Post-Translational Modifications of G Protein–Coupled Receptors Control Cellular Signaling Dynamics in Space and Time

1. E3 Ubiquitin Ligases and GPCRs

E3 ligases of the RING finger family are unique and simultaneously bind to both the charged E2 and the substrate, thereby facilitating the direct transfer of the ubiquitin moiety to the substrate. A notable member of RING family is Casitas B lineage lymphoma, which ubiquitinates protease-activated receptor-2 (Jacob et al., 2005). In contrast to RING-types, the HECT-type E3 ligases interact with the E2s, which facilitates the transfer ubiquitin to an active site cysteine residue within the E3 HECT catalytic domain. The ubiquitin is subsequently conjugated to lysine acceptor sites of the substrate protein. Of the HECT E3 ubiquitin ligases, the neural precursor cell expressed, developmentally downregulated (NEDD)-4 subfamily is best known to regulate GPCR trafficking. The NEDD4 family contains nine members, including NEDD4, NEDD4-2, atrophin-1–interacting protein-4 (AIP4), WW domain–containing E3 ubiquitin protein ligase (WWP) 1, WWP2, Sma- and Mad-related protein-specific E3 ubiquitin ligase proteins 1 and 2, and NEDD-like ubiquitin protein ligases 1 and 2 (Weber et al., 2019). All NEDD4 family members share a similar domain structure, including an amino-terminal C2 domain, three to four WW domains, and a carboxy-terminal catalytic HECT domain. The first authenticated examples of HECT-type E3 ligase–mediated ubiquitination of mammalian GPCRs include AIP4-mediated ubiquitination of the C-X-C chemokine receptor (CXCR) 4 (Marchese et al., 2003) and the β₂-adrenoceptor (Shenoy et al., 2001, 2008). In both cases, agonist-induced GPCR ubiquitination mediates endolysosomal trafficking and receptor degradation. Finally, the RING between RING type of E3 ubiquitin ligases regulate diverse cellular processes (Dove and Klevit, 2017) with the most notable being Parkin, which expedites the clearance of damaged mitochondria via a process called mitophagy (Lazarou et al., 2012). In addition, Parkin mediates endoplasmic reticulum (ER)–associated protein degradation (ERAD) of the class A orphan G protein–coupled receptor 37 (GPR37) that regulates ER stress in Parkinson’s disease (Berger et al., 2017). GPR37 also functions as an ER chaperone for the Wnt co‐receptor lipoprotein receptor-related protein 6 in neuronal progenitor cells, emphasizing the importance of regulating GPR37 function at the ER (Imai et al., 2001; Berger et al., 2017).

2. GPCR Regulation of E3 Ligase Activity

Mammalian GPCRs are differentially modified with ubiquitin in space and time. This suggest that distinct mechanisms likely exist to control the diverse functions of E3 ligases in regulating GPCR ubiquitination. In most studies, regulation of HECT domain–containing NEDD4 E3 ubiquitin ligase activity appears to occur through recruitment of the E3 ligase to the GPCR substrate either directly through noncanonical WW domain–mediated interactions, as demonstrated for the CXCR4 and E3 ligase AIP4 (Bhandari et al., 2009), or indirectly through interactions with adaptor proteins, mainly β-arrestin recruitment of NEDD4 to β₂-adrenoceptor (Shenoy et al., 2008) and metabotropic glutamate (mGlu)₇ receptor (Lee et al., 2019). The less studied, mammalian α-arrestin domain-containing proteins (ARRDCs) may also function as adaptors to recruit E3 ligases to GPCRs (Alvarez, 2008). Several studies indicate that NEDD4 E3 ligases are exquisitely regulated through release of autoinhibition, which can occur through allosteric interactions (Rotin and Kumar, 2009) or via ligand-induced phosphorylation (Persaud et al., 2014) specifically at tyrosine residues (Chen et al., 2017b). A recent study demonstrated that endothelial GPCRs can also regulate NEDD4 E3 ligase activity by release of autoinhibition. In this work, thrombin-activated PAR1 stimulates c-Src–mediated phosphorylation of NEDD4-2 at tyrosine (Y)⁴⁸⁵ located within the autoinhibitory 2,3- linker peptide between WW domains 2 and 3, leading to its activation and ubiquitination of PAR1 (Fig. 4C) (Grimsey et al., 2018). This ultimately results in activated PAR1-stimulated p38 MAP kinase activation and regulation of endothelial inflammatory responses (Grimsey et al., 2018). The purinergic P2Y1 receptors also required c-Src and NEDD4 tyrosine phosphorylation for endothelial inflammatory signaling (Grimsey et al., 2018).

3. Ubiquitin Linkages and GPCRs

GPCR ubiquitination occurs on intracellular loops and on the C-terminal tail (Komander and Rape, 2012). GPCRs can be modified at one or multiple lysine residues with either monoubiquitin, as shown for the yeast α-factor receptor Sterile 2 (Ste2 or Ste2p), and CXCR4 or via polyubiquitin chains such as K63-linked ubiquitin for PAR1 (Fig. 4, B and C) (Grimsey et al., 2015), which can differentially affect receptor function. Ubiquitin chains are formed through ubiquitin linkages at several lysine sites within the ubiquitin molecule, including well characterized branched K48-, K63-, and linear N-terminal methionine–linked ubiquitin, offering numerous possibilities of ubiquitin polymer assembly (Fig. 4B) (Peng et al., 2003). In general, monoubiquitin and K63-linked ubiquitin predominate as sorting signals for GPCRs within the endocytic pathway (Terrell et al., 1998; Gulia et al., 2017). However, for certain GPCRs, K63 ubiquitin linkage has been recently implicated in regulating signaling from endosomes (Grimsey et al., 2015). Ubiquitin K48 and K11 linkages serve as a potent proteasomal degradation signals, whereas K29 and K63 linkages function to target substrate proteins for degradation via autophagy (Mukhopadhyay and Riezman, 2007). However, as an internalization signal, a single ubiquitin is exceptionally weak, and ubiquitin is likely operational only as polyubiquitin chains (Barriere et al., 2006). In the case of proteasomal degradation, proteins bearing chains of at least four ubiquitin molecules are the preferred substrates of the 26S proteasome (Chau et al., 1989). Recently, linkage of ubiquitin moieties to nonlysine nucleophilic residues such serine, cysteine, and threonine residues, as well as the free amino group of the N-terminus of proteins, has been demonstrated (Fig. 4B) (McDowell and Philpott, 2016). However, conjugates generated from ubiquitination on nonlysine residues are thermodynamically less stable than those generated on canonical lysine residues (McClellan et al., 2019). A recent study showed that a lysine-deficient dopamine D₄ receptor was ubiquitinated on cytoplasmic serine and threonine residues that is important for regulating proteasomal degradation (Skieterska et al., 2015; Peeler et al., 2017). Whether nonlysine ubiquitination of GPCRs is common to other GPCRs remains to be determined.

