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| GPCRs signal to SH3-CPs via the 3 proline regions of β-arrestin 1. a, Activation of selected GPCRs recruited SH3-CPs to β-arrestin 1, which was examined using Co-IP. The bar graph presents quantitative analyses of interactions between β-arrestin 1 and different SH3-CPs after the activation of different receptors by their corresponding agonists (see Methods and Supplementary Fig. 5 for further details). Data are presented as means ± s.d. from three independent experiments (n = 3). *P < 0.05; **P < 0.01; ***P < 0.005 (agonist-treated cells and PBS buffer-treated cells), one-way ANOVA. b, Graphic representation of the specificity of β-arrestin 1 PRs in mediating GPCR-SH3-CPs coupling. c-e, Overexpression of GRK6 promotes β-arrestin1-mediated SH3-CP recruitment, whereas GRK2 overexpression exerted significant opposite effects. The results of quantitative analyses of Co-IP between β-arrestin 1 and SH3-CPs are shown as bar graphs, and representative western blots are shown in Supplementary Fig. 4d. f-h, Effects of the 'snuggly' conformation mutant (β 2AR ΔICL3 ) and the 'hanging' conformation mutants (the S355A and E369A mutants of β 2AR) on β-arrestin-1-mediated SH3-CP recruitment. Quantitative analyses of sequential co-IP for HEK293 cells transfected with Flag-β 2AR (or its mutants), HA-β-arrestin 1 and different SH3-CPs (see Methods and Supplementary Fig. 8d for further details). For c-e and f-h, values are presented as the mean ± s.d. of three independent experiments (n = 3). Significant differences between ISO-treated and untreated cells (**P < 0.01; ***P < 0.005; ns, no significant difference), different β 2AR mutants and wild-type β 2AR, or cells transfected with the selected GRK and pcDNA3.0 ( ## P < 0.01; ### P < 0.005) were analyzed by one-way ANOVA. i, A best-fit linear correlation of the receptor phospho-C-tail-induced chemical shifts at the F87F2Y site with the binding efficacy of the SRC SH3 domain at the P1 site of β-arrestin 1 (Supplementary Fig. 13b). j, Correlation of the receptor phospho-C-tail-induced chemical shifts at the L123F2Y site with the binding efficacy of SRC SH3 domain at the P2 site of β-arrestin 1 (Supplementary Fig. 13c). In i and j the error bar along the y axis for each point indicates the deviation of the pull-down assay results derived from three independent experiments (n = 3). k, Structural representation of the allosteric regulation between specific phospho-binding sites and remote PRs (PDB: 4JQI).
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Signals from 800 G-protein-coupled receptors (GPCRs) to many SH3 domain-containing proteins (SH3-CPs) regulate important physiological functions. These GPCRs may share a common pathway by signaling to SH3-CPs via agonist-dependent arrestin recruitment rather than through direct interactions. In the present study, 19F-NMR and cellular studies reveal...
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... mediates GPCR-SH3-CP coupling via three proline regions. Among the 825 GPCR sequences, 753 GPCRs (91.3%) do not contain the classic PR recognized by SH3-CPs (Supplementary Figs. 1-3; see also Supplementary Note 1). Although 72 GPCRs bearing the PR motifs were potentially able to directly interact with SH3-CPs, the remaining GPCRs might connect to SH3- CPs through other receptor-interacting proteins. We therefore postulated that many GPCRs might share a common pathway connected to a landscape of SH3-CPs via ...
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... CHeMICal BIoloGy type I PRs that are potential SH3-CP docking sites ( Fig. 1b and Supplementary Fig. ...
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... 1, Flag-tagged GPCRs and different SH3-CPs into HEK293 cells. After agonist stimulation, the β -arrestin-1-mediated recruitment of SH3-CPs was detected by co-immunoprecipitation (co-IP). Importantly, regardless of the G-protein subtype coupling or arrestin-binding avidity, each receptor used arrestin to recruit 1-3 of the 5 SH3-CPs tested ( Fig. 1a and Supplementary Fig. ...
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... C-tail or intracellular loops, have been shown to bind arrestin 10,11,28,36,37 . We examined the roles of dif- ferent GRK subtypes using the prototype β 2AR as a model to assess the contribution of the receptor-phospho barcode. Overexpression of GRK6, but not GRK2, effectively promoted arrestin-mediated β 2AR-SH3-CP coupling in the co-IP assay (Fig. ...
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... purified with M1-Flag beads and eluted with Flag peptide, and the β 2AR-β -arrestin 1 complex was then further immunoprecipitated with HA-agarose ( Supplementary Fig. 8d). Although β 2AR ΔICL3 activation did not significantly decrease the association of SRC and FYN with β -arrestin 1, the β 2AR phospho-site mutants abol- ished these interactions (Fig. 1f,g and Supplementary Fig. 8d). The β 2AR S355A mutant significantly reduced the interaction between PLCγ and β -arrestin 1 in response to ISO stimulation. Moreover, the E369A mutation of β 2AR, which is a potential common interac- tion site for both the 'hanging' and 'snuggly' conformations of the GRK6-phosphorylated-β 2AR-β -arrestin 1 complex, completely ...
