-Arr-1 Max. = 0.09 ± 0.00 (p < 0.0001), β-Arr-2 Max. = 0.09 ± 0.01 (p < 0.0001)] ( Fig. 5D & 5E). Similar to the decreases observed with peptide stimulation, β-arrestin-1/-2 recruitment was significantly reduced in both PAR4 Y340A -YFP [β-Arr-1 Max. = 0.09 ± 0.00 net BRET (p < 0.0001), β-Arr-2 Max. = 0.09 ± 0.00 net BRET (p < 0.0001)] and PAR4 F347A -YFP (β-Arr-1 Max. = 0.08 ± 0.00net BRET (p < 0.0001), β-Arr-2 Max. = 0.09 ± 0.00 net BRET (p < 0.0001)] mutants compared to wild-type receptor (β-Arr-1 Max. = 0.19 ± 0.01 net BRET, β-Arr-2 Max. = 0.24 ± 0.01 net BRET) when stimulated with thrombin ( Fig. 5J & 5K). Given the significant reduction in β-arrestin recruitment observed with both thrombin- and peptide-stimulated PAR4 containing these mutations, Tyr 340 and Phe 347 are likely key residues involved in the TM7/H8 interactions governing β-arrestin recruitment. Indeed, interaction between these two residues as a part of the DPxxYx 6 F motif in PAR4 appears to be important for both G protein activation and β-arrestin recruitment. Impact of CT phosphorylatable residue mutations on PAR4-stimulated G protein signalling β-arrestin-mediated desensitization of GPCR G protein signalling is a well-established phenomeon 38 – 41 . Within the PAR family of receptors, it has been demonstrated that PAR2 phosphorylation is a requirement for β-arrestin recruitment, ultimately leading to desensitization of the receptor 42 , 43 . In keeping with the well-established role of β-arrestins in GPCR desensitization, we previously demonstrated that of β-arrestin-1/-2 CRISPR/Cas9 knockout HEK-293 cells had both increased and prolonged calcium signalling following PAR2 stimulation 21 . Previously, it was reported that serine/threonine CT mutations in PAR1 increased G protein signalling, while analogous mutations to PAR4 had no impact on signalling 44 . To determine contribution of CT phosphorylation patterns on β-arrestin-mediated PAR4 receptor desensitization, we recorded calcium signalling in cells expressing two phosphorylation-site mutant receptors. PAR4 T363A/S366A/S369A -YFP-mediated calcium signalling (EC 50 = 14.0 ± 4.1 µM, Max. = 23.7 ± 3.3% A23187) was not significantly different than that elicited by the wild-type PAR4-YFP receptor (EC 50 = 21.1 ± 3.5 µM, Max. = 29.6 ± 1.0% A23187) in response to AYPGKF-NH 2 stimulation ( Fig. 6A ). Similarly, the EC 50 of thrombin-stimulated calcium signalling from PAR4 T363A/S366A/S369A -YFP (EC 50 = 0.8 ± 0.2 units/mL) was comparable to PAR4-YFP (EC 50 = 0.5 ± 0.2 units/mL). When Gα 13 signalling was assessed we observed a significant decrease in the net BRET recorded at the highest concentrations in response to both AYPGKF-NH 2 [EC 50 = 14.6 ± 4.7 µM, Max. = -0.11 ± 0.01 net BRET (p < 0.0001), net BRET at 300 µM = -0.12 ± 0.01 (p < 0.05), Fig. 6B ] or thrombin [EC 50 = 2.7 ± 0.9 units/mL, Max. = -0.11 ± 0.01 net BRET, net BRET at 300 µM = -0.09 ± 0.01 (p < 0.05), Fig. 6H ]. Unlike Gα q/11 and Gα 13 activation, we observed no significant differences in Gα oB activation with either agonist tested in PAR4 T363A/S366A/S369A -YFP signalling compared to wild-type [AYPGKF-NH 2 -EC 50 = 66.0 ± 35.8 µM, Max. = -0.15 ± 0.03 net BRET, net BRET at 300 µM = -0.13 ± 0.01, Fig. 6C ; Thrombin - EC 50 = 3.7 ± 2.9 units/mL, Max. = -0.13 ± 0.04 net BRET, net BRET at 300 µM = -0.09 ± 0.01, Fig. 6I ]. Download figure Open in new tab Fig. 6 CT phosphorylation barcode and phosphorylatable residue mutations decrease β-arrestin-1/-2 recruitment to both AYPGKF-NH 2 - and thrombin-stimulated PAR4 and phospho-null PAR4 CT mutation impacts appropriate G protein activation. G protein activation (A-C, H-J). Calcium signalling from wild-type PAR4-YFP, PAR4 T363A/S366A/S369A -YFP, and phospho-site null PAR4-YFP (PAR4 0P -YFP) receptors in response to AYPGKF-NH 2 (A) or thrombin (G) stimulation. Nonlinear regression curve fits are shown (mean ± S.E.) for three to four independent experiments ( n = 3-4 ). G protein activation (TRUPATH) was recorded in cells expressing wild-type or mutant PAR4-YFP receptors in response to either AYPGKF-NH 2 [HEK-293 cells; Gα 13 (B), Gα oB (C)] or thrombin [PAR1-KO-HEK-293 cells; Gα 13 (H), Gα oB (I)]. Nonlinear regression curve fits are shown (mean ± S.E.). Technical replicates were collected in triplicate for each experimental replicate ( n = 3-4 ). β -arrestin recruitment (D-E, J-K). Recruitment of β-arrestins to wild-type or mutant PAR4-YFP receptors following stimulation with AYPGKF- NH 2 [β-Arr-1 (D), β-Arr-2 (E)] or thrombin [β-Arr-1 (J), β-Arr-2 (K)]. Nonlinear regression curve fits are shown (mean ± S.E.) with triplicate data points for each concentration and receptor collected within each experiment. ( n = 3-4) . Summary data normalized to wild-type receptor (F, L) . For ease of comparison, signalling of mutant PAR4 receptors was normalized to signalling recorded from wild-type PAR4 receptor in response to AYPGKF-NH 2 (F) or thrombin (L) at the highest concentrations tested (300 µM AYPGKF-NH 2 , 10 units/mL thrombin) for each of the G protein activation and β-arrestin recruitment assays (A-E, G K). Significance was calculated on raw (non-normalized) values for each assay by T-test and is indicated (*p < 0.05 compared to wild-type PAR4-YFP receptor). When calcium signalling was recorded in the phosphorylation-site-null PAR4 0P -YFP receptor, peptide-stimulated calcium signalling was significantly decreased [EC 50 = 20.9 ± 15.3 µM, Max. = 10.3 ± 1.4% A23187, Signal at 300 µM = 10.9 ± 1.9 (p < 0.05), Fig. 6A ] compared to wild-type receptor at 300 µM (EC 50 = 21.4 ± 7.7 µM, Max. = 26.3 ± 2.1% A23187, Fig. 6A ). Interestingly, thrombin-stimulated signalling was unaffected (EC 50 = 1.2 ± 1.1 units/mL, Max. = 13.8 ± 2.6% A23187, Signal at 10 units/mL = 13.6 ± 2.4% A23187, Fig. 6G ) compared to wild-type receptor (EC 50 = 0.5 ± 0.1 units/mL, Max. = 19.4 ± 2.6%; Fig. 6G ). Therefore, unlike PAR2 which had significantly increased and prolonged calcium signalling in the absence of β-arrestins, removal of PAR4-phosphorylation sites does not significantly enhance calcium signalling, indeed it impairs Gα q/11 coupled calcium signaling in response to a peptide agonist. We were surprised to observe that, in addition to the decreased signalling observed with AYPGKF-NH 2 -stimulated Gα q/11 signalling, both Gα 13 and Gα oB signalling was also decreased in response to either AYPGKF-NH 2 [Gα 13 – EC 50 = n.d. , Max. = n.d. , net BRET at 300 µM = -0.10 ± 0.03 (p < 0.05), Fig. 6B ; Gα oB – EC 50 = n.d. , Max. = n.d. , net BRET at 300 µM = -0.11 ± 0.01 (p < 0.05), Fig. 6C ] or thrombin [Gα 13 – EC 50 = n.d. , Max. = n.d. , net BRET at 10 units/mL = - 0.06 ± 0.01 (p < 0.05), Fig. 6H ; Gα oB – EC 50 = n.d. , Max. = n.d. , net BRET at 10 units/mL= -0.06 ± 0.01 (p < 0.05), Fig. 6I ] stimulation of PAR4 0P -YFP compared to the wild-type receptor. Role for phosphorylation barcode motif and CT phosphorylation sites in differential recruitment of β-arrestins to PAR4 dependent on either AYPGKF-NH 2 or thrombin-revealed tethered ligand A role for phosphorylation-dependent GPCR-mediated recruitment of β-arrestins is well established 45 , 46 . The importance of phosphorylation codes/motifs in β-arrestin binding and signalling has also been indicated for many GPCRs including the β2-adrenergic receptor, rhodopsin receptor and vasopressin receptors 47 – 50 . A so-called “complete” phosphorylation barcode motif, involves three phosphorylatable residues interspaced by two residues [PxxxPxxP, wherein “P” denotes a phosphorylatable serine or threonine residue, and “x” denotes any other residue], which have been shown to be a key component of receptor CT/arrestin interaction 51 . Additionally, partial barcode motifs have been identified wherein a phosphorylatable residue spot within the motif is occupied by another residue, frequently an acidic residue 51 . Phosphorylation motifs in the PAR4 C-terminal tail were identified using the PhosCoFinder tool 51 which identified one complete phosphorylation motif -Thr 363 /Ser 366 /Ser 369 . Mutation of this motif to alanine (PAR4 T363A, S366A, S369A -YFP) significantly reduced β-arrestin recruitment to PAR4 in response to AYPGKF-NH 2 stimulation of the receptor [β-Arr-1 Max. = 0.10 ± 0.01 net BRET (p < 0.05), β-Arr-2 Max. = 0.18 ± 0.02 net BRET (p < 0.05), Fig. 6D ) compared to wild-type receptor (β-Arr-1 Max. = 0.15 ± 0.01 net BRET, β-Arr-2 Max. = 0.26 ± 0.02 net BRET, Fig. 6D ). Interestingly, in response to thrombin stimulation only β-arrestin-1 recruitment was significantly decreased [Max. = 0.07 ± 0.01 net BRET (p < 0.05), Fig. 6J ] compared to wild-type receptor (Max. = 0.14 ± 0.01 net BRET, Fig. 6J ); while β-arrestin-2 recruitment [Max. = 0.12 ± 0.02 net BRET (p = 0.11), Fig. 6J ] was not statistically different than thrombin-stimulated recruitment to wild-type receptor (Max. = 0.17 ± 0.02 net BRET, Fig. 6J ). Our data therefore implicate the CT barcode motif Thr 363 /Ser 366 /Ser 369 in peptide-stimulated recruitment of β-arrestin-1 and -2 to PAR4 and thrombin-stimulated β-arrestin-1 recruitment. Given that we do not observe a complete loss of β-arrestin-1/-2 recruitment with the complete barcode motif mutation (Thr 363 Ala/Ser 366 Ala/Ser 369 Ala), we generated a mutant PAR4 receptor wherein all of the CT serine/threonine residues are mutated to alanine (PAR4 0P -YFP) to determine if there was any further loss of recruitment. Recruitment of β-arrestin-1 to PAR4 0P -YFP was significantly reduced compared to wild-type receptor, however, was not significantly more detrimental than the barcode motif mutation alone [AYPGKF-NH 2 -β-Arr-1 Max. = 0.11 ± 0.00 net BRET (p < 0.0001 compared to wild-type; p = 0.70 compared to PAR4 T363A, S366A, S369A -YFP), Fig. 6E ; thrombin - β-Arr-1 Max. = 0.08 ± 0.01 net BRET, (p < 0.0001 compared to wild-type; p = 0.35 compared to PAR4 T363A, S366A, S369A -YFP), Fig. 6K ] ( Fig. 6F & 6L). Additionally, β-arrestin-2 recruitment in response to thrombin stimulation of PAR4 was significantly decreased compared to wild-type receptor, however, not more so than the barcode mutation alone [β-Arr-2 Max. = 0.10 ± 0.00 (p < 0.0001 compared to wild-type; p = 0.23 compared