Ubiquitin-driven G protein-coupled receptor inflammatory signaling at the endosome

Abstract

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-β-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes.

INTRODUCTION

G protein-coupled receptors (GPCRs) contain seven transmembrane α-helical domains, which are ubiquitously expressed across various cell types and control vast physiological tissue and organ responses. Given the widespread distribution and function of GPCRs, not unexpectedly, dysregulation of GPCR signaling has been implicated in disease, making this receptor family the largest target class for drugs approved by the US Food and Drug Administration (1, 2). GPCRs are generally diffusively distributed on the cell surface and, upon activation, rapidly engage heterotrimeric G proteins to elicit cellular responses. After activation, GPCRs are phosphorylated by GPCR kinases (GRKs) and directly bind β-arrestin proteins at the plasma membrane. β-Arrestins are multifaceted adaptor proteins that mediate GPCR desensitization by uncoupling the receptor from heterotrimeric G proteins, promote internalization through clathrin-coated pits, and function as scaffolds to facilitate kinase signaling (3–5). Internalized GPCRs are then either recycled back to the cell surface in a competent state to signal again or sorted to lysosomes and degraded, terminating the GPCR life cycle (6–8). This classical view has led to the assumption that GPCR signaling is mainly transduced at the plasma membrane (Fig. 1) and serves as the primary target for modulation by drugs.

Figure 1.

GPCR signaling at the plasma membrane and endosomes. In the classic view, GPCRs couple predominantly to heterotrimeric G proteins at the plasma membrane to elicit cellular responses. After activation, GPCRs are phosphorylated and ubiquitinated, internalized to endosomes, and either recycled back to the cell surface or sorted to multivesicular bodies (MVBs) and degraded in lysosomes. GPCRs also localize in intracellular compartments including endosomes where they are competent to signal. GPCR-G protein signaling at endosomes has been shown for some GPCRs that retain β-arrestin binding in a manner that does not preclude endosomal G protein signaling. Other internalized GPCRs form a stable complex with β-arrestin, inducing activation of various kinases at endosomes. New emerging studies indicate that ubiquitination of a subset of endothelial GPCRs promotes assembly and activation of a p38 endosomal signaling complex mediated by TAB2 and TAB1 that promotes endothelial inflammatory responses. Figure created with BioRender.com. GPCR, G protein-coupled receptor; TAB1/2, transforming growth factor β-activated kinase-binding protein 1/2.

COMPARTMENTALIZED SIGNALING

New insights into GPCR subcellular localization in functionally distinct membrane-bound compartments have recently emerged and shown to augment cellular signaling and physiological responses (9, 10). Indeed, GPCRs are not entirely static but rather move dynamically within and between compartments. At the plasma membrane, GPCRs localize to specialized microdomains such as caveolae, lipid rafts, and clathrin-coated pits that can modulate signal transduction. In addition, GPCRs have been shown to localize to intracellular compartments beyond the plasma membrane including the endoplasmic reticulum, Golgi, mitochondria, nuclear membrane, endosomes, and lysosomes. Intracellular GPCR signaling is best studied in endosomes where heterotrimeric G proteins and β-arrestins reside and function. GPCR-G protein endosomal signaling has been reported for several well-studied receptors including the β2-adrenergic receptor and parathyroid hormone receptor that couple to Gαs proteins and the Gαi-linked δ- and μ-opioid receptors (11–14). The use of genetically encoded nanobodies and conformational biosensors in living cells has elegantly demonstrated GPCR-G protein signaling at endosomes (Fig. 1) (11, 14, 15). Nanobodies are compact single heavy chain proteins with full antigen binding capacity that recognize active states of GPCRs and activated GPCR-Gα subunit signaling complexes (16). The expression of genetically encoded nanobodies fused to green fluorescent protein (GFP) in living cells has enabled detailed visualization of activated GPCRs and active GPCR-Gα signaling complexes at endosomes (11, 14, 15). Whereas the use of genetically encoded engineered GFP variant biosensors that sense endogenous signaling activity of kinases and second messengers targeted to specific subcellular organelles such as endosomes has permitted the detection of spatially organized or compartmentalization signaling dynamics in living cells (17).