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... reduced the interaction between PLCγ and β -arrestin 1 in response to ISO stimulation. Moreover, the E369A mutation of β 2AR, which is a potential common interac- tion site for both the 'hanging' and 'snuggly' conformations of the GRK6-phosphorylated-β 2AR-β -arrestin 1 complex, completely eliminated the interaction between β -arrestin 1 and PLCγ (Fig. 1h). Moreover, the inclusion of the GRK6-phosphorylated β 2AR C-tail (GRK6pp-β 2AR-CT), but not that of the GRK2-phosphorylated C-tail (GRK2Bpp-β 2AR-CT), biochemically recapitulated the recruitment of the SH3 domain to active arrestin based on in vitro reconstitution and a GST-pull-down assay ( Supplementary Figs. 8e and 11a). These ...
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... next incorporated the unnatural amino acid 3,5-difluoroty- rosine (F2Y) in specific positions of β -arrestin 1 and used 19 F-NMR to detect the conformational changes in the three PRs in response to different types of receptor phospho-C-tail stimuli ( Supplementary Figs. 9 and 11a,b). These synthesized receptor phospho-C-tails induced the formation of active arrestin conformations that largely mimicked the hanging conformation of the receptor-arrestin com- plex 10,15,28 . ...
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... synthesized receptor phospho-C-tails induced the formation of active arrestin conformations that largely mimicked the hanging conformation of the receptor-arrestin com- plex 10,15,28 . We used the F2Y technique because F2Y-incorporated arrestin causes minimal perturbations to the overall structure and provides a more significant chemical shift than traditional bromo-4(trifluoromethyl)acetanilide (BTFA)-labeled cysteine- negative arrestin ( Supplementary Fig. 10a-c). The increase in the 19 F-NMR signal was due to the sensitivity of the phenolic F2Y oxygen in response to environmental alterations ( Supplementary Fig. 10a-c). ...
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... used the F2Y technique because F2Y-incorporated arrestin causes minimal perturbations to the overall structure and provides a more significant chemical shift than traditional bromo-4(trifluoromethyl)acetanilide (BTFA)-labeled cysteine- negative arrestin ( Supplementary Fig. 10a-c). The increase in the 19 F-NMR signal was due to the sensitivity of the phenolic F2Y oxygen in response to environmental alterations ( Supplementary Fig. 10a-c). All F2Y-incorporated β -arrestin 1 probes were func- tional ( Supplementary Fig. 10d-f). ...
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... increase in the 19 F-NMR signal was due to the sensitivity of the phenolic F2Y oxygen in response to environmental alterations ( Supplementary Fig. 10a-c). All F2Y-incorporated β -arrestin 1 probes were func- tional ( Supplementary Fig. 10d-f). Intriguingly, the phospho-V2R- C-tail (V2Rpp) and the GRK6pp-β 2AR-CT induced prominent chemical shifts, whereas GRK2Bpp-β 2AR-CT produced small but significant downfield-shifted resonances at the F87 and L123 positions, representing structural rearrangements in P1 and P2 of β -arrestin 1 ( Supplementary Figs. ...
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... 1 probes were func- tional ( Supplementary Fig. 10d-f). Intriguingly, the phospho-V2R- C-tail (V2Rpp) and the GRK6pp-β 2AR-CT induced prominent chemical shifts, whereas GRK2Bpp-β 2AR-CT produced small but significant downfield-shifted resonances at the F87 and L123 positions, representing structural rearrangements in P1 and P2 of β -arrestin 1 ( Supplementary Figs. 9c and 11a,b). In contrast, only the V2R phospho-C-tail induced a conformational change at the P3 site (R177F2Y) (Supplementary Fig. 9c). Notably, GRK6pp-β 2AR-CT and the phospho-V2R-C-tail did not induce any chemical shifts at the F87, L123 or R177 positions in phospho-peptide-binding-defi- cient mutants of β -arrestin 1 ( Supplementary Fig. ...
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... contrast, only the V2R phospho-C-tail induced a conformational change at the P3 site (R177F2Y) (Supplementary Fig. 9c). Notably, GRK6pp-β 2AR-CT and the phospho-V2R-C-tail did not induce any chemical shifts at the F87, L123 or R177 positions in phospho-peptide-binding-defi- cient mutants of β -arrestin 1 ( Supplementary Fig. 12a-e). ...
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... ## P < 0.01; ### P < 0.005) were analyzed by one-way ANOVA. i, A best-fit linear correlation of the receptor phospho-C-tail-induced chemical shifts at the F87F2Y site with the binding efficacy of the SRC SH3 domain at the P1 site of β -arrestin 1 ( Supplementary Fig. 13b). j, Correlation of the receptor phospho-C-tail-induced chemical shifts at the L123F2Y site with the binding efficacy of SRC SH3 domain at the P2 site of β -arrestin 1 ( Supplementary Fig. 13c). ...