to PAR4 T363A/S366A/S369A -YFP), Fig. 6K & 6L]. Interestingly, we observed a further reduction of β-arrestin-2 recruitment to PAR4 in response to peptide stimulation in PAR4 0P -YFP compared to barcode motif mutation alone [β-Arr-2 Max. = 0.10 ± 0.01 (p < 0.0001 compared to wild-type; p < 0.05 compared to PAR4 T363A/S366A/S369A -YFP, Fig. 6E & 6F). These data may therefore highlight a role for agonist-dependent subtype selectivity, as was observed with the mutation of the complete phosphorylation barcode and reveal residues involved in arrestin-subtype selectivity in the CT of PAR4. These data, however, should be carefully followed up with further structural and functional studies to identify specific residues that enable this effect. Alphafold 3 predicted interactions between PAR4 and signaling effectors Finally we modeled PAR4 interactions with β-arrestin-1/-2, Gα q , Gα 11 , Gα 12 , Gα 13 , Gα i1 , and Gα o using AlphaFold 3 ( Fig. 7 ). The models revealed multiple contacts between β-arrestins and the phosphorylated C-tail of PAR4. Additionally, β-arrestins formed contacts with PAR4 intracellular loops and transmembrane domains, suggesting the capture of a core conformation. While the models showed interaction between PAR4 H8 residue (Ser 8.47 ) and β-arrestin-1, no H8 interactions were observed with β-arrestin-2. The models also predicted several interactions between PAR4 H8 and CT residues with Gα q . However, for Gα 11 , Gα i1 , Gα o , and Gα 12/13 , the predicted interactions were predominantly confined to the intracellular loops and transmembrane domain 6. The H8 residues identified through mutagenesis as critical for PAR4 interaction with signaling effectors may therefore mediate intermediate steps in these interactions that are not fully captured by our modeling approach. Further functional or structural studies will be needed to validate these predicted interactions. Download figure Open in new tab Figure 7. Alphafold3 prediction of PAR4 interaction with β-arrestins and G proteins. Tethered-ligand exposed PAR4 interactions with β-arrestin-1/-2, Gα q , Gα 11 , Gα 12 , Gα 13 , Gα o and Gα i1 were modeled using alphafold3. β-arrestin interactions were modeled with phosphorylated C-tail serine and threonine residues while G proteins were modeled without any post-translational modifications. Contacts within 3.5Å were identified and contacts are depicted as green pseudobonds and listed in the adjacent tables. In all cases the exposed tethered-ligand, docked in the orthosteric ligand binding pocket is depicted in blue. Ballesteros-Weinstein numbering (BW#) is from GPCRdb. Discussion Here we examined residues found within the H8 and CT as determinants of PAR4 coupling to several major signalling effectors including Gα q/11 , Gα 12/13 , Gα i/o , and β-arrestin. We find that residues in the PAR4 H8 (Lys 350 , Leu 355 , Phe 356 , Gln 357 ) are necessary for appropriate G protein activation and signalling, as well as β-arrestin-1/-2 recruitment. Additionally, we find that a TM7/H8 interaction (Tyr 340 /Phe 347 ) is important for receptor signalling activity. Further, we demonstrate that a phosphorylation barcode (Thr 363 /Ser 366 /Ser 369 ) is crucial for β-arrestin-1/-2 recruitment to PAR4. Interestingly, we also observed key differences in the relative importance of these sites, dependent on whether PAR4 was activated by the proteolytically-revealed tethered ligand or through agonist peptide stimulation. Thus, this study highlights the importance of H8 and CT residues in PAR4 signalling as well as underscores the subtle differences in effector coupling triggered by different modes of receptor activation. H8 R 352 AGLFQRS 359 Motif The H8 and C-terminal tail of GPCRs are key sites of recruitment, interaction, and activation of intracellular effectors, such as G proteins and β-arrestins. Structures of Gα s -, Gα i -, Gα o -, and Gα q -bound GPCRs show that residues within the H8 of adenosine 2A (A 2A R), β 2 AR, µ-opioid (MOR), adenosine A1 (A 1 R), serotonin 1B (5HT 1B R), rhodopsin, and 5-HT 2A receptors make contacts with the α and β (β1 or β2) subunits of their respective G proteins 52 , 53 . Conversely, studies of dopamine D1 and D2 receptor H8 chimeras have revealed that the H8 is not involved in dopamine receptor G protein coupling but is important for β-arrestin-mediated receptor desensitization of these receptors 54 . Previously, we demonstrated that deletion of an eight amino acid sequence in the H8 of PAR4 (R 352 AGLFQRS 359 ) abrogates Gα q/11 -mediated calcium signalling and significantly decreased β-arrestin-1/-2 recruitment 30 . In the current study we observed that mutation of this entire sequence to alanine (PAR4 R352-S359A -YFP) mimicked the abrogation of calcium signalling and reduced β-arrestin recruitment previously reported with the deletion mutant. Residues within the PAR1 H8 (S 375 SECQRYVYSILCC 388 ) were also shown to be necessary for Gα q signalling. Specifically, mutation of hydrophilic residues (Gln 379 Ala/Arg 380 Ala) significantly reduced Gα q -PLC-β-dependent inositol phosphate production