A function for β-arrestins in GPCR intracellular signaling has also been established. For certain receptors like the vasopressin receptor 2, GPCR-G protein endosomal signaling requires retention of β-arrestin binding to the receptor in a unique conformation that does not preclude G protein signaling (Fig. 1) (18–20). Whereas for other receptors, GPCRs bind tightly to β-arrestins forming a stable complex that cointernalizes with the receptor and generates endosomal signaling hotspots that sustain or initiate kinase signaling cascades (Fig. 1). This has been well documented for protease-activated receptor-2 (PAR2)-induced β-arrestin-1-stimulated extracellular signal-regulated kinase 1/2 (ERK1/2) endosomal signaling and the angiotensin II type 1 A receptor-mediated β-arrestin-2-promoted activation of c-Jun amino-terminal kinase 3 (21–24). The β-arrestin binding affinity for GPCRs is governed by GRK-dependent phosphorylation and is controlled by the abundance of phosphorylated sites as well as the position of specific phosphorylation sites that vary depending on the cellular context (4, 25). Different GPCR phosphorylation patterns induce distinct β-arrestin conformations that can dictate various β-arrestin-mediated functions such as desensitization, internalization, and signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin, which is likely to occur at least once in the GPCR life cycle (8). The function of ubiquitination has been studied mainly in the context of GPCR endosomal-lysosomal trafficking (6–8, 26). Although there are defined functions for phosphorylation in regulating GPCR signaling, the role of ubiquitination in receptor signaling is less clear. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes.

UBIQUITINATION AND P38 MAPK SIGNALING

Like phosphorylation, ubiquitination is a reversible and dynamic process balanced by the activity of ubiquitin ligases that attach ubiquitin moieties to substrate proteins with deubiquitinases, enzymes that remove ubiquitin from substrate proteins (27, 28). Ubiquitination of a subset of endogenous GPCRs including PAR1 and the purinergic P2Y1 receptor expressed in human-cultured endothelial cells was shown to initiate assembly of a p38 mitogen-activated protein kinase (MAPK) signaling complex on endosomes (Fig. 1) (29, 30). This work was prompted by earlier landmark studies demonstrating that ubiquitination of PAR1 and P2Y1 was not required for receptor sorting into multivesicular bodies and degradation in lysosomes unlike classic GPCRs (31–33). These studies suggested that GPCR ubiquitination is dispensable for lysosomal sorting and raised questions regarding the function of ubiquitination. Subsequent studies using primarily human-cultured endothelial cells showed that ubiquitination of endogenous PAR1 and P2Y1 was specifically required for p38 MAPK signaling but not ERK1/2 activation (29). Moreover, GPCR-stimulated ERK1/2 signaling is rapid and temporally distinct compared with p38 MAPK signaling, which occurs at a time when the activated receptor has internalized and localized primarily on endosomes. Remarkably, this work also identified a noncanonical pathway used by a subset of endothelial GPCRs including PAR1, P2Y1, histamine, and prostaglandin receptors that lead to autoactivation of the p38α isoform mediated by transforming growth factor β-activated kinase-binding protein 2 (TAB2) and TAB1 adaptor proteins (Fig. 1) (29, 30). TAB1 was shown to directly bind to p38α, triggering autophosphorylation and activation (34–36) and to coassociate with TAB2, an adaptor protein with two ubiquitin-binding motifs (37). In live cell imaging experiments, both GPCR ubiquitination and an ubiquitin-binding motif of TAB2 were found to be essential for corecruitment to endocytic vesicles after agonist simulation (29). In new work, PAR1 and prostaglandin receptor activation of p38 at endosomes were demonstrated using a genetically encoded p38 biosensor (38, 39). These studies provide evidence that ubiquitin-dependent assembly and activation of the GPCR and TAB2-TAB1-p38 signaling complex occur on endosomes.

