Minireview: Ubiquitination-regulated G Protein-Coupled Receptor Signaling and Trafficking

Abstract

G protein-coupled receptors (GPCRs) are the largest and most diverse superfamily of membrane proteins and mediate most cellular responses to hormones and neurotransmitters. Posttranslational modifications are considered the main regulators of all GPCRs. In addition to phosphorylation, glycosylation, and palmitoylation, increasing evidence as reviewed here reveals that ubiquitination also regulates the magnitude and temporospatial aspects of GPCR signaling. Posttranslational protein modification by ubiquitin is a key molecular mechanism governing proteins degradation. Ubiquitination mediates the covalent conjugation of ubiquitin, a highly conserved polypeptide of 76 amino acids, to protein substrates. This process is catalyzed by 3 enzymes acting in tandem: an E1, ubiquitin-activating enzyme; an E2, ubiquitin-carrying enzyme; and an E3, ubiquitin ligase. Ubiquitination is counteracted by deubiquitinating enzymes that deconjugate ubiquitin-modified proteins and rescue the substrate from proteasomal degradation. Although ubiquitination is known to target many GPCRs for lysosomal or proteasomal degradation, emerging findings define novel roles for the basal status of ubiquitination and for rapid deubiquitination and transubiquitination controlling cell surface expression and cellular responsiveness of some GPCRs. In this review, we highlight the classical and novel roles of ubiquitin in the regulation of GPCR function, signaling, and trafficking.

G protein-coupled receptors (GPCRs) are the largest superfamily of membrane proteins and mediate most cellular responses to hormones and neurotransmitters. In fact, GPCRs are a major target for drug discovery by virtue of their role in regulating a broad range of physiological responses and pathological conditions including cardiovascular and neurological disorders, immunological, endocrine, renal and pulmonary diseases, pain, and cancer (1). In vertebrates, GPCRs are now divided into 4 classes based on their sequence and primary structural similarity: rhodopsin (class A), secretin (class B), glutamate (class C), and frizzled (class 4) (2). GPCRs are among the essential nodes of communication between the internal and external environments of cells, transducing the information provided by stimuli into intracellular second messengers. On activation, GPCRs undergo conformational changes that facilitate activation of heterotrimeric guanine nucleotide binding proteins (G proteins) and also set in place a series of molecular interactions that allow for the following: 1) feedback regulation of G protein coupling; 2) receptor endocytosis; and 3) signaling through G protein-independent signal transduction pathways (3–9).

Ligand-stimulated GPCR activation, desensitization, internalization, and recycling proceed in a cyclical manner. This behavior provides a mechanism to protect cells against excessive stimulation, on the one hand, and guards cells against prolonged desensitization and hormone insensitivity on the other hand. Thus, the magnitude and duration of ligand-induced GPCR responses are tightly linked to the balance between signal generation and signal termination. The endocytic pathway tightly controls the activity of GPCRs. In most described situations this leads to terminating GPCR signaling. However, emerging information now reveals a new paradigm, whereby GPCR endocytosis promotes persistent signaling (10–15) and prolonged function (16).

Agonist-stimulated endocytosis can drive receptors toward divergent itineraries, including degradative routes that lead to GPCR down-regulation, and recycling pathways that promote receptor resensitization (17). Phosphorylation of the receptor and subsequent binding of β-arrestin prevent consequent interaction of receptors with G proteins, effectively terminating the G protein-mediated signal and initiating the endocytic process. GPCR phosphorylation occurs predominantly on Ser and Thr residues within the carboxyl receptor tail and the third intracellular loop. Agonist-activated GPCRs are rapidly phosphorylated by G protein-coupled receptor kinases (GRKs) (18) with ensuing translocation of arrestins to the plasma membrane (19). A conformational change in arrestin is induced on binding to GPCRs, exposing the carboxy-terminal domain, which interacts with clathrin and the β2-adaptin subunit of the clathrin adaptor adaptor protein complex-2, components of the endocytic machinery, thereby promoting endocytosis of arrestin-bound receptors (20–22). The endocytic activity of arrestin is also subject to dynamic regulation by dephosphorylation and ubiquitination as we discuss here.