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... A best-fit linear correlation of the receptor phospho-C-tail-induced chemical shifts at the F87F2Y site with the binding efficacy of the SRC SH3 domain at the P1 site of β -arrestin 1 ( Supplementary Fig. 13b). j, Correlation of the receptor phospho-C-tail-induced chemical shifts at the L123F2Y site with the binding efficacy of SRC SH3 domain at the P2 site of β -arrestin 1 ( Supplementary Fig. 13c). In i and j the error bar along the y axis for each point indicates the deviation of the pull-down assay results derived from three independent experiments (n = 3). ...
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... Fig. 9b-d). Additionally, the chem- ical shifts at F87F2Y and L123F2Y observed in response to the bind- ing of different receptor phospho-C tails correlated with the efficacy of the interaction between the SRC SH3 domain and specific β -arrestin 1 PR mutants, as determined by GST pull-down assays (R 2 = 0.9 and R 2 = 0.9, respectively; Fig. 1i,j and Supplementary Fig. 13a-c). This correlated linear trend indicated that arrestin receives and converts information from specific receptor-phosphate . b, Effects of the mutation of key residue contacts (V40E and P114E) on the 19 F-NMR spectra of β -arrestin 1 F87F2Y before and after GRK6pp-β 2AR-CT (GRK6pp) binding. c, Effect of ...
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... P1 and P2 (Supplementary Fig. 9b-d). Additionally, the chem- ical shifts at F87F2Y and L123F2Y observed in response to the bind- ing of different receptor phospho-C tails correlated with the efficacy of the interaction between the SRC SH3 domain and specific β -arrestin 1 PR mutants, as determined by GST pull-down assays (R 2 = 0.9 and R 2 = 0.9, respectively; Fig. 1i,j and Supplementary Fig. 13a-c). This correlated linear trend indicated that arrestin receives and converts information from specific receptor-phosphate . ...
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... receptor-phospho- binding sites and functionally relevant arrestin conformational states (specifically, phospho-binding site 5 coupling to P1 and P2 and phospho-binding sites 2-3 to the P3 region of β -arrestin 1). The magnitude of the 19 F-NMR chemical shift reflected the intrinsic effi- cacy of β -arrestin 1 for recruiting downstream SH3-CPs ( Fig. ...
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... binding site S5 connects to the P1 region of arrestin via three hydro- phobic core regions (Fig. 2a). In response to V2Rpp binding, only a slight reorganization of β -arrestin 1 residue contacts has been observed using crystallography 26 , including the relaxation of core 1 and core 3, as well as the assembly of core 2 ( Supplementary Fig. 14a). These small changes observed in crystal structures are attributed to the lowest energy structural states, which are often captured by crystallography. ...
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... mutated the connecting knobs V40 or P114 to the polar residue E, which maintains its ability to associate with the active receptor ( Supplementary Fig. 14b,c). The structural integrity of these mutants was further verified by circular dichroism (CD) spectroscopy ( Supplementary Fig. 14d). ...
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... mutated the connecting knobs V40 or P114 to the polar residue E, which maintains its ability to associate with the active receptor ( Supplementary Fig. 14b,c). The structural integrity of these mutants was further verified by circular dichroism (CD) spectroscopy ( Supplementary Fig. 14d). Although GRK6pp-β 2AR- CT stimulation caused a significant downfield shift (0.79 p.p.m.) at the F87F2Y position, the V40E and P114E mutations significantly decreased this chemical shift by 0.60 and 0.66 p.p.m., respectively. ...
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... conformational changes that occurred at the β -arrestin 1 P1 region (Fig. 2b). Moreover, V40E and P114E specifically disrupted the recruitment of the SRC SH3 domain to β -arrestin 1 in response to GRK6pp-β 2AR-CT but had no significant effect on the interaction with clathrin after V2Rpp binding via GST-pull-down assay (Supplementary Fig. 15a-d). In contrast, mutating these knobs to hydrophobic residues (for example, V40W and P40W) or mutating other residues proximal to the connecting region (for example, L108 and Y113) had no sig- nificant effect on the β -arrestin-1-SRC SH3 domain association (Supplementary Fig. ...
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... clathrin after V2Rpp binding via GST-pull-down assay (Supplementary Fig. 15a-d). In contrast, mutating these knobs to hydrophobic residues (for example, V40W and P40W) or mutating other residues proximal to the connecting region (for example, L108 and Y113) had no sig- nificant effect on the β -arrestin-1-SRC SH3 domain association (Supplementary Fig. ...