1. GPCR Biosynthesis and Cell Surface Expression

Constitutive or basal GPCR ubiquitination functions primarily to control receptor trafficking through the biosynthetic pathway. During biogenesis, newly synthesized GPCRs are folded in the ER with the assistance of chaperone proteins, undergo maturation in the Golgi, and then traffic to the plasma membrane (Fig. 5). However, misfolded or incompletely folded GPCRs are polyubiquitinated, retro-translocated from the ER to the cytosol via the ERAD quality control system, and then shuttled to the proteasome for proteolytic degradation (Fig. 5) (Petaja-Repo et al., 2001; Cook et al., 2003; Huang et al., 2006). Multiple GPCRs have been shown to be modified with K48 polyubiquitin chains during biogenesis and targeted to the 26S proteasome for degradation, often resulting from receptor mutations that can underlie the basis for disease, as reported for the visual GPCR rhodopsin and the vasopressin V₂ receptor (Conn et al., 2007; Robben et al., 2009; Athanasiou et al., 2018). This has prompted the development of small molecules or pharmacoperones that bind to GPCRs and correct misfolding of mutant receptors (Nakamura et al., 2010) and has been well described for the gonadotropin-releasing hormone receptor (Bernier et al., 2004; Conn et al., 2007). Thus, at the very beginning of the GPCR life cycle, ubiquitination has an important role in regulating receptor biosynthesis.

In addition to biosynthesis, basal ubiquitination of GPCRs is important for regulating receptor expression at the plasma membrane. This has been illustrated for several GPCRs including PAR1, CXCR7, and chemokine (C-C motif) receptor (CCR) 7 (Moriyoshi et al., 2004; Wolfe et al., 2007; Canals et al., 2012). In the case of PAR1, ubiquitination occurs basally and negatively regulates constitutive internalization, thereby increasing cell surface expression (Wolfe et al., 2007). Constitutive internalization of PAR1 is mediated by the clathrin AP-2, where the μ2-adaptin subunit of AP-2 binds to the C-tail of PAR1 via interaction with a classic tyrosine-based motif (Y⁴²⁰KKL⁴²³) rather than β-arrestins (Paing et al., 2006; Wolfe et al., 2007). Ubiquitination of PAR1 occurs on C-tail lysine residues that has been confirmed by site-directed mutagenesis (Wolfe et al., 2007) and mass spectrometry high-throughput discovery-based analysis, PhosphoSitePlus (Hornbeck et al., 2015), that reside within the tyrosine-based motif and precludes binding of the μ2-adaptin subunit (Hornbeck et al., 2015). Moreover, a lysineless ubiquitin-deficient PAR1 mutant displayed enhanced internalization that was reversed by the fusion of a single ubiquitin moiety to the C-terminal tail, suggesting that ubiquitination is important for retaining PAR1 on the cell surface (Wolfe et al., 2007). Similar to PAR1, basal ubiquitination of the chemokine receptor CXCR7 occurs on a lysine residue located within the C-tail region and is deubiquitinated after agonist activation through a process that requires phosphorylation and β-arrestin recruitment (Canals et al., 2012). The immune cell expressed CCR7 chemokine receptor is also constitutively modified with K63-linked polyubiquitination and regulates basal trafficking of CCR7 (Schaeuble et al., 2012). A mutant CCR7 defective in ubiquitination alters the spatial distribution of the receptor and impairs immune cell migration (Schaeuble et al., 2012). Thus, basal ubiquitination of GPCRs is important for the appropriate spatial subcellular localization, which has a critical role in governing cellular behavior.

2. Endocytosis of GPCRs

GPCR trafficking through the endocytic pathway is a highly conserved process that includes internalization, recycling, and lysosomal sorting and is regulated by ubiquitination (Fig. 5). A function for ubiquitination in GPCR endocytosis was first described for the yeast Saccharomyces cerevisiae α-mating factor GPCR Ste2 (Rohrer et al., 1993). Subsequent studies identified Rsp5, an ortholog of Nedd4-like HECT domain E3 ubiquitin ligases, as the key regulator of ligand-induced Ste2p ubiquitination, internalization, and targeting to vacuoles, an organelle equivalent to the mammalian lysosome (Hicke and Riezman, 1996; Dunn and Hicke, 2001). Unlike yeast, most mammalian GPCRs are internalized through clathrin-coated pits via a β-arrestin–dependent pathway. β-arrestins act as endocytic adaptors by binding directly to the clathrin heavy chain and to the β-adaptin subunit of AP-2 (Goodman et al., 1996; Laporte et al., 1999; Gaidarov and Keen, 1999). Interestingly, yeast lack β-arrestins and rather express a family of ARTs (arrestin-related trafficking proteins) that mediate recruitment of E3 ligases to facilitate internalization of several membrane-spanning proteins, including Ste2 (Alvaro et al., 2014). However, mammalian GPCRs do not require ubiquitination for efficient endocytosis in most cases; in fact, the prevention of ubiquitination has minimal or no effect on endocytosis of a number of GPCRs, including the β₂-adrenoceptor, DOR, and neurokinin 1 (NK₁) receptor (Shenoy et al., 2001; Tanowitz and Von Zastrow, 2002; Hanyaloglu et al., 2005). However, not all GPCRs require β-arrestins for endocytosis, and this is best exemplified for PAR1 (Paing et al., 2002). Instead of β-arrestins, PAR1 requires the clathrin adaptors AP-2 and epsin-1 for efficient internalization (Chen et al., 2011). In this case, AP-2 recognizes activated PAR1 phosphorylation sites within the C-tail region rather than the tyrosine-based motif, whereas epsin-1 requires both the ubiquitin-binding motifs of epsin-1 and PAR1 ubiquitination to facilitate efficient endocytosis (Chen et al., 2011).

