The family of G protein‐coupled receptors (GPCRs) constitutes the largest class of signalling receptors in the human genome, controlling vast physiological responses and are the target of many drugs. After activation, GPCRs are rapidly desensitized by phosphorylation and β‐arrestin binding. Most classic GPCRs are internalized through a clathrin, dynamin and β‐arrestin‐dependent pathway and then recycled back to the cell surface or sorted to lysosomes for degradation. Given the vast number and diversity of GPCRs, different mechanisms are likely to exist to precisely regulate the magnitude, duration and spatial aspects of receptor signalling. The G protein‐coupled protease‐activated receptors (PARs) provide elegant examples of GPCRs that are regulated by distinct desensitization and endocytic sorting mechanisms, processes that are critically important for the spatial and temporal fidelity of PAR signalling. PARs are irreversibly activated through proteolytic cleavage and transmit cellular responses to extracellular proteases. Activated PAR₁ internalizes through a clathrin‐ and dynamin‐dependent pathway independent of β‐arrestins. Interestingly, PAR₁ is basally ubiquitinated and deubiquitinated after activation and traffics from endosomes to lysosomes independent of ubiquitination. In contrast, β‐arrestins mediate activated PAR₂ internalization and function as scaffolds that promote signalling from endocytic vesicles. Moreover, activated PAR₂ is modified with ubiquitin, which facilitates lysosomal degradation. Activated PARs also adopt distinct active conformations that signal to diverse effectors and are likely regulated by different mechanisms. Thus, the identification of the molecular machinery important for PAR signal regulation will enable the development of new strategies to manipulate receptor signalling and will provide novel targets for the development of drugs.

G protein–coupled receptors (GPCRs) are a large family comprising >800 signaling receptors that regulate numerous cellular and physiologic responses. GPCRs have been implicated in numerous diseases and represent the largest class of drug targets. Although advances in GPCR structure and pharmacology have improved drug discovery, the regulation of GPCR function by diverse post-translational modifications (PTMs) has received minimal attention. Over 200 PTMs are known to exist in mammalian cells, yet only a few have been reported for GPCRs. Early studies revealed phosphorylation as a major regulator of GPCR signaling, whereas later reports implicated a function for ubiquitination, glycosylation, and palmitoylation in GPCR biology. Although our knowledge of GPCR phosphorylation is extensive, our knowledge of the modifying enzymes, regulation, and function of other GPCR PTMs is limited. In this review we provide a comprehensive overview of GPCR post-translational modifications with a greater focus on new discoveries. We discuss the subcellular location and regulatory mechanisms that control post-translational modifications of GPCRs. The functional implications of newly discovered GPCR PTMs on receptor folding, biosynthesis, endocytic trafficking, dimerization, compartmentalized signaling, and biased signaling are also provided. Methods to detect and study GPCR PTMs as well as PTM crosstalk are further highlighted. Finally, we conclude with a discussion of the implications of GPCR PTMs in human disease and their importance for drug discovery. Significance Statement Post-translational modification of G protein–coupled receptors (GPCRs) controls all aspects of receptor function; however, the detection and study of diverse types of GPCR modifications are limited. A thorough understanding of the role and mechanisms by which diverse post-translational modifications regulate GPCR signaling and trafficking is essential for understanding dysregulated mechanisms in disease and for improving and refining drug development for GPCRs.

The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)–0, –I, –II, and –III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor–regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III–dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4–ESCRT-III interacting protein, bound to a YPX₃L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPX_nL motifs.