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... path connecting to the P2 region. Diverging by approximately 50 degrees compared to the P1 region, the P2 region is connected to receptor-phospho-site-S5 binding site through the β 3 sheet and loop 3 of β -arrestin 1 (Figs. 1k and 2e). Hydrophobic interactions dominate the connection between phospho-site 5 and the P2 region, which also contains three hydrophobic cores ( Supplementary Fig. 16e). Disruption of these hydrophobic cores with the V34R or P36E mutation significantly decreased the downfield shifts at the P2 probe L123F2Y position and decreased SRC ...
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... and 2e). Hydrophobic interactions dominate the connection between phospho-site 5 and the P2 region, which also contains three hydrophobic cores ( Supplementary Fig. 16e). Disruption of these hydrophobic cores with the V34R or P36E mutation significantly decreased the downfield shifts at the P2 probe L123F2Y position and decreased SRC recruitment in response to GRK6pp-β 2AR-CT engagement ( Fig. 2f and Supplementary Fig. 16a-d). ...
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... interactions dominate the connection between phospho-site 5 and the P2 region, which also contains three hydrophobic cores ( Supplementary Fig. 16e). Disruption of these hydrophobic cores with the V34R or P36E mutation significantly decreased the downfield shifts at the P2 probe L123F2Y position and decreased SRC recruitment in response to GRK6pp-β 2AR-CT engagement ( Fig. 2f and Supplementary Fig. 16a-d). In contrast, these muta- tions had no significant effect on V2Rpp-induced clathrin bind- ing to arrestin, their recruitment to the active β 2AR or the CD spectrum, suggesting their structural integrity (Supplementary Figs. ...
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... com- plexes consisting of F2Y-incorporated β -arrestin 1, SRC and the receptor phospho-tail (in a 1:1:3 molar ratio) were reconstituted in vitro and subjected to 19 F-NMR analysis. Binding of the SRC induced significant upfield shifts at F87F2Y and L123F2Y but not at R177F2Y (Supplementary Fig. 17). We examined the effect of the Statistical differences were determined by one-way ANOVA. ...
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... CHeMICal BIoloGy paramagnetic relaxation agent chromium acetylacetone (Cr(iii)) to further characterize the environment of F87F2Y and L123F2Y. The signal-to-noise (S/N) ratios for 19 F-NMR spectra at different F2Y incorporation sites were determined ( Supplementary Figs. 18-21). Specifically, although the 19 F-NMR spectra revealed that all three F2Y sensors located in different PRs were more susceptible to line broadening by Cr(iii) after incubation with GRK6pp-β 2AR-CT, only two specific sites in P1 and P2, including F87F2Y and L123F2Y in the ternary GRK6pp-β 2AR-CT-β -arrestin-1-SRC complex, were less ...
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... the 19 F-NMR spectra revealed that all three F2Y sensors located in different PRs were more susceptible to line broadening by Cr(iii) after incubation with GRK6pp-β 2AR-CT, only two specific sites in P1 and P2, including F87F2Y and L123F2Y in the ternary GRK6pp-β 2AR-CT-β -arrestin-1-SRC complex, were less accessible to solvent (Fig. 3a-c and Supplementary Figs. 18-20). In contrast, although the N375F2Y site, a hallmark 19 F-NMR probe that reports on the conformational change at the arrestin C-tail, was dislodged in response to receptor interactions and was sensi- tive to line broadening by Cr after clathrin association 11 , significant solvent-accessible protection at this site was not observed ...
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... In contrast, although the N375F2Y site, a hallmark 19 F-NMR probe that reports on the conformational change at the arrestin C-tail, was dislodged in response to receptor interactions and was sensi- tive to line broadening by Cr after clathrin association 11 , significant solvent-accessible protection at this site was not observed after the formation of the ternary GRK6pp-β 2AR-CT-β -arrestin-1-SRC complex ( Fig. 3d and Supplementary Fig. 21). Thus, SRC recog- nized specific PR conformational states in β -arrestin 1 but not dis- lodgement of the C-tail. ...
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... and kinase domains form a highly organized, inactive structure 40,41 . The tethering of these ordered, inactive structures occurs via interac- tions between the SH3 domain and the linker regions between the SH2 and the kinase domains. Both the P1 and P2 regions of active arrestin are protected from solvents after SRC association (Fig. 3a,b and Supplementary Figs. 18 and 19). Therefore, the PRs of active β -arrestin 1 might interact with the SRC SH3 domain, inhibiting its interaction with the SH2-kinase linker, thereby resulting in SRC activation via the disassembly of its regulatory ...
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... the fluorescence donor at the N terminus of SRC and a fluorescein arsenical acceptor specifically located at the intra- domain of the SRC proteins. β 2AR activation by ISO increased the intermolecular BRET between the β -arrestin-1-YFP and rLuc wild-type SRC and between different N-rLuc-SRC-FlAsH biosen- sors, consistent with the co-IP results ( Fig. 1a and Supplementary Fig. 27b). Intriguingly, β 2AR activation led to decreased intramo- lecular BRET in rLuc-SRC-β 5-FlAsH-α D but had no significant effect on rLuc-SRC-β B-FlAsH-α A (Fig. 5d,e), suggesting that the kinase domain shifted away from the N-terminal SH3 domain of SRC. Notably, these conformational changes were abolished by ...