Subsequent steps of endocytic sorting play a critical role in dictating the receptor signaling response after the initial desensitization event. The sorting of internalized receptors between lysosomal and recycling pathways produces essentially opposite effects on cell signaling. Endocytic trafficking to lysosomes represents a major mechanism by which many GPCRs and various other signaling receptors are down-regulated following ligand-induced activation (23–26). Some GPCRs are driven along an alternate route that facilitates the efficient recycling of plasma membrane receptors after ligand-induced endocytosis. The recycling pathway can promote rapid recovery (or resensitization) of cellular responsiveness (27–29). The molecular mechanisms that regulate trafficking of mammalian GPCRs are essential to receptor signaling, desensitization, and resensitization yet are incompletely understood. Posttranslational modifications are considered the primary regulator of all GPCRs. In addition to phosphorylation, glycosylation, and palmitoylation (30), emerging evidence increasingly identifies a role for posttranslational modification by ubiquitin that regulates the magnitude, duration, and spatial components of GPCR signaling. In this review, we discuss the classical and novel roles of ubiquitin in the regulation of GPCR function, signaling, and trafficking.

Posttranslational Modifications: Ubiquitination

Ubiquitination is the enzymatic posttranslational modification process that mediates the covalent conjugation of ubiquitin, a highly conserved polypeptide of 76 amino acids, to protein substrates (31). This reaction is catalyzed by 3 enzymes acting in tandem. First, ubiquitin is activated by an ubiquitin-activating enzyme (known also as E1) in an ATP-dependent process. The initial step involves production of a ubiquitin-adenylate intermediate, followed by the transfer of ubiquitin to the E1 active site cysteine residue and release of AMP. This step results in a thiol-ester linkage between the C-terminal carboxyl glycine of ubiquitin and the E1 cysteine sulfhydryl group. Second, a ubiquitin-conjugating enzyme or ubiquitin-carrier enzyme (UBCs or E2) accepts ubiquitin from E1 by a transthiolation reaction, again involving the carboxyl terminus of ubiquitin. Finally, an ubiquitin protein ligase (E3) transfers ubiquitin from the E2 enzyme to the ϵ-amino group of a lysine residue on the substrate (32, 33). Initially it was thought that each ubiquitin moiety attaches to a single internal Lys on the target protein. Later, Hershko (34) and then Varshavsky (35) and their respective colleagues demonstrated the formation of a polyubiquitin chain anchored to a single internal Lys residue (reviewed in Ref. 36) (Figure 1).

Figure 1.

The posttranslational modification by ubiquitin is carried out by a set of 3 enzymes, E1, E2, and E3. Ubiquitin is first activated by ubiquitin-activating enzyme E1 and transferred to its active site. This transfer requires ATP. The ubiquitin molecule is then passed on to the second enzyme of the complex, E2 (ubiquitin-conjugating enzyme), before reaching the final enzyme, E3, the ubiquitin protein ligase, which recognizes, binds the target substrate, and labels it with the ubiquitin. The process can be repeated until a short chain is formed. PPi, pyrophosphate; SH, sulfydryl; UB, ubiquitin.