INFLAMMATORY SIGNALING

The physiological relevance of the GPCR ubiquitin-driven TAB2-TAB1-p38 signaling pathway has been linked to vascular inflammation including upregulation of cytokine expression and endothelial barrier disruption in vitro (Fig. 2) (29, 30, 40). The PAR1-p38 signaling axis was also shown to regulate vascular leakage in vivo in a mouse model (29). GPCR regulation of endothelial barrier permeability is mediated by RhoA signaling, a key regulator of actin stress fibers and actomyosin contractility (41, 42). Interestingly, noncanonical GPCR-p38 signaling does not integrate with the RhoA pathway but rather appears to function in parallel to regulate endothelial barrier disruption (Fig. 2) (43). Moreover, a recent study showed that thrombin-activated p38 signaling converged on mitogen-activated protein kinase-activated protein kinase 2 (MK2) and MK3 induced heat shock protein 27 (HSP27) phosphorylation, which altered HSP27 oligomerization and the dynamics of endothelial barrier recovery (43). To identify effectors of noncanonical p38 MAPK signaling, a global phosphoproteomic mass spectrometry analysis of thrombin-stimulated PAR1 signaling was conducted in endothelial cells treated with or without a p38 kinase inhibitor (44). This analysis identified 5491 phosphopeptides, 2,317 phosphoproteins, and 4 different dynamic phosphoproteome profiles induced by thrombin that were dependent on p38 MAPK endosomal signaling (44). Of relevance was the discovery that the p38α isoform directly regulated phosphorylation of ERK1/2 and α-catenin, a component of adherens junctions that controls barrier integrity. This study provides a rich source of proteins and pathways that are specific to GPCR-induced p38 endosomal signaling in human-cultured endothelial cells and provide new opportunities for future investigations.

Figure 2.

Ubiquitin-driven GPCR inflammatory endosomal signaling. A subset of endothelial GPCRs promote ubiquitin-mediated p38 endosomal inflammatory signaling, which is best exemplified by PAR1. Thrombin binds to and activates PAR1 by cleavage of an N-terminal arginine (R) 41 site, unveiling a new N-terminus that acts as a tethered ligand to activate the receptor. Activated PAR1 couples to heterotrimeric G proteins at the plasma membrane, eliciting a variety of intracellular responses including RhoA signaling, Ca²⁺ mobilization, diacylglycerol (DAG) production, protein kinase C (PKC), RhoA, and RhoA kinase (ROCK) activation that promote myosin light chain (MLC) phosphorylation, actomyosin contractility, and endothelial barrier disruption. In addition, thrombin-stimulated PAR1 activation of p38 MAPK inflammatory signaling is induced by c-Src-mediated phosphorylation and activation of the E3 ligase NEDD4-2. Activated NEDD4-2 induces ubiquitination of PAR1 that promotes assembly and activation of an endosomal signaling complex mediated by TAB2-TAB1-induced p38α isoform autoactivation. Ubiquitin-induced thrombin-activated PAR1-p38 signaling axis contributes to endothelial barrier disruption and induction of interleukin-6 (IL-6) cytokine production. New studies have identified two deubiquitinases, CYLD and USP34, as important for controlling thrombin-induced p38 activation. However, the mechanism by which CYLD and USP34 regulates endosomal GPCR-p38 MAPK signaling is not known. Figure created with BioRender.com. CYLD, cylindromatosis; NEDD4, neural precursor cell expressed, developmentally downregulated-4; PAR1, protease-activated receptor-1; TAB1/2, transforming growth factor β-activated kinase-binding protein 2; USP34, ubiquitin-specific protease-34.