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... in the distinct, functionally related con- formational states of arrestin directed by phosphorylated GPCR barcodes. Allosteric coupling between selective receptor-phospho- site binding and the specific conformational states of three PRs located within β -arrestin 1 determine the selectivity and intrinsic efficacy of downstream SH3-CP recruitment (Figs. 1b,i-k and 6). Notably, recent works have identified two functionally important conformational modes of the receptor-arrestin complex, namely, the snuggly and hanging conformations 10,15,28 . Although the use of the synthesized receptor-phospho-C-tail in this study primarily reflected the hanging conformation of the receptor-arrestin com- plex, the ...
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... western blot quantification analysis has been successfully used to assess the DR5:DISC stoichiometry, to measure the SV proteins and lipids, and to determine the enzyme catalytic activity of MKP3 and STEP 58-60 . To determine the concentration of the β -arrestin 1 or its mutant in the GST-SRC-SH3 containing complex, a calibration curve was established first (Supplementary Fig. 13a) and quantitative analysis of western blots were then performed 11,58 . Protein samples alongside a serial concentration of purified recombinant His-β -arrestin 1 proteins within the linear range were subjected to SDS-PAGE in precast 12% polyacrylamide gels (Bio-Rad), which was followed by transferring and blocking. ...
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... This is important because βarrs are multifunctional proteins. Their binding sites with diverse downstream signaling proteins are often located in the disordered CT or various loop regions [40][41][42][43] . Among the different conformational states of βarr1, these regions may exhibit subtle differences that affect the binding site accessibility or affinity. ...
Arrestins (βarrs) are functionally versatile proteins that play critical roles in the G-protein-coupled receptor (GPCR) signaling pathways. While it is well established that the phosphorylated receptor tail plays a central role in βarr activation, emerging evidence highlights the contribution from membrane lipids. However, detailed molecular mechanisms of βarr activation by different binding partners remain elusive. In this work, we present a comprehensive study of the structural changes in critical regions of βarr1 during activation using ¹⁹F NMR spectroscopy. We show that phosphopeptides derived from different classes of GPCRs display different βarr1 activation abilities, whereas binding of the membrane phosphoinositide PIP2 stabilizes a distinct partially activated conformational state. Our results further unveil a sparsely-populated activation intermediate as well as complex cross-talks between different binding partners, implying a highly multifaceted conformational energy landscape of βarr1 that can be intricately modulated during signaling.
... For example, AVP acts on physiological salt and water balance Ocampo Daza et al., 2012;Wacker and Ludwig, 2019), and this function is primarily dependent on its binding to the vasopressin receptor AVPR2 (or VTR2C; Theofanopoulou et al., 2021), a Class-A G protein-coupled receptor (GPCR). Extracellular signals from GPCRs promote structural rearrangements in their cytoplasmic regions, which are then recognized by transducer G proteins and regulate intracellular messengers (Yang et al., 2018). ...
The current study focuses on the investigation of AVPR2 (VTR2C) protein-coupled receptor variants specific to different primate taxa. AVPR2 is activated by the neurohormone AVP, which modulates physiological processes, including water homeostasis. Our findings reveal positive selection at three AVPR2 sites at positions 190, 250, and 346. Variation at position 250 is associated with human Congenital Nephrogenic Diabetes Insipidus (cNDI), a condition characterized by excessive water loss. Other 13 functional sites with potential adaptive relevance include positions 185, 202, 204, and 252 associated with cNDI. We identified SH3-binding motifs in AVPR2’s ICL3 and N-terminus domains, with some losses observed in clades of Cercopithecidae, Callitrichinae, and Atelidae. SH3-binding motifs are crucial in regulating cellular physiology, indicating that the differences may be adaptive. Co-evolution was found between AVPR2 residues and those in the AVP signal peptide/Neurophysin-2 and AQP2, other molecules in the same signaling cascade. No significant correlation was found between these Primates’ taxon-specific variants and the bioclimatic variables of the areas where they live. Distinct co-evolving amino acid sequences in functional sites were found in Platyrrhini and Catarrhini, which may have adaptive implications involving glucocorticoid hormones, suggesting varied selective pressures. Further studies are required to confirm these results.
... C5a interacts with PI3K to regulate neutrophil aggregation by down-regulating the expression of CXCR4 in neutrophils and inhibiting the homing effect of SDF-1 (CXCL12). phosphoric acid sites 5 on GPCRs were coupled to P1 and P2, phosphoric acid sites 2-3 were coupled to P3, and phosphoric acid binding sites 5 induced conformational changes in regions P1 and P2 through the propagation paths of V40!P114!F87 and P36!V34!F123, respectively (221). The different GPCRs phosphorylation sites regulated by different GRKs lead to different b-arrestin conformations and thus different signal transduction functions, which is known as the "phosphorylation barcode hypothesis" (229-231). ...