3. Lysosomal Sorting of GPCRs

The best characterized function of ubiquitination is to target activated receptors to lysosomes for degradation (Fig. 5). The β₂-adrenoceptor was the first mammalian GPCR shown to exhibit ligand-dependent ubiquitination and degradation (Shenoy et al., 2001), and this was closely followed by a report of ligand-induced ubiquitination and degradation of the CXCR4 chemokine receptor (Marchese and Benovic, 2001). Initiation of ligand-induced ubiquitination of GPCRs occurs at the plasma membrane, generally requiring receptor phosphorylation and β-arrestin recruitment. Isoproterenol-stimulated β₂-adrenoceptor is rapidly phosphorylated, which enhances β-arrestin–mediated recruitment of the NEDD4 E3 ubiquitin ligase (Shenoy et al., 2001, 2008). Mass spectrometry analysis of agonist-induced β₂-adrenoceptor ubiquitination revealed the major sites for polyubiquitination reside in ICL3 at K²⁶³ and K²⁷⁰ and in the C-tail at K³⁴⁸, K³⁷², and K³⁷⁵ also modified with polyubiquitination (Xiao and Shenoy, 2011), later identified to be K63-type ubiquitin linkages (El Ayadi et al., 2018). A β₂-adrenoceptor variant in which all the phosphorylation sites are mutated showed impaired ubiquitination as well as significantly reduced β-arrestin interaction (DeWire et al., 2007). In contrast to the β₂-adrenoceptor, CXCR4 displays ligand-induced monoubiquitination (Marchese and Benovic, 2001). The CXCR4 receptor contains two serine residues, S³²⁴ and S³²⁵, located within the C-tail degradation motif, which are rapidly phosphorylated by agonist activation (Busillo et al., 2010). Agonist-induced ubiquitination of CXCR4 mediated by the E3 ubiquitin ligase AIP4 targets CXCR4 for lysosomal degradation (Marchese et al., 2003). Similarly, thrombin activation of PAR1 results in rapid modification with K63-ubiquitin linkages mediated by the recruitment and activation of NEDD4-2 initiated at the plasma membrane (Fig. 4C) (Grimsey et al., 2015, 2018). Although ligand-induced ubiquitination of GPCRs ultimately controls lysosomal degradation, the time scales for GPCR degradation are vastly different. Activated PAR1 is sorted rapidly to lysosomes and degraded within minutes (Trejo et al., 1998; Trejo and Coughlin, 1999), whereas CXCR4 ubiquitination and degradation occurs much later, within 3–6 hours (Marchese and Benovic, 2001). In contrast, β₂-adrenoceptor is rapidly ubiquitinated, but lysosomal degradation is protracted and occurs after prolonged 6–24 hours of isoproterenol stimulation (Shenoy et al., 2008). Ubiquitination of most classic GPCRs facilitates engagement with the endosomal sorting complex required for transport (ESCRT)-0, -I, -II, and -III machinery. The ubiquitin-binding ESCRT components function sequentially to sort GPCRs from endosomes to multivesicular bodies (MVBs) or lysosomes for degradation. The importance of ubiquitin and ESCRTs in receptor lysosomal degradation has been illustrated for several GPCRs. As discussed above, ubiquitination of CXCR4 facilitates lysosomal sorting and requires AIP4-mediated ubiquitination of the ESCRT-0 protein, hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), and vacuolar protein sorting 4 (Vps4), an ATPases associated with diverse cellular activities (AAA)-ATPase (Marchese et al., 2003). Agonist-induced ubiquitination of PAR2 also requires HRS for lysosomal degradation (Hasdemir et al., 2007). Although ubiquitination plays an important role in GPCR lysosomal degradation, there are examples of receptors that can efficiently sort to lysosomes in a ubiquitin-independent manner, as exemplified by DOR. A ubiquitination-deficient DOR mutant is efficiently sorted to the limiting membrane of intralumenal vesicles (ILVs) of MVBs, where extensive proteolytic fragmentation of the receptor ectodomain occurs (Henry et al., 2011). Sorting of DOR to ILVs or MVBs requires HRS and Vps4 but not the ESCRT-I component, tumor susceptibility gene 101 (Hislop et al., 2004), indicating that ubiquitin-independent receptor sorting requires some but not all components of the ubiquitin-binding ESCRT machinery. Ubiquitination of μ-opioid receptor (MOR) mediates ESCRT-dependent degradation by controlling receptor distribution between the limiting endosome membrane and lumen but is not required for receptor delivery to the proteolytic compartments. Instead, this is dictated by the MOR C-terminal tail and is independent of receptor ubiquitination (Hislop et al., 2011). In contrast, the calcitonin-like receptor is not ubiquitinated after activation but nonetheless is sorted and degraded in the lysosomes via an ESCRT-0–dependent pathway, confirming that ubiquitination is not obligatory for GPCRs to enter the ESCRT pathway (Cottrell et al., 2007).

An alternative pathway for GPCR lysosomal sorting that bypasses the requirement for both receptor ubiquitination and ubiquitin-binding ESCRTs has been described for PAR1 and the purinergic P2Y1 receptor. Apoptosis-linked gene-2 (ALG-2)-interacting protein X (ALIX), an ESCRT-III–interacting protein, interacts directly with a highly conserved YPX₃L motif present the second intracellular loop of PAR1 and the P2Y1 receptor via its central V domain to facilitate receptor lysosomal sorting. ALIX also directly binds to the ESCRT‐III complex, allowing receptors to bypass the ubiquitin‐binding ESCRTs and sort directly into ILVs of MVBs (Dores et al., 2012a, 2016). In recent work, ALIX activity was shown to be regulated by the ARRDC3, which facilitates ALIX ubiquitination and dimerization by the WWP2 HECT domain–containing E3 ubiquitin ligase (Gullapalli et al., 2006; Dores et al., 2012a,b, 2015, 2016). ALIX, ARRDC3, and WWP2 are essential for targeting activated PAR1 and P2Y₁ to MVBs or lysosomes via an ECSRT-III charged MVB protein 4– and Vps4-dependent pathway (Dores et al., 2016). Besides PAR1 and P2Y₁, six other mammalian GPCRs were found to possess conserved ALIX YPX_nL binding motifs within their second ICL2, including the α_1B-adrenoreceptor, angiotensin type 2 (AT₂) receptor, galanin receptor, histamine H₂ receptors, neuropeptide FF receptors, and neuropeptide S receptor (Dores et al., 2012a). Both ALIX and ARRDC3 exploit diverse pathways to capture receptors for endolysosomal sorting (Tian et al., 2016), suggesting that a vast number of other GPCRs may also be regulated by the ALIX-ARRDC3 pathway. Together, these studies illustrate that GPCRs have the capacity to use ubiquitin directly or indirectly to facilitate trafficking through the endolysosomal pathway.

1. Ubiquitin and Plasma Membrane GPCR Signaling

Ubiquitin-driven GPCR signaling was recently shown for mGlu₇ receptor induction of extracellular signal–regulated protein kinase (ERK) 1/2 signaling in hippocampal neurons (Lee et al., 2019). In this scenario, agonist stimulation of mGlu₇ receptor enables β-arrestin recruitment of NEDD4, which forms a complex at the plasma membrane and facilitates ubiquitination of the receptor. Both NEDD4 and β-arrestin are required for activated mGlu₇ receptor-dependent ERK1/2 signaling, whereas induction of c-jun N-terminal kinase signaling occurs independently of NEDD4-mediated ubiquitination (Lee et al., 2019), suggesting that ubiquitin-driven mGlu₇ receptor signaling is specific to ERK1/2. Similarly, activation of the chemokine receptor CXCR2 by interleukin-8 promotes ubiquitin-mediated proinflammatory signaling and proangiogenic responses in leukocytes and endothelial cells (Leclair et al., 2014). In this study, Leclair et al. mapped the site of CXCR2 ubiquitination to a single C-tail localized lysine K³²⁷ residue and showed that an ubiquitination-deficient CXCR2 mutant with a K³²⁷ to arginine (R) conversion failed to recruit β-arrestin-2 at the plasma membrane and blocked intracellular signaling including ERK1/2 phosphorylation (Leclair et al., 2014), suggesting that receptor ubiquitination is necessary for triggering signaling cascades. Agonist-induced ubiquitination of the parathyroid hormone receptor (PTHR) also requires phosphorylation and β-arrestin binding, but here ubiquitination appears to function selectively in promoting p38 MAP kinase activation and not cAMP accumulation (Zhang et al., 2018). The chemokine receptor CXCR4 has also been shown to mediate ubiquitin-dependent signaling; however, in this case the effect is indirect via modulation of signal transducing adapter molecule (STAM)-1, an ESCRT-0 component (Malik et al., 2012). In this study, agonist-induced CXCR4-mediated ERK1/2 signaling required the E3 ubiquitin ligase AIP4, which facilitates ubiquitination of STAM-1. Interestingly, CXCR4-promoted ERK1/2 activation is governed by a discrete subpopulation of STAM-1 and AIP4 localized to caveolae microdomains at the plasma membrane (Malik et al., 2012). This work expands the role of AIP4 and STAM-1 beyond regulation of CXCR4 lysosomal trafficking (Malik and Marchese, 2010) and provides a new link for ubiquitin-driven GPCR signaling between the trafficking machinery and signal propagation from the plasma membrane.