Protease-activated receptor-1 (PAR1) is a guanine nucleotide-binding (G) protein-coupled receptor that elicits cellular responses to coagulant and anticoagulant proteases. Activation of PAR1 by the coagulant protease thrombin results in Ras homolog gene family member A (RhoA) activation, disassembly of adherens junctions, and disruption of the endothelial barrier. In contrast, activation of PAR1 with the anticoagulant protease activated protein C (APC) results in activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and endothelial barrier protection. We previously showed that APC cytoprotective signaling requires the compartmentalization of PAR1 in caveolar microdomains. However, the mechanism by which APC-activated PAR1 promotes cytoprotective signaling in human endothelial cells remains poorly understood. Here we show that APC-activated PAR1 cytoprotective signaling is mediated by β-arrestin recruitment and activation of the dishevelled-2 (Dvl-2) scaffold and not by G protein α inhibiting activity polypeptide 2 (Gα _i ) signaling. In human endothelial cells, PAR1 and β-arrestins form a preassembled complex and cosegregate in caveolin-1–enriched fractions. Remarkably, we found that depletion of β-arrestin expression by RNA interference resulted in the loss of APC-induced Rac1 activation but not of thrombin-stimulated RhoA signaling. APC also failed to protect against thrombin-induced endothelial barrier permeability in cells deficient in β-arrestin expression. We further demonstrate that APC activation of PAR1 results in β-arrestin–dependent recruitment of Dvl-2, which is critical for Rac1 signaling and endothelial barrier protection but not for thrombin-induced RhoA signaling. Our findings identify a role for β-arrestin and Dvl-2 scaffolds in APC-activated PAR1 cytoprotective signaling in human endothelial cells.

Coagulation is a host defense system that limits the spread of pathogens. Coagulation proteases, such as thrombin, also activate cells by cleaving PARs. In this study, we analyzed the role of PAR-1 in coxsackievirus B3–induced (CVB3-induced) myocarditis and influenza A infection. CVB3-infected Par1^–/– mice expressed reduced levels of IFN-β and CXCL10 during the early phase of infection compared with Par1^+/+ mice that resulted in higher viral loads and cardiac injury at day 8 after infection. Inhibition of either tissue factor or thrombin in WT mice also significantly increased CVB3 levels in the heart and cardiac injury compared with controls. BM transplantation experiments demonstrated that PAR-1 in nonhematopoietic cells protected mice from CVB3 infection. Transgenic mice overexpressing PAR-1 in cardiomyocytes had reduced CVB3-induced myocarditis. We found that cooperative signaling between PAR-1 and TLR3 in mouse cardiac fibroblasts enhanced activation of p38 and induction of IFN-β and CXCL10 expression. Par1^–/– mice also had decreased CXCL10 expression and increased viral levels in the lung after influenza A infection compared with Par1^+/+ mice. Our results indicate that the tissue factor/thrombin/PAR-1 pathway enhances IFN-β expression and contributes to the innate immune response during single-stranded RNA viral infection.

Protease-activated receptor-1 (PAR ₁ ) is a G-protein–coupled receptor uniquely activated by proteolysis. Thrombin, a coagulant protease, induces inflammatory responses and endothelial barrier permeability through the activation of PAR ₁ . Activated protein C (APC), an anti-coagulant protease, also activates PAR ₁ . However, unlike thrombin, APC elicits anti-inflammatory responses and protects against endothelial barrier dysfunction induced by thrombin. We found that thrombin and APC signaling were lost in PAR ₁ -deficient endothelial cells, indicating that PAR ₁ is the major effector of protease signaling. To delineate the mechanism responsible for protease-selective signaling by PAR ₁ , we examined the effect of APC and thrombin on the activation of RhoA and Rac1, small GTPases that differentially regulate endothelial barrier permeability. Thrombin caused robust RhoA signaling but not Rac1 activation, whereas APC stimulated a marked increase in Rac1 activation but not RhoA signaling, consistent with the opposing functions of these proteases on endothelial barrier integrity. Strikingly, APC signaling and endothelial barrier protection effects were abolished in cells lacking caveolin-1, whereas thrombin signaling remained intact. These findings suggest that compartmentalization of PAR ₁ in caveolae is critical for APC selective signaling to Rac1 activation and endothelial barrier protection. We further report that APC induces PAR ₁ phosphorylation and desensitizes endothelial cells to thrombin signaling but promotes limited receptor cleavage and negligible internalization and degradation even after prolonged APC exposure. Thus, APC selective signaling and endothelial barrier protective effects are mediated through compartmentalization of PAR ₁ in caveolae and a novel mechanism of PAR ₁ signal regulation.