... b-arrestin recruited by phosphorylated GPCRs through Clathrin-mediated endocytosis is the main pathway for the internalization of GPCRs, which is important for both receptor recycling and intracellular signal transduction (244). Src are a kind of kinases that contain the SH3 domain and recognize the specific PRs conformation in b-arrestin without recognizing the C-terminal shift of GPCRs (221). The binding of b-arrestin to GPCR can also induce clathrin and adaptor protein 2 (AP-2) aggregation by increasing c-Src kinase activity (245)(246)(247). ...
Sepsis is defined as a life-threatening dysfunction due to a dysregulated host response to infection. It is a common and complex syndrome and is the leading cause of death in intensive care units. The lungs are most vulnerable to the challenge of sepsis, and the incidence of respiratory dysfunction has been reported to be up to 70%, in which neutrophils play a major role. Neutrophils are the first line of defense against infection, and they are regarded as the most responsive cells in sepsis. Normally, neutrophils recognize chemokines including the bacterial product N-formyl-methionyl-leucyl-phenylalanine (fMLP), complement 5a (C5a), and lipid molecules Leukotriene B4 (LTB4) and C-X-C motif chemokine ligand 8 (CXCL8), and enter the site of infection through mobilization, rolling, adhesion, migration, and chemotaxis. However, numerous studies have confirmed that despite the high levels of chemokines in septic patients and mice at the site of infection, the neutrophils cannot migrate to the proper target location, but instead they accumulate in the lungs, releasing histones, DNA, and proteases that mediate tissue damage and induce acute respiratory distress syndrome (ARDS). This is closely related to impaired neutrophil migration in sepsis, but the mechanism involved is still unclear. Many studies have shown that chemokine receptor dysregulation is an important cause of impaired neutrophil migration, and the vast majority of these chemokine receptors belong to the G protein-coupled receptors (GPCRs). In this review, we summarize the signaling pathways by which neutrophil GPCR regulates chemotaxis and the mechanisms by which abnormal GPCR function in sepsis leads to impaired neutrophil chemotaxis, which can further cause ARDS. Several potential targets for intervention are proposed to improve neutrophil chemotaxis, and we hope that this review may provide insights for clinical practitioners.
... Typically, activation of the SRC is seen in GPCR signaling via arrestin. Compared with the administration of β-arrestin1 alone, β-arrestin1 binding to phosphorylated β 2 -AR significantly increased the phosphorylation of SRC [9]. Biased activation of AT1R-β-arrestin1 promotes acute catecholamine secretion by recruiting transient receptor potential cation channel subfamily C 3 [88]. ...
G protein-coupled receptors (GPCRs) are the largest family of membrane proteins in the human body and are responsible for accurately transmitting extracellular information to cells. Arrestin is an important member of the GPCR signaling pathway. The main function of arrestin is to assist receptor desensitization, endocytosis and signal transduction. In these processes, the recognition and binding of arrestin to phosphorylated GPCRs is fundamental. However, the mechanism by which arrestin recognizes phosphorylated GPCRs is not fully understood. The GPCR phosphorylation recognition “bar code model” and “flute” model describe the basic process of receptor phosphorylation recognition in terms of receptor phosphorylation sites, arrestin structural changes and downstream signaling. These two models suggest that GPCR phosphorylation recognition is a process involving multiple factors. This process can be described by a “QR code” model in which ligands, GPCRs, G protein-coupled receptor kinase, arrestin, and phosphorylation sites work together to determine the biological functions of phosphorylated receptors.
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... It is now clear that β-arrestins are more flexible regulators, and that they have the capacity to promote as well as attenuate signaling from the plasma membrane and endomembranes (Calebiro et al., 2009;DeFea et al., 2000;Feinstein et al., 2013;Ferrandon et al., 2009;Irannejad et al., 2013;McDonald et al., 2000;Terrillon and Bouvier, 2004). Furthermore, it is now also widely recognized that β-arrestins can have different effects on downstream signaling depending on GPCR-specific differences in the composition, stability, and/or conformation of the complex that they form with GPCRs (Asher et al., 2022;Latorraca et al., 2020;Lee et al., 2016;Mayer et al., 2019;Nobles et al., 2011;Nuber et al., 2016;Yang et al., 2018;Yang et al., 2015). In light of the high level of diversity and flexibility that is presently recognized to exist at the GPCR/β-arrestin interface, we wondered if release of the β-arrestin CT is sufficient to explain the diversity of downstream effects conferred by GPCR transit through CCPs. ...