2. Ubiquitination and Endosomal GPCR Signaling

Several recent studies established that agonist-induced ubiquitination of endothelial GPCRs promotes p38 MAP kinase activation on endosomes via a noncanonical pathway (Fig. 5) (Grimsey et al., 2015, 2018, 2019). The activation and ubiquitination of PAR1 by NEDD4-2 initiates the recruitment of transforming growth factor-β–activated protein kinase 1–binding protein (TAB) 2 via an association with the TAB2 ubiquitin-binding domain, which specifically interacts with K63-linked ubiquitin (Kulathu et al., 2009). TAB2 is known to associate with TAB1 (Bouwmeester et al., 2004). TAB1 has also been shown to directly bind specifically to the p38α isoform, inducing a conformation change resulting in autophosphorylation and activation through a noncanonical pathway (Ge et al., 2002; DeNicola et al., 2013). Importantly, TAB1-dependent activation of p38α induced by PAR1 bypasses the requirement for upstream MAP2K of the canonical three-tiered kinase cascade. Moreover, although it is presumed that GPCRs activate p38 MAP kinase through the three-tiered kinase cascade, there is very limited supportive evidence, and rarely has the role of MAP kinase kinases been directly tested (Goldsmith and Dhanasekaran, 2007). The ubiquitin-driven PAR1 signaling pathway is specific to p38, as thrombin activation of ERK1/2 proceeds through the canonical three-tiered kinase cascade (Grimsey et al., 2015). Similar to PAR1, ubiquitination of the purinergic P2Y1 receptor also mediates p38 activation through a TAB1-TAB2–dependent pathway (Grimsey et al., 2015), indicating that this pathway is used by multiple GPCRs. Indeed, multiple endothelial GPCRs agonists including histamine (H₁ or H₂ receptor) and prostaglandin EP₂ (EP₄ prostanoid receptor) also activate p38 MAPK through a noncanonical TAB1-dependent pathway (Grimsey et al., 2019). This work was further advanced by showing that TAB1-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro and that p38 signaling was required for PAR1-induced vascular leakage in vivo (Grimsey et al., 2015). Dysfunction of the endothelial barrier is a hallmark of vascular inflammation and suggests that ubiquitin-driven p38 proinflammatory signaling is a common pathway used broadly by GPCRs at least in the context of the vascular endothelium.

3. Ubiquitination and Biased Signaling

Activation of the same GPCR by two or more distinct ligands can elicit different distinct responses, is referred to as biased agonism, and is an important emerging area for drug discovery. Several previous studies indicate that post-translational modification of GPCRs with ubiquitin is uniquely influenced by biased agonists and likely contributes to differential responses. A well studied example is μ-opioid receptor activation with morphine versus DAMGO that resulted in differential recruitment and utilization of β-arrestins and displayed remarkable differences in receptor ubiquitination. DAMGO stimulated robust ubiquitination of μ-opioid receptor, whereas morphine-induced receptor ubiquitination was negligible (Groer et al., 2011). As stated above, isoproterenol stimulates β₂-adrenoceptor ubiquitination via a β-arrestin–NEDD4–mediated pathway (Shenoy et al., 2008); however, β₂-adrenoceptor ubiquitination induced by the biased ligand carvedilol is mediated by membrane-associated RING-CH-type finger 2 (MARCH2), a RING-type E3 ligase, in the place of NEDD4 (Han et al., 2012). Clearly, biased ligands have the capacity to differentially regulate the ubiquitination machinery as well as GPCR ubiquitination and are important to consider for understanding the molecular basis of biased signaling and future drug discovery.

1. Constitutive GPCR Deubiquitiation

An emerging role for DUBs is in control of GPCR transport to the cell surface and thereby in preventing ubiquitin-proteasomal degradation (Fig. 5). The quality control machinery in the ER is stringent and prefers to err on the side of rapid degradation of proteins that, when given time, would fold into a properly functionally active protein. This has been shown for the GPCR adenosine A_2A receptor. Deubiquitination of adenosine A_2A receptor by USP4 relaxes quality control in the ER, enhances cell surface expression, and rescues the receptor from proteasomal degradation (Milojevic et al., 2006). DUBs show remarkably substrate specificity. USP4 binds to the adenosine A_2A receptor C terminus and deubiquitinates the receptor but does not act on mGlu₅ receptor, another GPCR that tends to accumulate intracellularly (Milojevic et al., 2006). Another role for DUBs in regulating GPCR cell surface expression occurs via regulation of receptor recycling as exemplified by Frizzled-4, a seven-transmembrane receptor for Wnt ligands (Mukai et al., 2010). Constitutive ubiquitination of Frizzled-4 promotes internalization and lysosomal degradation, whereas deubiquitination mediated by USP8 leads to recycling and increased surface expression, events that occur independently of stimulation with Wnt ligands (Mukai et al., 2010). Thus, the balance of ubiquitination and deubiquitination mediated by DUBs switch the receptor’s fate from lysosomal degradation to recycling and enhanced cellular resensitization.

2. Deubiquitination of Agonist-Activated GPCRs

The regulation of agonist-stimulated GPCR ubiquitination is ultimately important for regulating the biologic function of the receptor but has not been extensively studied. Shenoy et al. identified USP33 in a yeast-two hybrid screen with β-arrestin (Shenoy et al., 2009), suggesting that β-arrestins have a dual function to recruit not only the E3 ubiquitin ligases but also DUBs to regulate GPCR function. In a follow-up study, UPS33 as well as its homolog USP20 were shown to reverse agonist-activated β₂-adrenoceptor ubiquitination and thereby switched the receptor fate from lysosomal degradation to recycling, resulting in enhanced cellular resensitization (Berthouze et al., 2009). In addition, phosphorylation of USP20 induced by β₁-adrenoceptor is required for efficient lysosomal degradation of the receptor (Yu et al., 2019). The chemokine receptor CXCR4 has been shown to associate with USP14 in an agonist-dependent manner, which causes a decrease in receptor ubiquitination and lysosomal degradation (Mines et al., 2009). Interestingly, depletion of USP14 expression also results in loss of chemokine (C-X-C motif) ligand 12–CXCR4–induced cell migration but not of ERK1/2 signaling, suggesting that ubiquitin positively modulates certain aspects of CXCR4 signaling (Mines et al., 2009). Another study examined the effect of USP8 on CXCR4 ubiquitation and reported that loss of USP8 expression enhanced CXCR4 expression by preventing degradation without altering CXCR4 ubiquitination status and ERK1/2 signaling (Berlin et al., 2010). USP8 appears to modulate chemokine (C-X-C motif) ligand 12–CXCR4–induced ubiquitination of HRS, a component of ESCRT-0, which is mediated by AIP4 to control CXCR4 lysosomal trafficking (Berlin et al., 2010).