by approximately 25% in response to both thrombin and peptide activation of PAR1 55 . When this mutation was combined with intracellular loop 1 mutant, Lys 135 Ala, a 40-50% reduction was observed in Gα q -PLC-β activity, highlighting the importance of an ICL1/H8 activation motif in PAR1-mediated Gα q activation 55 . Mutational studies of the rhodopsin receptor revealed a different mechanism wherein Arg 314 /Asn 315 mutation, located at analogous sites to PAR1 Gln 379 /Arg 380 (8.51/8.52), had no impact on Gα t activation; however, mutation of hydrophobic residues (Phe 313 , Met 8.54 ) significantly reduced Gα t activation 26,56 . Interestingly, mutation of the analogous PAR1 residues (Cys 378 , Val 382 ) did not impact Gα q -coupling suggesting a role for H8 in G protein subtype selectivity 55 . Similar to findings with rhodopsin, mutation of hydrophobic residues (Leu 404 , Phe 408 , and Phe 412 ) in the H8 of cannabinoid 1 receptor (CB 1 ) decreased G protein activation, while mutation of basic residue had no effect 57 . Here we identified that H8 residues Leu 355 -Gln 357 regulated PAR4 G protein activation. Interestingly, we observed that mutation of these residues impacts on both peptide- and enzyme-activated PAR4 mediated Gα q/11 -coupled calcium signalling. Therefore PAR4, like PAR1, requires a H8 glutamine for efficient Gα q/11 activation. Like in CB 1 , mutation of leucine may remove a necessary hydrophobic residue for G protein binding and activation 57 . Following a similar strategy, we investigated β-arrestin recruitment to the segmental mutations of the R 352 AGLFQRS 359 motif. As with calcium signalling, β-arrestin-1/-2 recruitment was significantly reduced with PAR4 Leu 355 -Gln 357 Ala mutation in response to both thrombin and peptide stimulation. Both peptide- and thrombin-stimulated β-arrestin-1 recruitment to PAR4 was decreased with Arg 358 -Ser 359 Ala mutation however, only peptide-stimulated β-arrestin-2 recruitment was diminished in this mutant. Further, we observed a reduction in thrombin-stimulated β-arrestin-2 recruitment to PAR4 with Arg 352 -Gly 354 Ala mutation. Interestingly, Arg 352 -Gly 354 Ala mutation enhanced both β-arrestin-1 and -2 recruitment following peptide stimulation. Thus, the overall decrease observed with Leu 355 -Gln 357 Ala mutation may represent a global phenotype since this mutation also decreased PAR4 calcium signalling, but there may be a role for other residues within the R 352 AGLFQRS 359 motif in arrestin-subtype selectivity and agonist-dependent differences in modulation of β-arrestin-1/-2 recruitment. Residues within the H8 has been implicated in regulating β-arrestin recruitment to other GPCRs. For example H8 domain swap experiments in the dopamine D1 (D1R) and D2 (D2R) receptors revealed a role for H8 in β-arrestin-1/-2-mediated desensitization in D1R, but not D2R 54 . The H8 of the rhodopsin receptor has also been implicated in mediating phosphate sensing through changes in conformational dynamics during the “pre-binding” state preceding CT phosphorylated residues interaction with the polar core of visual arrestin 58 . Therefore, our findings here should be followed up with studies evaluating whether mutations perturb interactions of the PAR4 H8 with β-arrestins directly or whether mutations in H8 impact conformational dynamics of H8 resulting in the diminished β-arrestin recruitment observed. The H8 of many GPCRs are known to interact with other regions of the GPCR or the plasma membrane to stabilize receptor conformations which are crucial to maintain both inactive and active conformations as well as mediate effector interaction 36 , 55 , 58 – 60 . In our current investigation of the H8 R 352 AGLFQRS 359 sequence we have not extensively evaluated the potential loss of intramolecular contacts with other regions of the receptor and this remains an area for future investigation. H8 Lys 350 and CT Lys 367 The PAR4 CT contains two lysine residues – Lys 350 in the H8 and Lys 367 distal to H8. Mutation of Lys 350 to alanine significantly decreased PAR4-mediated Gα q/11 , Gα 13 , and Gα oB activation and β-arrestin-1/-2 recruitment following peptide stimulation and Gα q/11 and Gα oB with thrombin activation of PAR4. Interestingly, Lys 367 Ala mutation also significantly reduced peptide-stimulated calcium signalling from PAR4, however, not as significantly as H8 Lys 350 Ala mutation and with no detrimental impact on peptide- or thrombin-stimulated β-arrestin recruitment. While the mechanism underlying the contribution of Lys 350 to Gα q/11 , Gα13, and Gα oB binding were not investigated in this study, there is a clear and supported role for charged H8 lysine residue in direct G protein interactions demonstrated in other class A GPCRs. Mutational studies with the muscarinic 3 receptor (M3R) revealed lysine within the N-terminal domain of H8 made contacts with the α4/β6 loop of Gα q/11 35 . Further, studies with µ-opioid receptor (µOR) implicate H8 lysine as having decreased solvent accessibility upon G protein binding following activation 33 . Similar to our