E3 ubiquitin ligases and GPCRs

The human genome encodes 2 ubiquitin activating enzymes (E1), at least 38 ubiquitin-conjugating enzymes (E2), and 600 to 1000 ubiquitin protein ligases (E3) (37–39). Ubiquitination is a highly specific process at all levels. E1s select the correct ubiquitin-like protein for their respective pathways and display remarkable specificity. For example, despite the fact that ubiquitin and NEDD8 (ubiquitin-like protein) share 57% sequence identity and have markedly similar structures, NEDD8 is poorly employed by the ubiquitin conjugation system (40). This specificity is obvious because E1 also transfers the activated ubiquitin-like protein to its cognate E2, thereby coupling the ubiquitin-like protein to its cognate downstream pathway (33). E2 and E3 work in a combinatorial manner to generate different forms of substrate modification (32, 41). E3 ligases recognize the substrate protein and impart specificity to the reaction (38, 42). Many E2s have the capacity to determine ubiquitin chain topology. This probably requires a noncovalent interaction between E2 and the acceptor ubiquitin, which exposes a specific Lys on the acceptor ubiquitin to the active site of the E2 charged with the donor ubiquitin. Depending on the recognized surface of ubiquitin, chains of different linkage are assembled and the modified proteins will be assigned distinct fates (39). Based on the sequence homology of their E2-binding domains, there are 3 major types of E3s in eukaryotes, defined by the presence of either a homologous to the E6-AP carboxyl terminus (HECT), a really interesting new gene (RING) (38), or a U-box domain (43). To date, few E3 ligases attaching ubiquitin to specific GPCR have been identified. Nedd4 and especially Nedd4-2, HECT domain-containing ligases, are well known for their role in ubiquitinating ion channels, such as the amiloride-sensitive ENaC, and nonmembrane substrates mostly via direct interaction with the substrate. The substrate's specificity of the HECT-type E3 is dictated by protein-protein interaction domains (43). It is now known that Nedd4 ubiquitinates the β₂-adrenergic receptor (B2AR) and regulates its lysosomal trafficking (44). However, B2AR ubiquitination by Nedd4 is effective only when β-arrestin2 is present as an adaptor, suggesting that β-arrestin2 binds at least 2 E3 ubiquitin ligases: Mdm2 (a RING E3 ubiquitin ligase) and Nedd4. These findings show dissimilar function in B2AR regulation: Mdm2 mediates β-arrestin ubiquitination (45), which defines the initial step in receptor endocytosis, whereas Nedd4 mediates receptor ubiquitination that targets the B2AR to lysosomal compartments (44). Other RING E3 ubiquitin ligases involved in GPCR ubiquitination include c-Cbl and AIP4. c-Cbl mediates ubiquitination, degradation, and down-regulation of the human protease-activated receptor (PAR) 2, while AIP4 is able to bind ubiquitin to C-X-C chemokine receptor-4 (CXCR4) (46) and δ-opioid receptors (DOR) (47). Interestingly, Hislop and colleagues uncovered a distinct function of AIP4-mediated ubiquitination in regulating the proteolytic processing of DOR without affecting its endocytic sorting to lysosomes (47).

Recently, the HECT E3 ubiquitin ligase WWP2 was identified as a critical regulator of sphingosine-1-phosphate receptor (S1PR) (lysophospholipid receptor) degradation induced by the inhibitor FTY720P (48). In the same way, RING E3 ubiquitin ligases and their role in the regulation of several GPCRs have been identified. Dorfin, for instance, mediates calcium-sensing receptor ubiquitination and degradation (49). Seven-in-absentia homolog 1A mediates ubiquitination and degradation of group 1 metabotropic glutamate receptors (50), cullin3 with BTB Protein KLHL12 as adaptor targets the dopamine D4 receptor for ubiquitination (51), and mahogunin ubiquitinates the melanocortin 2 receptor (52).