In contrast to most GPCRs, metabotropic GABA_B receptor is insensitive to agonist-induced internalization but undergoes constitutive ubiquitination at the cell surface followed by internalization and lysosomal degradation. Overexpression of USP14 decreased GABA_B1 receptor ubiquitination, which appears to occur at a postendocytic site, and consequently regulates lysosomal degradation independently of USP14 catalytic activity (Lahaie et al., 2016), suggesting that GPCR deubiquitination occurs at multiple subcellular locations. Indeed, the subcellular localization of DUBs is an important spatial-temporal regulatory mechanism for ubiquitinated proteins, especially for signaling receptors, but remains poorly understood for GPCRs (Coyne and Wing, 2016). Although PAR2 is proteolytically activated like PAR1, ubiquitination of activated PAR2 is mediated by a Casitas B lineage lymphoma, a RING-type E3 ligase, rather than NEDD4 HECT E3 ligase, as has been demonstrated for PAR1 and numerous other GPCRs (Grimsey et al., 2015; Jean-Charles et al., 2016). In addition, agonist-induced ubiquitination of PAR2 is required for lysosomal degradation, unlike PAR1 (Hasdemir et al., 2009). To understand how ubiquitination regulates PAR2 trafficking and signaling, which occurs from the plasma membrane via G proteins and on endosomes via β-arrestins, one study focused on the function of two endosomal DUBs, associated molecule with the Src homology 3 domain of STAM (AMSH) and ubiquitin-specific protease Y (UBPY), also known as USP8. This study showed that deubiquitination of activated PAR2 is mediated by both AMSH and UBPY and occurs in the endocytic pathway. Moreover, perturbation of either AMSH or UBPY function results in accumulation of ubiquitinated PAR2 in endosomes and slowed lysosomal degradation but failed to alter activated PAR2–β-arrestin association or β-arrestin–dependent signaling (Hasdemir et al., 2009). Given the preponderance of ubiquitinated receptors, there is no doubt that DUBs will have important roles in regulating receptor function by modulating the spatial and temporal dynamics of receptor signaling. However, most studies to date have failed to use comprehensive approaches to identify and study the physiologically relevant DUBs that control cellular responses.

1. N-Glycosylation of GPCRs

N-glycosylation is initiated in the ER and occurs cotranslationally by the actions of an oligosaccharide transferase (Fig. 6A). The N-linked glycan structure then undergoes extensive trimming during protein transport from the Golgi to the plasma membrane resulting in significant heterogeneity of the glycan structure. N-glycosylation is one of the most common types of glycosylation, where a complex glycan structure is linked to the amide nitrogen on the side chain of an asparagine (N) residue at the consensus sequence N-X-serine (S)/threonine (T), where X is any amino acid other than proline. N-linked glycan structures are often capped with negatively charged sialic acids. The vast majority of mammalian GPCRs contain at least one N-linked glycosylation consensus sites (N-X-S/T) present in the extracellular N-terminal domain (Wheatley and Hawtin, 1999; Wheatley et al., 2012). GPCRs also often contain N-linked glycosylation N-X-S/T sites in ECL2 (Fig. 6B). PAR1 contains five N-linked glycosylation sites—three in the N-terminus and two in ECL2—and is subjected to extensive glycosylation (Fig. 6C) (Soto and Trejo, 2010). Treatment of cells with tunicamycin, an inhibitor of glycosylation, causes a marked mobility shift of PAR1 to ∼39 kDa, its predicted size based on amino acid sequence (Fig. 6C). Interestingly, continuous agonist stimulation caused degradation of mature PAR1 (Fig. 6C), whereas inhibition of protein synthesis with cycloheximide resulted in loss of nascent forms of PAR1 but not the mature form (Fig. 6C). In-depth analysis of GPCRs using computational methods revealed that the consensus N-X-S/T sequence is present on ECL2 (66%), ECL1 (14%), and ECL3 (20%) (Lanctot et al., 2005). Overall, N-glycosylation of GPCR is abundant and occurs at the extracellular N-terminal domain as well as on ECL2 in most receptors and is capable of performing various GPCR functions.

2. O-Linked Glycosylation of GPCRs.

O-glycosylation is initiated by the transfer a N-acetylgalactosamine (GalNAc) to the hydroxyl group of serine or threonine residues, rarely on tyrosine, and occurs in the Golgi after protein folding (Stanley et al., 2009) (Figs. 6A and 7). This reaction is catalyzed by 20 different GalNAc transferases, which can produce eight different core structures (Mulloy et al., 2015). Elongation occurs by the addition of monosaccharides to yield higher-order linear and branched glycan structures, which are capped with negatively charged sialic acids. Unlike N-linked glycosylation, O-glycosylation occurs at serine or threonine residues, usually in stretches rich in hydroxy amino acids, but there is no consensus sequence. Currently, over 60 GPCRs have detected O-linked glycosylation sites within the N-terminal domain, whereas more than 350 GPCRs have predicted O-glycosylation sites based on the use of the NetOGlyc 4.0 model for prediction of O-glycosylation (Steentoft et al., 2013). The quantity and quality of glycosylation depends both on the GPCR itself and on the cell type expressing the protein. However, validation of these predicted sites on the GPCRs and the regulatory functions of O-glycosylation is only beginning to emerge.