β-arrestins are master regulators of cellular signaling that operate by desensitizing ligand-activated G protein-coupled receptors (GPCRs) at the plasma membrane and promoting their subsequent endocytosis. The endocytic activity of β-arrestins is ligand-dependent, triggered by GPCR binding, and increasingly recognized to have a multitude of downstream signaling and trafficking consequences that are specifically programmed by the bound GPCR. However, only one biochemical 'mode' for GPCR-mediated triggering of the endocytic activity is presently known- displacement of the β-arrestin C-terminus (CT) to expose CCP-binding determinants that are masked in the inactive state. Here we revise this view by uncovering a second mode of GPCR-triggered endocytic activity that is independent of the β-arrestin CT and, instead, requires the cytosolic base of the β-arrestin C-lobe (CLB). We further show each of the discrete endocytic modes is triggered in a receptor-specific manner, with GPCRs that bind β-arrestin transiently ('class A') primarily triggering the CLB-dependent mode and GPCRs that bind more stably ('class B') triggering both the CT and CLB -dependent modes in combination. Moreover, we show that different modes have opposing effects on the net signaling output of receptors- with the CLB-dependent mode promoting rapid signal desensitization and the CT-dependent mode enabling prolonged signaling. Together, these results fundamentally revise understanding of how β-arrestins operate as efficient endocytic adaptors while facilitating diversity and flexibility in the control of cell signaling.
... These receptors detect molecules, such as hormones, neurotransmitters, chemokines, or odorants [13]. The binding of these ligands induces conformational changes in the transmembrane and intracellular domains of the receptor, allowing its interaction with heterotrimeric G proteins or with arrestins [14,15]. After activation, GPCRs act as guanine nucleotide exchange factors (GEFs) for the α subunits of heterotrimeric G proteins, catalyzing the release of GDP and the binding of GTP for G-protein activation. ...
G protein-coupled receptors (GPCRs) are involved in several physiological processes, and they represent the largest family of drug targets to date. However, the presence and function of these receptors are poorly described in human spermatozoa. Here, we aimed to identify and characterize the GPCRs present in human spermatozoa and perform an in silico analysis to understand their potential role in sperm functions. The human sperm proteome, including proteomic studies in which the criteria used for protein identification was set as <5% FDR and a minimum of 2 peptides match per protein, was crossed with the list of GPCRs retrieved from GLASS and GPCRdb databases. A total of 71 GPCRs were identified in human spermatozoa, of which 7 had selective expression in male tissues (epididymis, seminal vesicles, and testis), and 9 were associated with male infertility defects in mice. Additionally, ADRA2A, AGTR1, AGTR2, FZD3, and GLP1R were already associated with sperm-specific functions such as sperm capacitation, acrosome reaction, and motility, representing potential targets to modulate and improve sperm function. Finally, the protein-protein interaction network for the human sperm GPCRs revealed that 24 GPCRs interact with 49 proteins involved in crucial processes for sperm formation, maturation, and fertilization. This approach allowed the identification of 8 relevant GPCRs (ADGRE5, ADGRL2, GLP1R, AGTR2, CELSR2, FZD3, CELSR3, and GABBR1) present in human spermatozoa that can be the subject of further investigation to be used even as potential modulatory targets to treat male infertility or to develop new non-hormonal male contraceptives.
... Induced BRET change (ratio 540nm/480nm) many cases, β-arrestins function as a scaffold for c-SRC-mediated activation of MAPKs (27)(28)(29). Alternatively, c-SRC may be directly activated by binding to GPCR in the absence of β-arrestin (30). ...
... Previous studies have shown that proline-rich motifs (PXXP) in the third intracellular loop and the carboxyl terminus of GPCRs are involved in the recruitment of SH3-domain containing proteins (SH3-CPs), like c-SRC (27,29). Interestingly, we identified such a proline-rich motif in the carboxyl terminus of the 5-HT 7 R and aimed at dissecting its putative role in c-SRC and ERK activation. ...
... It is known that β-arrestins may function as a scaffold for c-SRC-mediated activation of ERK, as previously demonstrated (29). Alternatively, some GPCRs can interact directly with the tyrosine kinase c-SRC in the absence of β-arrestin (30). ...
Significance
Transmembrane signaling through G protein–coupled receptors (GPCRs), originally described as requiring coupling to intracellular G proteins, also uses G protein–independent pathways through β-arrestin recruitment. Biased ligands, by favoring one of the multiple bioactive conformations of GPCRs, allow selective signaling through either of these pathways. Here, we identified Serodolin as the first β-arrestin–biased agonist of the serotonin 5-HT 7 receptor. This new ligand, while acting as an inverse agonist on G s signaling, selectively induces ERK activation in a β-arrestin–dependent way. Importantly, we report that Serodolin decreases pain intensity caused by thermal, mechanical, or inflammatory stimuli. Our findings suggest that targeting the 5-HT 7 R with β-arrestin–biased ligand could be a valid alternative strategy to the use of opioids for the relief of pain.
... Gi-Dependent SRC Activation Contributes to Progesterone/ GPR126-Mediated Cellular Function. Increasing evidence has highlighted the importance of SRC in tumorigenesis and in Gi-coupled receptor signaling (60)(61)(62)(63)(64)(65)(66). We therefore sought to determine the involvement of SRC signaling in progesteronestimulated cell proliferation through GPR126-Gi activation in BC cells. ...