REGULATORY MECHANISMS

New studies have begun to delineate the molecular mechanisms that control ubiquitin-mediated GPCR inflammatory endosomal signaling. There are over 600 E3 ubiquitin ligases codified in the human proteome, however, a much smaller 14-member family of neural precursor cell expressed, developmentally downregulated-4 (NEDD4) homologous to E6AP carboxyl terminus (HECT) domain-containing E3 ligases have been linked to GPCR ubiquitination (45). Not surprising, the HECT domain containing E3 ligase NEDD4-2 was identified in an siRNA screen of NEDD4 family members as a key mediator of PAR1 and P2Y1 ubiquitination and shown to regulate TAB2-TAB1-p38 signaling in endothelial cells (Fig. 2) (29). Like other GPCRs, the mechanisms that control E3 ligase activity following GPCR activation are largely unknown. The NEDD4 E3 ligase domain architecture includes an N-terminal C2 domain, two to four WW domains, and a catalytic C-terminal HECT domain and are generally autoinhibited. In a foundational study, endothelial PAR1 and P2Y1 were shown to stimulate NEDD4-2 E3 ligase activity via a Gq- and G12/13-dependent c-Src-mediated phosphorylation of a critical tyrosine residue present within the WW domains 2,3-linker peptide region releasing autoinhibition (Fig. 2) (40). An siRNA knockdown-rescue strategy was further taken to demonstrate a prerequisite for NEDD4 tyrosine phosphorylation in thrombin-induced PAR1-mediated endothelial barrier permeability (40). This study documented the mechanism by which endogenous GPCRs expressed in endothelial cells stimulate E3 ligase activity by unlocking the autoinhibited state of NEDD4-2 to activate p38 MAPK signaling and control endothelial inflammatory responses.

Ubiquitination and deubiquitination of vital substrate proteins are crucial for ensuring cells function optimally. Deubiquitinating enzymes (DUBs), a family of 100 members, are responsible for removing, editing, and recycling ubiquitin from substrate proteins including GPCRs (8, 28). Although several studies have demonstrated the roles for DUBs in regulating GPCR function (8, 45), a significant knowledge gap exists regarding the identification of the physiologically relevant DUBs that regulate GPCR function in appropriate cellular contexts. A recent study utilized a comprehensive unbiased siRNA library screen targeting 96 human DUBs to identify specific DUBs that control GPCR-p38 MAPK signaling in endothelial cells (46). The library screen identified and validated the function of cylindromatosis (CYLD) and ubiquitin-specific protease (USP34) as important for regulating thrombin-activated PAR1-induced p38 MAPK signaling in endothelial cells (Fig. 2). Interestingly, however, only CYLD was found to mediate thrombin-induced endothelial barrier permeability (46). Whereas CYLD and USP34 were both shown to alter thrombin-stimulated inflammatory cytokine expression, albeit in opposite directions (46). This work indicates that specific DUBs identified through an unbiased screen distinctly regulate GPCR-induced p38-mediated inflammatory responses and emphasize the importance of using a systematic wide-scale approach to interrogate gene function to identify relevant and novel proteins for specific biological functions.

CONCLUSIONS

In summary, GPCRs reside in distinct membrane-bound compartments and signal to different effectors at the plasma membrane and endosomes to promote specific physiological responses. A greater understanding of the cellular location of GPCR signaling is critical for gaining important insights of where signaling pathways might be altered in disease. In addition, understanding the full repertoire of GPCR cellular signaling offers an opportunity to develop drugs that can target specific pathways to improve drug efficacy and limit unwanted side effects.

GRANTS

This work was supported by NIH/National Heart, Lung, and Blood Institute (NHLBI) F31 HL158213 (to N.C.), NIH/National Institute of General Medical Sciences (NIGMS) T32 GM007752 (to N.C.), NIH/NIGMS K12 GM068524 (to J.M.P.), NIH/NHLBI R01 HL163931-S1 (to J.M.P.), NIH/NHLBI R01 HL163931 (to J.T.), and NIH/NIGMS R35 GM127121 (to J.T.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.T. conceived and designed research; N.C., J.M.P., and J.T. prepared figures; N.C., J.M.P., and J.T. drafted manuscript; N.C., J.M.P., and J.T. edited and revised manuscript; N.C., J.M.P., and J.T. approved final version of manuscript.