1. GPCR Biosynthesis and Cell Surface Expression

The functional effects of N-glycosylation on GPCRs generally control biosynthesis and cell surface expression (Fig. 7). However, for certain receptors, there seem to be no detectable deficits in receptor function if N-glycosylation is blocked, as has been described for the muscarinic M₂ receptor, H₂ histamine receptor, dopamine D₁ receptor, class A orphan GPR61, α₁-adrenoreceptor, vasopressin V₂ receptor, PTHR, and others (van Koppen and Nathanson, 1990; Fukushima et al., 1995; Kozielewicz et al., 2017), whereas for many other GPCRs, disrupting N-glycosylation or expression of aglycosylated mutant perturbs receptor surface expression, as shown for the β₂-adrenoceptor, angiotensin type 1 (AT₁) receptor, dopamine D₅ receptor, smoothened (SMO), PAR2, GPR176, and others (Karpa et al., 1999; Lanctôt et al., 1999; Compton et al., 2002; Michineau et al., 2004; Marada et al., 2015; Wang et al., 2020). Notably, mutation of all angiotensin AT₁ receptor N-glycosylation sites resulted in loss of plasma membrane expression, where the nonglycosylated receptors accumulated in the ER (Deslauriers et al., 1999). However, preservation of AT₁ receptor Asn¹⁷⁶ in the second extracellular loop enabled surface expression similar to wild-type receptors. A glycosylation-defective gonadotropin-releasing hormone receptor also displayed decreased expression (Davidson et al., 1995). Other studies showed that the final processing for N-glycans for DOR occurs in the trans-Golgi network, whereas O-linked glycosylation is mediated in the trans-Golgi cisternae (Petaja-Repo et al., 2000). Two N-glycosylation sites in the N-terminus of DOR were subsequently shown to enhance transport through the ER but resulted in loss of receptor surface expression due to increased internalization and lysosomal degradation (Markkanen and Petäjä-Repo, 2008). Similarly, the β₂-adrenoceptor N-terminus harbors two N-glycosylation sites that have been implicated in receptor trafficking to cell surface but not in ligand binding or G protein coupling (Rands et al., 1990; He et al., 2002). A more recent report indicates that a mutant purinergic P2Y₂ receptor deficient in glycosylation undergoes ER-associated proteasomal degradation pathway, possibly due to retention in ER lipid rafts and failure of traffic to the Golgi (Nakagawa et al., 2017). A similar observation was made for α_1D-adrenoreceptor, where a glycosylation-deficient mutant displays impaired plasma membrane expression likely because of degradation via ERAD (Janezic et al., 2020). Collectively, substantial evidence suggests that N-glycosylation of GPCRs during maturation in the biosynthetic pathway is essential to achieve optimal cell surface expression.

2. GPCR Plasma Membrane Compartmentalization and Internalization

Once GPCRs reach the cell surface, they can partition into plasma membrane subdomains, including clathrin-coated pits and caveolar microdomains (Guo et al., 2015), which appears to be governed in part by N-glycosylation. The sphingosine 1-phosphate (S1P₁) receptor is modified at an N-terminal site with N-glycosylation, and a mutant receptor lacking glycosylation fails to efficiently partition in caveolin-enriched microdomains (Kohno et al., 2002), suggesting that N-glycans may function in plasma membrane compartmentalization. Another study examining the role of N-glycosylation in trafficking of the dopamine D₂ and D₃ receptor showed that glycosylation regulates not only receptor transit through the biosynthetic pathway but also receptor uptake within microdomains at the plasma membrane (Min et al., 2015). Specifically, glycosylation on the N-terminus was shown to mediate internalization of dopamine D₂ receptor through caveolae, whereas glycosylation of dopamine D3 receptor mediates internalization via clathrin-coated pits, which is regulated through direct interactions with caveolin-1 and clathrin (Min et al., 2015). These new findings support a role for N-glycans in mediating GPCR internalization via clathrin-mediated and caveolae-dependent pathways, the major internalization routes of GPCRs (Guo et al., 2015).

In addition to the dopamine D₂ and D₃ receptor, N-glycan functions have been linked to the chemokine receptor CCR7 internalization (Hauser et al., 2016). Using N-glycosylation prediction software NetNGlyc 4.0, two potential N‐glycosylation sites were identified: one in the N-terminus and one within ECL3 of human CCR7. Surprisingly, only the CCR7 receptor variant with mutation of the N-terminal site had a significantly reduced endocytic rate, whereas the ECL3 mutant variant behaved similar to the wild type (Hauser et al., 2016). S1P₁ receptor containing a mutation in an N-terminal N-glycosylation site also exhibited impaired endocytosis (Kohno et al., 2002). Thus, N-glycosylation of S1P₁ receptor is required not only for association with caveolae but also for agonist-induced internalization (Kohno et al., 2002). In contrast, a glycosylation-deficient NK₁ receptor displayed enhanced internalization after agonist-stimulated compared with wild-type glycosylated NK₁ receptor, suggesting that glycosylation may function to retain NK₁ receptor at the cell surface (Tansky et al., 2007). In many cases, N-glycosylation has multiple purposes in controlling biosynthesis, export to the cell surface, and internalization through clathrin-coated pits. This has been exemplified for PAR1, which is extensively modified by N-linked glycosylation on both the N-terminus and ECL2 (Fig. 6C). Although N-terminal glycosylation was shown to function in export to the cell surface, glycosylation of PAR1 at ECL2 caused a modest impact on agonist-induced internalization, whereas constitutive internalization remained intact (Soto and Trejo, 2010).

1. N-Glycosylation, Ligand Binding, and GPCR Signaling

In early studies of mammalian GPCRs, perturbation of N-glycosylation resulted in either nonfunctional receptors or receptors with severe functional deficits in ligand binding, as was demonstrated for the thyrotropin receptor, parathyroid hormone, shekel somatostatin receptor type 3, and rhodopsin (Russo et al., 1991; Kaushal et al., 1994; Nehring et al., 2000). In the case of PAR1, loss of N-glycosylation at ECL2 caused a marked increase in signaling compared with wild-type receptor (Soto and Trejo, 2010), suggesting that the lack of glycosylation in this region allows the ligand to bind the receptor in a manner that induces an active receptor conformation that is more efficient in coupling to G protein signaling. Glycosylation may hinder stabilization of the active conformation of the loop or may orientate the loop to prevent ligand access to binding pocket. Similarly, glycosylation of the human chemokine receptor CCR7 at the N-terminus and ECL3 reduces responsiveness, where the lack of glycosylation enhances chemokine signaling (Hauser et al., 2016). In contrast, N‐glycosylation of CXCR4 at the N-terminus is necessary for high‐affinity binding of the chemokine ligand (Wang et al., 2004). Constitutive signaling by GPR30, an emerging player in breast cancer and cardiometabolic regulation, is regulated by N-glycosylation. A recent study demonstrated that one of the three N-glycosylation sites in the N-terminus is important for receptor-stimulated ERK1/2 activity (Gonzalez de Valdivia et al., 2019), suggesting that a single site is critical for receptor structure and activity. In addition to diffusible ligands, recent work suggests that GPCRs can act as mechanosensors activated by mechanical stimulus that appears to be governed by N-glycans (Langenhan et al., 2016). In this case, activation of the β₂-adrenoceptor expressed in endothelial cells occurs during infection with the bacteria meningococcus, where the filamentous structures appear to trigger receptor signaling by exerting direct mechanical traction forces via the exposed N-glycans (N⁶ and N¹⁵) present in the N-terminus of β₂-adrenoceptor (Virion et al., 2019). This is the first example of a glycan-dependent mode of allosteric mechanical activation of a GPCR.