Typical and atypical ubiquitin chains

Ubiquitin can be attached to proteins as a single unit on one or multiple sites, yielding monoubiquitinated and multimonoubiquitinated proteins, respectively (Figure 2). Ubiquitin contains 7 Lys residues (Lys⁶, Lys¹¹, Lys²⁷, Lys²⁹, Lys³³, Lys⁴⁸, or Lys⁶³) and an amino-terminal Met (Met¹) that can serve as acceptor sites for additional ubiquitin molecules generating polyubiquitinated proteins. Thus, ubiquitin chains can be connected by at least 8 different homotypic linkages, as well as by a range of atypical chains (heterologous, forked, or mixed) (39, 53–55) (Figure 2). However, the most common polyubiquitin chains are formed through linkages at K-48 and K-63. Extensive studies on Lys⁴⁸-linked and Lys⁶³-linked chains established essential roles in proteasomal degradation for Lys⁴⁸ and in cell signaling for Lys⁶³-linked chains and monoubiquitin conjugation. In comparison, rather little is known about the remaining atypical chain types (56). More recent reports show that Lys⁶³-linked chains can drive degradation, whereas Lys⁴⁸-linked chains may function nonproteolytically, for example, in the inactivation of the transcriptional activator Met-4 (57). This suggests that the functions of ubiquitination depend not only on chain topology but also on other factors such as the timing and reversibility of the reaction, enzyme or substrate localization, or interactions between E3s and effectors. Notably, ubiquitin can also link to the amino group in the N-terminal methionine (Met¹) of the acceptor ubiquitin, leading to the formation of linear ubiquitin chains (58). It is clear that all ubiquitin linkage types coexist in all cell types analyzed to date (56). The different linkage types are classified as having either “compact” or “open” conformations. In the compact conformation of Lys⁶-linked, Lys¹¹-linked, and Lys⁴⁸-linked chains, the distal and the proximal ubiquitin moieties form an intramolecular interface, whereas in the open conformation of Lys⁶³-linked and Met¹-linked polymers, the only contact is the linkage point. Such conformation diversity may explain how proteins and enzymes discriminate between different linkage types (56). However, ubiquitination is a complex process and multiple acceptor sites could be ubiquitinated. Interestingly, the anchor of ubiquitin to Met1 facilitates the formation of Lys⁴⁸ polyubiquitin chains in MyoD, a tissue-specific transcriptional activator (59). Met1, the acceptor site of the ubiquitin, generates a head-to-tail, or linear, interubiquitin linkage. The ubiquitin linkage in this specific acceptor site is crucial in various pathways. Furthermore, recent data suggest that physiological signaling requires cooperation between different ubiquitin linkage types (60). Lys⁶³-linked or Lys⁴⁸-linked polyubiquitin chains can be discriminated by different experimental techniques, using K63R and K48R mutants of ubiquitin, or specific Lys⁶³ and Lys⁴⁸ ubiquitin chain antibodies. These approaches were employed to determinate different linkage types and showed that Lys⁶³-linked polyubiquitins are the dominant form in ubiquitinated human κ-opioid receptor (KOR) on agonist stimulation (61), and Lys⁴⁸-linked chains were detected in the parathyroid hormone receptor (PTHR) on agonist and antagonist stimulation (62).

Figure 2.

Typical and atypical ubiquitin chains: ubiquitin can be attached to proteins as a single unit on one or multiple sites, yielding monoubiquitinated and multimonoubiquitinated proteins, respectively. Ubiquitin contains 7 Lys residues (Lys⁶, Lys¹¹, Lys²⁷, Lys²⁹, Lys³³, Lys⁴⁸, or Lys⁶³) that can serve as acceptor sites for additional ubiquitin molecules generating poly-ubiquitinated proteins with distinct types of ubiquitin conjugation. The scheme shows the most representative ubiquitin chains.

In some instances, efficient protein polyubiquitination requires additional conjugation factors, so-called E4 enzymes, that support the elongation of ubiquitin chains (53, 63, 64). Although lysines are the most frequent substrate sites for attachment of ubiquitin and ubiquitin-like proteins in the case of GPCRs, conjugation can sometimes occur on other residues such as the free α-NH₂ group of an N-terminal residue of a protein (65, 66). There are only a limited number of studies identifying the specific target sites of GPCR ubiquitination. It has been established that mutation of the 3 carboxyl tail lysines abrogated chemokine receptor CXCR4 ubiquitination and lysosomal sorting but did not affect receptor endocytosis (67). In the same way, the lysines residues (Lys³³⁸, Lys³⁴⁹, and Lys³⁷⁸) in the carboxy-terminal domain of the human KOR, and lysines residues (Lys⁴²¹ and Lys⁴²²) in the carboxyl tail of the PAR1, are the sites of receptor ubiquitination. However, the carboxyl tail of receptors is not the only domain where GPCRs may be ubiquitinated. Mutation of a single lysine, located in the third intracellular loop of the vasopressin V2 receptor (V2R), abrogated its ubiquitination and degradation (68), and ubiquitin modification in μ-opioid receptors (MOR) was identified in the first cytoplasmic loop as being the critical location for ubiquitin-dependent control of MOR down-regulation (69). In the case of the B2AR, the lysines in its carboxyl tail were reported to be the main sites of protein ubiquitination (70). However, mutation of these lysines did not eliminate receptor ubiquitination. Recently, studies based on both molecular and proteomic approaches (mass spectrometry) show that ubiquitination of the B2AR occurs on both the third intracellular loop and the carboxyl tail (71).