observations with PAR4 Lys 350 Ala mutation (8.53), Lys 320 Gln (8.52) mutation in MCH 1 R decreased MCH-stimulated, Gα q -mediated calcium mobilization 24 . It is important to note that H8 lysine mutations in several GPCRs, including the melanin-concentrating hormone (MCH) receptor 1 (MCH 1 R) and bradykinin B 2 receptor (B 2 R), resulted in reduced plasma membrane expression 24 , 25 . To ensure that reduction in G protein activation was not due to poor membrane localization of lysine mutant PAR4 receptor, we examined and observed by confocal microscopy that PAR4 Lys 350 Ala, Lys 367 Ala, and Lys 350 Ala/Lys 367 Ala double mutant expressed appropriately on the cell membrane. As previously stated, mutation of Lys 350 decreased both peptide- and thrombin-stimulated β-arrestin-1/-2 recruitment, while Lys 367 Ala had no effect on agonist-stimulated recruitment. Additionally, combined mutation was no more detrimental than Lys 350 Ala mutation alone. Previous studies of the thyrotropin-releasing hormone (TRH) receptor (TRH 1 R) and B 2 R revealed that H8 lysine residues were important in mediating GRK interaction and CT phosphorylation of the receptors. Mutation of TRH 1 R H8 Lys 326 was found to significantly reduce CT phosphorylation and receptor internalization, which could be partially overcome with overexpression of G protein receptor kinase 2 (GRK2) 61 . Similarly, H8 lysine mutation to proline in B 2 R (Lys 315/8.53 ; analogous to Lys 350/8.53 residue in PAR4) decreased agonist-stimulated CT phosphorylation and agonist-dependent internalization, which could be recovered in part by overexpression of GRK2 or GRK3 25 . Thus, alterations in GRK-mediated receptor phosphorylation may be responsible for the reductions observed with Lys 350 mutation and should be investigated in future studies. DPxxYx 6 F motif Many Class A GPCRs possess a canonical NPxxYX 5,6 F motif/domain, spanning TM7 and H8, including Asn 7.49 , Pro 7.50 , Tyr 7.53 , and H8 Phe 8.50 . This motif is involved in stabilizing the inactive state of Class A GPCRs. In crystal structures of inactive state GPCRs, the side chain of Tyr 7.53 points towards helices I, II, or VIII, whereas, structures of the active state have Tyr 7.53 changing rotamer conformation to face the interior of the transmembrane bundle, pointing towards helices VI and III 62 . Additionally, an inactive-state stabilizing water molecule network involving the TM7 NPxxY, TM3 E/DRY, and TM6 WXPF/Y motifs may be partially constituted by Tyr 7.53 37 . Previous studies of rhodopsin and the β 2 -adrenergic receptor have demonstrated that mutations in this motif alter ligand affinity, receptor plasma membrane expression, G protein coupling, and interactions with small G proteins, such as RhoA, dependent on the receptor studied 26 – 29 . In our mutational study, we observed a significant loss of Gα q/11 , Gα 13 , and Gα oB signalling with mutation of Tyr 340 -Ala and of Gα q/11 and Gα oB with Phe347-Ala mutations, irrespective of the stimulating PAR4-activating agonist. In all cases there was no impact on appropriate cell membrane localization ( Supplementary Fig. 1 ) of the receptor. Functional studies have highlighted that the absence of side-chain interaction between Tyr 7.53 and Phe 8.50 increases the population of active rhodopsin (Meta II state) with no concomitant increase in transducin (Gα t ) activation 26 , 37 . Further, these studies show that hydrophobic side chain interaction between Tyr 7.53 and Phe 8.50 constitutes a constraint that becomes necessarily perturbed during receptor interaction to allow for conformational rearrangement of H8 and G protein binding 26 , 37 . Mutation of rhodopsin H8 Phe 313 (8.50) to alanine caused a corresponding loss of Gα t activation with rhodopsin 26 . Further, perturbation of this interaction, as well an interaction between TM3 3.46 and TM6 6.37 residues, enables Tyr 7.53 to make a new contact with TM6 6.37 residue and form an interaction with the α5 helix of the G protein, which has been demonstrated in several Class A GPCRs (rhodopsin, M2R, µOR, β 2 AR, and A 2A R) 63 . Mutation of Tyr 7.53 in the V2 vasopressin receptor (V2R) also significantly reduces the receptor-mediated activation of both Gα s and Gα q 63 . While it is not possible in this study to resolve if a change in the active-state population of PAR4 or a perturbation in the conformational rearrangement of TM7/H8 interaction is responsible for the loss of signalling observed, it is clear that these residues, especially Tyr 340 , have a role in Gα q/11 , Gα 13 , and Gα oB signalling downstream of PAR4 activation. In addition to substantial reductions in G protein activation, we observed significant decreases in both thrombin- and peptide-stimulated recruitment of β-arrestin-1/-2 to PAR4 DPxxYx 6 F mutants, Tyr 340 Ala and Phe 347 Ala. The NPxxYx 5,6 F motif has been shown for several Class A GPCRs to be involved in interactions with β-arrestins. Mutations of this motif are thought to perturb GRK interactions with the GPCR and thus decrease GRK-mediated phosphorylation 33 – 35 . In studies of the