2. N-Linked Glycosylation and GPCR Biased Signaling

GPCRs are dynamic molecules that assume different conformational states. Consequently, different ligands can stabilize unique active conformations of the same GPCR and facilitate activation of distinct signaling effectors such as G proteins or β-arrestins (Walker et al., 2003; Kenakin and Christopoulos, 2013). This process is termed biased agonism or functional selectivity. New work suggests that N-glycosylation can control GPCR biased signaling. The first report to show a role for N-glycosylation in controlling biased signaling was demonstrated for PAR1. In this study, PAR1 N-glycosylation at ECL2 was shown to direct differential coupling of PAR1 to Gq versus G12/13 signaling (Soto et al., 2015). A fully glycosylated activated PAR1 wild type displayed greater efficacy at coupling to G12/13-dependent Ras homolog family member A signaling than the glycosylation-deficient mutant. In contrast, activation of PAR1 mutant lacking glycosylation at ECL2 exhibited a greater capacity to elicit Gq signaling compared with G12/13 signaling. Both PAR1 wild type and glycosylation-deficient mutant were equally effective at coupling to Gi signaling and β-arrestin recruitment. These findings suggest that N-glycosylation at ECL2 contributes to the stabilization of an active PAR1 state that preferentially couples to G12/13 versus Gq and defines a previously unappreciated function for N-glycosylation of GPCRs in regulating G protein signaling bias (Soto et al., 2015). Similarly, N-glycosylation of SMO, a GPCR that contains seven predicted glycosylation sites, functions as the signal transducer of the Hedgehog pathway and can bias SMO signaling. SMO signals via a noncanonical pathway mediated by Gαi as well as through a canonical route mediated by Gli transcriptional factors. In this study, an SMO mutant rendered glycosylation deficient via mutation of four N-glycosylation sites failed to induce a noncanonical signal through Gαi, whereas it retained normal receptor trafficking, ligand binding, and canonical Gli signaling (Marada et al., 2015). These studies demonstrate that modification of PAR1 and SMO with N-glycosylation can regulate biased signaling.

1. O-Glycosylation and GPCR Cell Surface Expression

The study of O-glycosylation is challenging; nonetheless, mass spectrometry has been used to detect O-glycans on rhodopsin (Nakagawa et al., 2001) and human opsins (Nakagawa et al., 2001), but in neither case was the function determined. In more recent work, using an improved prediction algorithm for O-glycosylation (NetOGlyc 4.0), five potential O-glycosylated sites were predicted to reside in the N-terminal domain of human DOR, with three sites—S⁶, S²⁵, and S²⁹—experimentally validated and shown to regulate DOR transport to the cell surface (Lackman et al., 2018). This study further identified the enzyme GalNAc-transferase 2, one of 20 GalNAc transferase isoforms expressed in mammalian cells, as the specific regulator of DOR O-glycosylation using a human embryonic kidney (HEK293) cell knockout system (Lackman et al., 2018). However, it should be noted that GPCRs are often modified with both N- and O-glycosylation simultaneously, which may perform different and overlapping functions (Sadeghi and Birnbaumer, 1999; Park et al., 2017; Goth et al., 2018; Salom et al., 2019).

2. O-Glycosylation and GPCR Ligand Binding

Similar to N-glycosylation, modification of the N-terminus of GPCRs with O-glycosylation influences ligand binding. O-linked glycosylation of the DP₂ and EP₂ prostanoid receptors is important for maintaining high-affinity ligand-binding activity (Morii and Watanabe, 1992), as was similarly demonstrated for N-linked glycosylation of the same receptors. The chemokine receptor system controls fundamental biologic processes such as inflammation and cell migration; however, there are far more ligands then receptors, and various ligands bind to multiple receptors; thus, understanding critical features of ligands and receptors that dictate ligand-binding specificity is important. One such determinant that contributes to the specificity of chemokine binding is the post-translational modification of the CCR5 chemokine receptor by the addition of O-linked glycans and tyrosine sulfates (Bannert et al., 2001). These modifications provide not only a larger and potentially more flexible binding surface but also supply an array of negative charges that allow electrostatic interactions with the generally positive receptor-binding interface of the chemokines (Bannert et al., 2001). Although there is no consensus sequence for O-glycosylation, a high prevalence of serine, threonine, and tyrosine residues in the N-terminal domain of chemokine receptors suggests that O-glycosylation may function broadly to modulate chemokine receptor function.

3. O-Glycosylation, GPCR N-Terminal Cleavage, and Signaling

In recent work, O-glycosylation has been implicated in N-terminal cleavage of certain GPCRs (Fig. 7) (Goth et al., 2017, 2018; Park et al., 2017). Interestingly, almost all GPCRs reported to undergo N-terminal cleavage possess identified or predicted O-glycosylation modifications in close proximity to the reported cleavage sites (Goth et al., 2018). Two studies examined the role and function of O-glycosylation on β₁-adrenoreceptor N-terminal cleavage and signaling (Goth et al., 2017; Park et al., 2017). Using in vitro O-glycosylation assays, synthetic peptides representing the β₁-adrenoreceptor N-terminus and recombinantly expressed GalNAc-transferase 2 identified O-glycosylation at several serine residues. Moreover, loss of β₁-adrenoreceptor O-glycosylation in cells, using GalNAc-transferase 2–deficient cells, resulted in a decrease in isoproterenol-induced Gs-mediated cAMP formation (Goth et al., 2017), suggesting that O-glycosylation is important for signaling. In contrast, other work showed that different serine residues of the β₁-adrenoreceptor N-terminus are O-glycosylated in GalNAc-transferase 2–expressing CHO cells (Park et al., 2017). This study also demonstrated that β₁-adrenoreceptor N-terminal cleavage controlled by O-glycosylation functions alters the balance of β₁-adrenoreceptor signaling between the Gs/cAMP and ERK signaling, with a preference for cAMP signaling (Park et al., 2017). N-terminal proteolysis of PAR2 by neutrophil elastase is also inhibited by the presence of O-glycosylation (King et al., 2017), indicating control of proteolytic activation of certain GPCRs. Finally, modulation of CCR7 on immune cells with multiple sialic acids attached to both the N- and O-linked glycans is important for maintaining immune cell responsiveness and immune cell trafficking (Kiermaier et al., 2016) (Figs. 6 and 7). Thus, similar to N-glycosylation, O-glycosylation has important functions in regulating GPCR biology.

1. Enzymology of GPCR Palmitoylation

A family of palmitoyl acyl transferases (PATs) catalyze the attachment of a palmitoyl group to cytosolic cysteine (C) residues and include at least 23 enzymes (Fig. 8, A and C). PATs contain a conserved D-H-H-C (Asp-His-His-Cys) cysteine-rich domain, designated as DHHC 1–23, that mediates the transfer of palmitoyl to substrate proteins (Fukata et al., 2006; Korycka et al., 2012; De and Sadhukhan, 2018). DHHC proteins are localized in the ER and Golgi, and some are targeted to the plasma membrane (Ohno et al., 2006; Petäjä-Repo et al., 2006; Korycka et al., 2012). Several GPCRs have been shown to be palmitoylated by various DHHCs in different subcellular compartments. GPCR palmitoylation in the ER-Golgi has been shown for DOR, β₂-adrenoceptor, PAR2, CCR5, thyrotropin receptor, and vasopressin V_1A receptor, whereas other GPCRs appear to be palmitoylated at the plasma membrane, including DOR and S1P₁ receptor (Petäjä-Repo et al., 2006; Adams et al., 2011; Adachi et al., 2016; Badawy et al., 2017). The substrate specificity of DHHCs is due in part to subcellular localization (Roth et al., 2006; Linder and Deschenes, 2007; Ohno et al., 2012). Palmitoylation is a reversible process, and depalmitoylation of substrate proteins is catalyzed by the action of acyl-protein thioesterase (APT) 1, APT2, and APT1-like and palmitoyl-protein thioesterase-1 and -2 (Fig. 8, A and C) (Linder and Deschenes, 2003). Currently, only APT1 and APT2 have been reported to mediate depalmitoylation of β₂-adrenoceptor and melanocortin MC₁ receptor, respectively (Adachi et al., 2016; Chen et al., 2019).