Ubiquitin-binding Domain Proteins: Chaperones for Trafficking Ubiquitinated GPCRs

Ubiquitination through alternative lysine sites is now considered a protein-modification code. Similar to the detection of phosphorylated proteins via phospho-binding domains recognizing phosphorylated Ser, Thr, or Tyr, ubiquitin modification of target proteins is recognized by a variety of ubiquitin-binding domain (UBD) -containing proteins or ubiquitin receptors that comprise at least one UBD within their structure. Effector proteins with UBDs translate the modifications into specific outcomes (72). The number of identified UBDs is growing, with more than 20 different families identified to date. Most of the UBDs fold into α-helical-based structures, including the ubiquitin-associated domain (UBA), ubiquitin interacting motif (UIM), DIUM (double-sided ubiquitin-interacting motif), MIU (motif interacting with ubiquitin), CUE (coupling of ubiquitin conjugation to ER degradation), and GAT (Golgi-associated γ-adaptin homologous and target of myb [TOM]) domains. Nonhelical UBDs are also frequent and include different ubiquitin-binding zinc fingers. Although structurally quite different, UBDs share a feature of noncovalent binding to the ubiquitin moiety formed by the hydrophobic patch including and surrounding Ile⁴⁴ (73). The UBDs interact with monoubiquitinated or polyubiquitinated chains to recruit the ubiquitinated protein into cellular signaling networks. Some UBDs display selectivity toward different types of ubiquitin conjugations and function in multiple cellular processes, including protein degradation by proteasomes, protein trafficking to lysosomes, DNA repair, cell-cycle progression, autophagy, and gene transcription (72). On the basis of the apparent correlation between linkage specificity and cellular functionality, it has been proposed that different ubiquitin chain conformations can be recognized by distinct UBDs. However, in vitro experiments have been unable to identify selectivity of 30 UBA domains toward any type of linkage (74). The sorting of most ubiquitinated receptors from endosomes to lysosomes occurs via interaction with endosomal-sorting-complex-required-for-transport (ESCRT) proteins. The ESCRT machinery comprises 4 distinct ESCRT complexes that function sequentially to coordinate the sorting of ubiquitinated receptors into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs) (75). ESCRT-dependent sorting of ubiquitin-modified receptors are recruited into an early endosomal subdomain enriched in hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component, also known as ESCRT-0, and clathrin (76). HRS interacts directly with polyubiquitinated receptors, monoubiquitinated receptors (23), and ESCRT-I, which also bind to ubiquitin and ESCRT-II. The ESCRT-III complex is then recruited, assembles on endosomal membranes, and facilitates ILV scission (77). Several ubiquitinated GPCRs, including the chemokine receptor CXCR4 (46), ocular albinism type 1 receptor (78), and PAR2 (79), are sorted by this mechanism. Furthermore, degradation of the DOR requires HRS and Vps4 functions (80). However, not all GPCRs require direct ubiquitination for MVB sorting and lysosomal degradation (81). For example, a ubiquitination-deficient mutant of DOR and the calcitonin-like receptor, a GPCR that is not ubiquitinated, traffic to MVBs/lysosomes like wild-type receptors (80, 82). Therefore, it remains to be determined whether a signaling receptor can bypass the requirement for both ubiquitination and ubiquitin-binding components of the ESCRT machinery and sort to MVBs/lysosomes (83). The association of other UBD-containing proteins with GPCRs has been studied. The Integrin-Associated Protein IAP (CD47) to cytoskeleton (PLIC-2), also called ubiquitilin 2, has been identified as a negative regulator of GPCR/arrestin clustering in clathrin-coated pits (CCPs). The PLIC proteins contain an amino-terminal ubiquitin-like domain and a carboxy-terminal UBA domain. PLIC-2 inhibited clustering of GPCR-arrestin complexes in CCPs, mediated by its ubiquitin-like domain and delayed endocytosis of the V2R and B2AR, but not of other membrane cargo like the epidermal growth factor receptor or transferrin receptor that are tyrosine-kinase receptors (84). It has been suggested that PLIC-2 could mediate this function by regulating the availability of a UIM-containing protein, such as Eps15 and/or epsin, that facilitates cargo recruitment into CCPs. Studies in yeast of UPDs epsin and Ede1 during receptor internalization showed that epsin and Ede1 ubiquitin-binding is dispensable for internalization of the GPCR Ste2, which uses ubiquitin internalization signals. However, variants of Ste2 carrying both ubiquitin-dependent and ubiquitin-independent internalization signals (Figure 3) were equally reliant on the epsin UIMs, indicating that UIM function is not restricted to ubiquitinated receptors (85). Conversely, recent studies in HEK293 cells show that ubiquitination of PAR1 and the UIM of epsin-1 are required for epsin1-dependent internalization of activated PAR1 (86). Furthermore, activation of PAR1 promotes epsin-1 deubiquitination, suggesting that epsin-1 deubiquitination may increase its endocytic activity toward ubiquitinated PAR1 to facilitate internalization.