β 2 -adrenergic receptor (β 2 AR), mutation of TM7 Tyr 326 (7.53) decreased GRK-mediated phosphorylation and receptor sequestration, which was reversible through overexpression of GRKs 2-6 27 , 64 , 65 . Similar to our observation in the PAR4 Tyr 340 Ala and Phe 347 Ala mutations, β-arrestin recruitment is significantly reduced in both β 2 AR and α 1B AR receptors with TM7 Tyr 7.53 mutations 66 . Whether changes in GRK-mediated receptor phosphorylation of PAR4 accounts for the reduction in β-arrestin recruitment observed with Tyr 340 Ala and Phe 347 Ala mutations should be the focus of further study. Phosphorylation-site mutations A role for phosphorylation-dependent GPCR-mediated recruitment of β-arrestins is well established 45 , 46 . The importance of a phosphorylation barcode motif in β-arrestin binding and signalling has also been demonstrated for many GPCRs including the β2-adrenergic receptor, rhodopsin receptor, chemokine receptors 4 & 7, vasopressin, and angiotensin II receptor type 1 receptors 47 – 50 . It has been reported that 52.3% of Rhodopsin family GPCRs possess either a full or partial phosphorylation barcode 51 . These GPCRs can be further divided into two classes based on their β-arrestin-1 and -2 binding characteristics. Class A GPCRs bind β-arrestin-2 more tightly than β-arrestin-1 and are typified by receptors such as α1- and β2-adrenergic, μ-opioid, and dopamine D 1 A receptors. Class B GPCRs bind β-arrestins-1/-2 equally well (e.g. angiotensin II type 1A receptor, neurotensin receptor 1, vasopressin-2 receptor, thyrotropin-releasing hormone receptor, and substance P receptor) 67 . All class A β-arrestin-binding GPCRs have, at most, one complete phosphorylation barcode (PxxPxxP, as previously defined) 51 . Interestingly, our data consistently show more robust recruitment of β-arrestin-2 versus β-arrestin-1 and the PAR4 CT possesses only one such complete phosphorylation barcode. Thus, PAR4 may be considered a class A β-arrestin-binding GPCR. Further, when the complete phosphorylation barcode is mutated (Thr 363 Ala/Ser 366 Ala/Ser 369 Ala), we observe significantly reduced β-arresin-1/-2 recruitment in response to both peptide and thrombin activation of the receptor compared to wild-type receptor. While some phosphorylation barcodes may contribute to β-arrestin recruitment to activated GPCRs, other phosphorylated residues may play a role in β-arrestin subtype-specificity, the recruitment of other downstream signalling partner (such as SNX27 for β 2 AR) and mediate of affinity of β-arrestin binding to GPCRs 47 , 48 , 51 , 68 . We therefore evaluated β-arrestin recruitment to a CT phospho-null PAR4 receptor. Interestingly, only peptide-stimulated β-arrestin-2 recruitment was further reduced compared to Thr 363 Ala/Ser 366 Ala/Ser 369 Ala mutation alone. Thus, these data support a predominant role for the complete phosphorylation barcode in β-arrestin recruitment to activated PAR4. When we evaluate the impact of reduced β-arrestin-1/-2 recruitment to PAR4 on G protein signalling, we observed no significant increase or prolongation in agonist-stimulated calcium signalling compared to wild-type receptor. This is in stark contrast to what we previously observed with PAR2-mediated signalling wherein, maximal calcium signalling increases and demonstrates prolonged kinetics compared to conditions when β-arrestin recruitment is impaired 21 . These data are in agreement with previously published mutational studies of serine/threonine residues in the PAR4 CT which had no effect on increasing PAR4 G protein signalling 44 . This suggests that while β-arrestin recruitment is reduced in the PAR4 phosphorylation barcode motif mutant, the level of recruitment may be sufficient to desensitize the calcium signalling pathway. Alternatively, there may be other mechanisms enabling PAR4 desensitization such as ubiquitination and internalization as observed with PAR1 and PAR2 69 . Intriguingly, we observed decreased activation of G protein signalling in the phosphorylation null PAR4 mutant (but not phosphorylation barcode mutated PAR4) which suggest that one or more of these CT residues may serve a functional role in G protein activation, the mechanisms of which are not readily apparent but may involve direct interactions and warrant future investigation. Conclusion Overall, our findings unveil crucial molecular mechanisms governing PAR4-stimulated G protein and β-arrestin-1/-2 signalling and regulation. Further, these data highlight important divergences in the relative importance of H8 and CT residues on PAR4 signalling, dependent on peptide- or enzyme revealed tethered-ligand activation of the receptor. Differential activation of PAR4 by different enzymes may similarly exhibit differential regulation of effector interactions with PAR4. Alphafold 3 modelling further predicted additional interactions that may govern stable interactions between PAR4 and signaling effectors. Ultimately, understanding the molecular mechanisms governing effector interactions, signalling, and signal regulation at PAR4 provides novel insights