2. Basal and Agonist-Induced GPCR Palmitoylation

The majority of newly synthesized GPCRs are subjected to palmitoylation basally during biosynthesis. This appears to be the case for PAR1, where the detection of palmitoylation occurs basally and is not further modified after agonist stimulation (Fig. 8B). However, a few GPCRs appear to be palmitoylated after agonist stimulation. This has been demonstrated for the β₂-adrenoceptor, which is basally palmitoylated at C³⁴¹, whereas agonist-induced palmitoylation and depalmitoylation occurs predominantly on C²⁶⁵ (O’Dowd et al., 1989; Adachi et al., 2016). Several other GPCRs undergo agonist-induced palmitoylation and depalmitoylation, including but not limited to the vasopressin V_1A receptor, dopamine D₁ receptor, α_1B-adrenoreceptor, 5-hydroxytryptamine (HT)₄ receptor, S1P₁ receptor, α_2A-adrenoreceptor, and muscarinic M₂ receptor (Ponimaskin et al., 2001; Badawy et al., 2017; Naumenko and Ponimaskin, 2018).

1. GPCR Surface Expression

A role for palmitoylation in regulating GPCR surface expression has been demonstrated for several GPCRs (Fig. 9). Mutation of three C-tail cysteine residues of the chemokine CCR5 receptor resulted in retention largely in the ER and Golgi complex (Blanpain et al., 2001; Percherancier et al., 2001). The authors further showed that the nonpalmitoylated CCR5 mutant displays impaired diffusion properties within the ER. Similarly, defects in palmitoylation caused a marked loss of endogenous PAR2 expression at the cell surface (Adams et al., 2011) as well as diminished surface expression of the thyrotropin receptor, vasopressin V₂ receptor, adenosine A₁ receptor, histamine H₂ receptor, and the dopamine D₁ and D₂ receptors (Schülein et al., 1996; Sadeghi et al., 1997; Tanaka et al., 1998; Fukushima et al., 2001; Ebersole et al., 2015). The mechanism by which defects in palmitoylation diminish GPCR surface expression is attributed mainly to receptor misfolding and proteasomal degradation. This has been shown for deficiencies in palmitoylation of CCR5 and adenosine A₁ receptor, which enhance degradation (Gao et al., 1999; Percherancier et al., 2001). Follicle-stimulating hormone receptor contains three cytosolic cysteine residues; however, mutation of a single C²⁶⁹ was sufficient to impair cell surface expression, likely due to misfolding and degradation (Uribe et al., 2008). These findings are consistent with an important role for palmitoylation in facilitating the proper folding and maturation of GPCRs.

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Fig. 9.

Model of GPCR regulation by palmitoylation. GPCRs are palmitoylated during biosynthesis and can occur at the endoplasmic reticulum (ER), endoplasmic-reticulum–Golgi intermediate compartment (ERGIC), Golgi and the plasma membrane, where DHHC PATs are known to be localized. GPCR palmitoylation regulates partitioning into membrane microdomains enriched in cholesterol such as lipid rafts and caveolae. GPCR palmitoylation has also been implicated in receptor dimerization as well as G protein coupling. Palmitoylation of GPCRs can further influence β-arrestin (β-arr) recruitment and receptor internalization. GPCR palmitoylation is also important for regulating receptor recycling and thereby prevents lysosomal degradation.

2. GPCR Dimerization and Lipid Rafts

GPCRs partition into lipid raft plasma membrane microdomains enriched in cholesterol and is regulated by palmitoylation (Figs. 8C and 9) (Barnett-Norris et al., 2005; Villar et al., 2016). The serotonin 5-HT_1A receptor defective in palmitoylation showed decreased association with lipid rafts (Papoucheva et al., 2004; Renner et al., 2007). Similarly, dopamine D₁ receptor (Tiu et al., 2020) and cannabinoid receptor type 1 (Oddi et al., 2012, 2018) mutants deficient in palmitoylation exhibited impaired lipid raft association. Interestingly, the crystal structure of the human β₂-adrenoceptor revealed a receptor dimer complex, where lipid-mediated contacts via palmitic acid and cholesterol are the major interactions (Cherezov et al., 2007). In addition, palmitoylation of several other GPCRs has been shown to promote lipid raft association and dimerization, including the MOR (Zheng et al., 2012), rhodopsin (Seno and Hayashi, 2017), and the serotonin 5-HT_1A receptor (Kobe et al., 2008). These results indicate that for certain GPCRs, palmitoylation facilitates receptor compartmentalization in lipid rafts and dimerization.

3. GPCR Internalization, Recycling, and Lysosomal Degradation

In addition to GPCR plasma membrane localization, palmitoylation has been shown to regulate GPCR internalization, recycling, and lysosomal degradation (Fig. 9). Several studies have documented a role for palmitoylation in GPCR internalization. Defects in cannabinoid receptor type 1 palmitoylation inhibited agonist-induced internalization and coassociation with caveolin 1 (Oddi et al., 2017). Similarly, defects in palmitoylation of the prostanoid thromboxane A₂ receptor, PAR2, and thyrotropin receptor perturbed agonist-induced β-arrestin recruitment and receptor internalization (Tanaka et al., 1998; Reid and Kinsella, 2007; Adams et al., 2011). In contrast, a vasopressin V_1A receptor mutant deficient in palmitoylation displayed an increased rate of agonist-induced internalization without affecting intracellular signaling (Hawtin et al., 2001). Similarly, a palmitoylation-deficient dopamine D₁ receptor mutant exhibited an enhanced rate of internalization compared with wild type and showed preferentially internalization via a clathrin-dependent pathway over caveolae (Kong et al., 2011).

Once internalized, GPCRs are either recycled back to the cell surface or targeted to lysosomes for degradation. Although palmitoylation of PAR2 is required for efficient internalization and lysosomal degradation (Adams et al., 2011), palmitoylation has an opposing function for PAR1, as a palmitoylation-deficient PAR1 mutant exhibited an enhanced rate of internalization and lysosomal degradation (Canto and Trejo, 2013). The defects in trafficking caused by alteration of PAR1 palmitoylation are due to inappropriate utilization of C-tail tyrosine-based sorting motifs for endocytic adaptor proteins (Canto and Trejo, 2013). In the absence of palmitoylation, PAR1 sorting motifs appear to be more accessible to the clathrin adaptor binding proteins AP-2 and adaptor protein complex 3, which accelerate the rate of internalization from the plasma membrane as well as enhanced sorting from endosomes to lysosomes and degradation (Canto and Trejo, 2013). Similarly, a CCR5 palmitoylation mutant exhibits rapid lysosomal degradation and a reduced half-life (Percherancier et al., 2001).