Figure 3.

GPCR ubiquitin-dependent and ubiquitin-independent MVB sorting: ubiquitin targets agonist-activated GPCRs to lysosomes for degradation via the highly conserved ESCRT machinery. However, several GPCRs that are not ubiquitinated have been shown to sort to lysosomes independent of ubiquitination. The adaptor protein ALIX links the GPCR to ESCRT III and facilitates sorting to MVB/lysosomes. Ub, ubiquitin.

Ubiquitin controls the endocytic pathway and serves as an internalization signal

The plasma membrane abundance of GPCRs results from the balance between 2 pathways: one that delivers properly folded receptors to the cell surface, and the other that removes receptors by endocytosis, either temporarily (by internalization followed by recycling) or permanently (by internalization followed by subsequent degradation). It has recently been reported that the covalent modification of GPCRs by ubiquitin plays an important role on these processes.

Initial studies on yeast showed that GPCR internalization is mediated by direct ubiquitination (87, 88). A single ubiquitin moiety binding to Ste2 was sufficient to induce receptor internalization (89). Interestingly, ubiquitination of the cytoplasmic residues is not required for angiotensin 1 receptor internalization, in contrast to results obtained in yeast. These results suggest that endocytosis of mammalian angiotensin 1 receptor occurs by a different mechanism in mammals than in yeast (90).

However, the attachment of Lys⁶³-linked ubiquitin chains facilitates endocytosis of many integral membrane proteins (91). Wolfe and colleagues revealed for the first time a direct and novel function for ubiquitination in internalization of a mammalian GPCR (92). They demonstrated that lysine-mutant PAR1, which is defective in ubiquitination, exhibited increased constitutive (ligand-independent) endocytosis. These results suggested that PAR1 is basally ubiquitinated and deubiquitinated after activation, and that ubiquitination negatively regulates PAR1 constitutive internalization. Recent studies with similar mutations of CXCR4, B2AR, and V2R demonstrate that mammalian GPCR ubiquitination is not required for receptor internalization (45, 67, 68).

Role of ubiquitin in the lysosomal sorting

As described above, abrogation of ubiquitination does not affect agonist-induced receptor internalization, but substantially increases receptor half-life (45, 67, 68). Although ubiquitination was originally characterized by its role in protein degradation carried out by the multiprotease complex, 26S proteasome (non-lysosomal-dependent), a nontraditional function of ubiquitin, especially in lysosomal degradation of membrane receptors, has been reported recently. CXCR4 provides a clear example of ubiquitin-dependent lysosomal sorting of a mammalian GPCR. A CXCR4 mutant defective in ubiquitination internalized at a normal rate but was not degraded in lysosomes (67). In the same way, it was observed that lysosomal trafficking correlated with B2AR ubiquitination. Agonist-stimulated B2AR sorted to late endosomes/lysosomes (71). Mutation of all lysines to arginines (zero-lysine β2-adrenergic receptor, 0K-B2AR) prevented the covalent attachment of ubiquitin, as well as receptor degradation (45). Only a negligible portion of the internalized 0K-B2AR localized to lysosomes, whereas most of the 0K-B2AR recycled to the plasma membrane (71).