into how this receptor functions and may aid in the development of novel therapeutics targeting PAR4. Materials and Methods Chemicals and Other Reagents Thrombin from human plasma (catalogue no. 605195) was purchased from Millipore-Sigma (St. Louis, MI). AYPGKF-NH 2 (> 95% purity by HPLC/MS) was purchased from Genscript (Piscataway, NJ). Agonists were prepared in 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES, 25 mM). All other chemicals or reagents were purchased from Millipore-Sigma, Thermo Fisher Scientific, or BioShop Canada, Inc. (Burlington, Ontario, Canada), unless otherwise stated. Molecular Cloning and Constructs The plasmid encoding the human PAR4 receptor with an in frame enhanced yellow fluorescent protein fusion tag (PAR4-YFP) has been previously described 30 . eYFP fused PAR4 has been utilized in several previous studies where we have demonstrated that PAR4-YFP couples to the known PAR4 signalling pathways appropriately 13 , 17 , 17 , 30 . QuikChange XL Multi Site-Directed Mutagenesis kit (Agilent Technologies, Mississauga, ON, Canada) was used to generate all H8 and the C-terminal tail PAR4 mutants described in this study. Additionally, wild-type and mutant PAR4 constructs were generated with C-terminal HA tags (2x HA-Stop) for use in TRUPATH signalling assays. All constructs were verified by Sanger sequencing (London Regional Genomics Centre, University of Western Ontario). Cell Lines and Culture Conditions All media and cell culture reagents were purchased from Thermo Fisher Scientific (Waltham, MA). Human embryonic kidney (HEK) cells (HEK-293; ATCC) and PAR1-knockout HEK-293 70 cell lines were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% sodium pyruvate, and penicillin streptomycin solution (50,000 units penicillin, 50,000 μg streptomycin). Since trypsin activates PAR4, cells were routinely sub-cultured using enzyme-free isotonic phosphate-buffered saline (PBS) containing EDTA (1 mM). Cells were transfected with PAR4-YFP or mutated PAR4-YFP receptor vectors using X-tremeGENE 9 (Millipore-Sigma). Transiently transfected cells were always assayed or imaged at 48 hours post-transfection to ensure consistent levels of protein expression. Calcium Signalling Agonist-stimulated calcium signalling was recorded in HEK-293 and PAR1-knockout HEK-293 cells, as previously described 13 , 71 , 72 . Cells were detached in enzyme-free cell dissociation buffer, pelleted, and resuspended in Fluo-4 NW (no wash) calcium indicator dye (Thermo Fisher Scientific). Following a 30-minute incubation at ambient temperature, intracellular fluorescence (excitation 480 nm; emission recorded at 530 nm) was monitored before and after addition of agonists (thrombin, PAR1-knockout HEK-293; or AYPGKF-NH 2 , HEK-293) on a PTI spectrophotometer (Photon Technology International, Birmingham, NJ). Responses were normalized to the fluorescence obtained with calcium ionophore (A23187, 3 μM; Sigma-Aldrich). Measurement of G protein activation with TRUPATH G protein activation in response to thrombin or AYPGKF-NH 2 -mediated activation of PAR4 was recorded with wild-type and mutant PAR4 constructs using TRUPATH biosensors (Addgene, Kit# 1000000163) 22 . Cells were plated to a density of 2.1-2.3 x 10 6 cells/dish in 10 cm dishes and transfected using a 1:1:1:1 DNA ratio of receptor:Gα-RLuc8:Gβ:Gγ-GFP2 (750ng/construct), using X-tremeGENE 9 (12 µl) (Millipore-Sigma) in OptiMEM (Gibco-ThermoFisher, Waltham, MA) with the appropriate Gα-RLuc8:Gβ:Gγ-GFP2 pairings reported in Olsen et. al, 2020 22 . 24 hours post-transfection, cells were mechanically lifted and replated into white 96-well plates (Greiner Bio-One, Monroe, NC; Corning; Oneonta, NY). At 48 hours, cell media was removed and replaced with 60 μL of assay buffer (1x HBSS + 20 mM HEPES, pH 7.4), followed by a 10 μL addition of freshly prepared 50 μM coelenterazine 400a (Nanolight Technologies, Pinetop, AZ). After a five-minute equilibration period, cells were treated with 30 μL of drug for an additional 5 minutes. Plates were then read in an LB940 Mithras plate reader (Berthold Technologies, Oak Ridge, TN) with 395 nm (RLuc8-coelenterazine 400a) and 510 nm (GFP2) emission filters, at 1 second/well integration times. BRET2 ratios were computed as the ratio of the GFP2 emission to RLuc8 emission. BRET Detection of β-arrestin-1/-2 Recruitment BRET assays were employed to detect agonist-stimulated β-arrestin-1/-2 recruitment to PAR4-YFP and mutant PAR4-YFP constructs in HEK-293 cells as described 13 , 17 , 30 . PAR4-YFP or mutant PAR4-YFP constructs (1 µg) and Renilla luciferase-tagged β-arrestin-1 or -2 (β-arr-1 and -2-Rluc; 0.1 µg) were transiently transfected for 48 hours. Cells were plated in white 96-well culture plates (Corning; Oneonta, NY) and recruitment of β-arresitin-1/-2 to PAR4 were detected by measuring the BRET signal 20 minutes after agonist stimulation and the addition of 5 μM h-coelenterazine prior to BRET recording (NanoLight Technology, Pinetop, AZ) on a Mithras LB940 plate reader (Berthold Technologies, Bad Wildbad, Germany), as previously described 13 , 