... GPR126, may have distinct biased signaling properties that constitute the fine-tuning signaling network for these receptors with diverse functions. Recently, different biased properties of the G protein subtype or arrestins of GPCR ligands have been suggested to have great therapeutic potential, such as that for angiotensin type I receptors, opioid receptors, and adrenergic receptors (66,(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(86)(87)(88)(89)(90). Therefore, understanding the preferences of GPR126 may facilitate future therapeutic development targeting this particular receptor. ...
Significance
The steroid hormone progesterone is highly involved in different physiological–pathophysiological processes, including bone formation and cancer progression. Understanding the working mechanisms, especially identifying the receptors of progesterone hormones, is of great value. In the present study, we identified GPR126 as a membrane receptor for both progesterone and 17-hydroxyprogesterone and triggered its downstream G protein signaling. We further characterized the residues of GPR126 that interact with these two ligands and found that progesterone promoted the progression of a triple-negative breast cancer model through GPR126-dependent Gi-SRC signaling. Therefore, developing antagonists targeting GPR126-Gi may provide an alternative therapeutic option for patients with triple-negative breast cancer.
... PTH1R signals primarily through coupling to Gs and Gq proteins, which activate the cAMP-PKA and DAG/Ca 2+ -PKC signaling pathways, respectively (21). The activated PTH1R also recruits β-arrestins, which not only regulate receptor desensitization and internalization but also redirect the signaling to a G protein-independent pathway by interacting with multiple downstream effector molecules (38)(39)(40)(41)(42)(43). Our results from both in vitro and in vivo studies demonstrated a predominant role of Gq-and β-arrestin-1-mediated signaling in the anti-inflammatory effects of ABL through PTH1R in epididymitis and orchitis. ...
Significance
Currently, male infertility has become a public health issue. The inflammations in the epididymis and testis are estimated to account for 6 to 15% of male infertility and thus have attracted increasing attention. Conventional therapies for epididymitis and orchitis using antibiotics have limitations in terms of side effects and ineffectiveness for nonbacterial inflammations, thus highlighting an urgent need for novel therapeutic strategies. Here, the screening of secretin family G protein–coupled receptors (GPCRs) enabled us to identify that activation of PTH1R by a Food and Drug Administration–approved osteoporosis drug abaloparatide effectively counteracts epididymitis and orchitis through selective transducer-mediated pathways. Our results offer potential therapeutics to treat epididymitis and orchitis by targeting GPCRs other than traditional antibiotic therapies.
... βarrs have been demonstrated to act like typical scaffold proteins that bring together the components of MAPK cascade to facilitate signal transduction (13,14). Surprisingly, recent studies showed that in addition to scaffolding functions, GPCR-βarr complexes may serve as allosteric modulators for downstream signaling partners such as kinase Src (22,23). However, it is unclear whether βarrs can also allosterically activate C-Raf. ...
... Here we show that βarr1, activated via either phosphorylated GPCR or the GPCR surrogate V2Rpp (a vasopressin receptor 2 phosphorylated C-terminal peptide with eight phosphates), binds C-Raf and allosterically activates it. Therefore, GPCR-βarr complexes function not just as typical scaffold proteins for the C-GPCR-βarr1 complexes allosterically activate C-Raf by interacting with its amino-terminus GPCR-βarr complexes have been demonstrated to allosterically activate signaling partners such as the tyrosine kinase Src (22,23). To investigate whether GPCR-βarr complexes allosterically regulate C-Raf activation, we used an enzyme-coupled fluorescence assay to measure the C-Raf activity in real time ( Fig. 2A). ...
... Previously, βarrs have been considered adaptors and scaffold proteins that link the receptors to different signaling partners and bring various signaling components into close contact to facilitate the signal transduction process (15). However, recent studies have shown that βarr1 can play a more active role in signaling by allosterically activating its binding partner, Src kinase (22,23). However, for the GPCR-βarrdependent MAPK signaling pathway, the mechanistic details of how C-Raf is regulated to initiate the signaling cascade remained unclear. ...
G protein-coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric G-proteins and β-arrestins (βarrs). In a βarr-dependent signaling pathway, βarrs link GPCRs to various downstream signaling partners, such as the Raf–MEK–ERK mitogen-activated protein kinase (MAPK) cascade. Agonist-stimulated GPCR–βarr complexes have been shown to interact with C-Raf and are thought to initiate the MAPK pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, βarrs can allosterically activate downstream targets, such as the non-receptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR–βarr1 complexes in vitro. Furthermore, we show that βarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates βarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C-terminus and βarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between βarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and βarr1 bind to the same region on C-Raf. Furthermore, we found that βarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR–βarr complexes directly allosterically activate C-Raf by binding to its amino-terminus. This work provides novel insights into how βarrs regulate effector molecules to activate downstream signaling pathways.