30 . Confocal Microscopy HEK-293 cells transiently transfected with PAR4-YFP or mutant PAR4-YFP were sub-cultured onto glass coverslips (Thermo Fisher Scientific) and analyzed by confocal microscopy to ensure appropriate cell surface expression. Cells were fixed with 4% w/v paraformaldehyde solution (methanol-free; Fisher Scientific), stained with DAPI for nuclear staining, and the subcellular receptor localization was assessed by imaging eYFP expression with an Olympus FV1000 (Centre Valley, PA). Alphafold3 modelling of PAR4 effector complexes Interactions of PAR4 with signaling effectors was modelled using the Alphafold3 server ( https://alphafoldserver.com ). The highest ranked models were visualized in ChimeraX and contacts within 3.5Å were identified using the “alphafold contacts” command. In the case of modelling PAR4-β-arrestin-1/-2 contacts, the CT Ser and Thr residues were phosphorylated. No post-translational modifications were applied when modeling G protein interactions. The list of amino acid sequences ( Table 1 ) and all Alphafold models and associated data are available in the supplementary information. View this table: View inline View popup Table 1. PAR4, β-arrestin and G protein sequences used for Alphafold3 prediction of protein structures. Statistical Analysis Curve fitting (three-parameter, nonlinear regression) and statistical analysis was completed with Prism 7 software (GraphPad Software, La Jolla, CA). Statistical significance of EC 50 shifts was calculated using the extra sum of squares analysis 13 , 21 , 73 . TRUPATH BRET were conducted on all mutants and wild-type receptor simultaneously thus the PAR4-YFP curves within all Gα 13 and Gα oB graphs is repeated for reference (data and curves shown in black, circles). Net BRET, calcium signalling, and TRUPATH data obtained with AYPGKF-NH 2 (300 µM) or thrombin (10 units/mL) were used to compare the maximal signal achieved in our study for comparison of mutant PAR4 constructs to wild-type PAR4. This has been especially important in cases where a mutant did not signal in a manner that enable robust curve-fitting for comparison. Data obtained with AYPGKF-NH 2 (300 µM) or thrombin (10 units/mL) was normalized compared to wild-type PAR4 receptor for all mutants and is shown as a column graph within figures; however, statistical significance was calculated by T-test on the raw data values and is reported on column graphs of normalized findings for ease of the reader. Previously, we demonstrated that β-arrestin recruitment to PAR4 does not saturate upon stimulation with AYPGKF-NH 2 and thrombin 17 , 21 , 30 ; thus, statistical significance of maximal net BRET (300 µM AYPGKF-NH 2 , 10 units/mL thrombin) was determined using t-test (*p < 0.05) and all data within the text represents the net BRET signal achieved at the highest concentration of agonists tested. Data are expressed as mean ± S.E. throughout the text, table, and figure legends. Funding These studies were funded by grants from the Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council of Canada. P.E.T. was the recipient of a QEII Graduate Scholarship in Science and Technology. Competing interests The authors have no conflict of interest related to findings described in this manuscript. Data and material availability All data supporting the findings in this study are included in the manuscript or supplementary materials. Original data or materials are available from the corresponding author upon reasonable request. Supplementary data Supplementary Figure 1. Confocal micrographs of wild-type and mutant PAR4-YFP expression Supplementary Figure 2. Thrombin stimulated PAR4 Gα 11 TRUPATH activation Supplementary Figure 3. AYPGKF-NH 2 stimulated PAR4 Gα 11 TRUPATH activation. Supplementary Table 1. Sequences of receptor and effectors for Alphafold3 modeling. Supplementary data Zip folder. PAR4 H8 and CT regulation of signaling SI AF models Download figure Open in new tab Supplementary Figure 1. Confocal micrographs of PAR4-YFP and mutant receptor cellular expression. Confocal microscopy was employed to determine appropriate cell membrane expression of wild-type and mutant PAR4-YFP constructs. We observed membrane expression with wild-type and all mutant PAR4 receptor constructs. Scale bar is 20 µm. ( n = 3 ) Download figure Open in new tab Supplementary Figure 2. Gα 11 activation (TRUPATH) by thrombin-stimulated wild-type (WT) and mutant PAR4 receptors. Nonlinear regression curve fits are shown (mean ± S.E.) for at least three independent experiments ( n = 3 ) in PAR1-KO-HEK-293 cells. Download figure Open in new tab Supplementary Figure 3. Gα 11 activation (TRUPATH) by AYPGKF-NH 2 -stimulated wild-type (WT) and mutant PAR4 receptors. Nonlinear regression curve fits are shown (mean ± S.E.) for at least three independent experiments ( n = 3 ) in PAR1-KO-HEK-293 cells. Acknowledgments We thank Dr. Michel Bouvier for providing the Renilla luciferase-tagged β-arrestin-1 and -2 constructs. The TRUPATH assay kit was a kind gift from Dr. Bryan Roth. 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