Other well-studied GPCRs require agonist-dependent ubiquitination for subsequent steps of intracellular receptor trafficking involving proper receptor sorting to lysosomes. Both PAR2 and the neurokinin-1 receptor are ubiquitinated in response to chronic stimulation with the agonist and alterations in the correct ubiquitination prevent lysosomal trafficking and degradation (93, 94). Even neurokinin-1 receptor recycled to the plasma membrane suggests that receptors lacking ubiquitin tags preferentially traffic through recycling vesicles (94). Interestingly, ligand-stimulated lysosomal degradation of the platelet-activating factor receptor was reported to be ubiquitin-dependent, although receptor ubiquitination itself was not completely agonist dependent (95). Thus, ubiquitin is best known to target agonist-activated GPCRs to lysosomes for degradation via the highly conserved ESCRT machinery Figure 3. Nevertheless, as discussed earlier, a ubiquitination-deficient mutant of the murine DOR and the calcitonin-like receptor that is not ubiquitinated sort to lysosomes independent of ubiquitination. Surprisingly, these receptors require a ubiquitin-binding component of the ESCRT machinery, suggesting that not all GPCRs demand direct ubiquitination for interaction with the canonical ESCRT pathway. Conversely, PAR1 is ubiquitinated after agonist stimulation but is efficiently sorted from endosomes to MVBs/lysosomes and degraded independent of ubiquitination and the ubiquitin-binding ESCRT-0 and ESCRT-I proteins, HRS and Tsg101 (77, 92, 96). Further studies showed that PAR1 sorted to ILVs of MVBs through a pathway involving ESCRT-III components, which do not contain any known UBD (Figure 3). It was demonstrated that ALIX, an ESCRT-III-interacting protein, bound to a YPX₃L motif within the second intracellular loop of PAR1, mediated ubiquitin-independent ESCRT-III/lysosomal sorting (83). In addition, 7 other mammalian GPCRs, such as α_1B adrenergic receptor, angiotensin AT2, galanin (GAL2) histamine (H2), neuropeptide FF (NPFF2), neuropeptide S (NPS receptor), and P2Y1 purinergic receptors (P2YR), contain conserved YPX₃L motifs within their second intracellular loop, raising the possibility that ALIX mediates lysosomal sorting without ubiquitin-protein modification, expanding the diversity of mechanism that control trafficking of mammalian GPCRs (76).

Proteolytic functions of the ubiquitin modification: regulation of proteasomal degradation

The process of ubiquitin targeting to proteasomes for degradation of cytosolic proteins has been well studied. The 26S proteasome is a 2-component complex consisting of a 20S proteolytic core complex and two 19S regulatory complexes. The proteasome is able to cleave basic, acidic, and hydrophobic amino acids within proteins. The polyubiquitinated proteins are recognized by the 19S subunits. The ubiquitin units are recycled through the action of ubiquitin hydrolases, and the targeted protein substrates are degraded by the 20S catalytic core complex. A ubiquitin chain of at least 4 units in length is required for recognition (97). Lys⁴⁸-linked chains are the most abundant linkage when the proteasome is inhibited, so their role in proteasomal targeting was first assigned to Lys⁴⁸-linked chains. However, early experiments established that other linkages could be recognized by the proteasome.

Three types of opioid receptor, δ, μ, κ, have been cloned and characterized extensively (98–101). Li and associates showed that Lys⁶³-linked polyubiquitination down-regulated the human KOR (hKOR) (61, 102), the demonstration of GPCR ubiquitination. They found that agonist-promoted Lys⁶³-linked polyubiquitin was the dominant form of hKOR ubiquitination. Ubiquitin modification occurs after receptor phosphorylation but before internalization. Interestingly, agonist-triggered ubiquitination plays a facultative regulatory, but not obligatory, role in agonist-induced down-regulation because a lysine-deficient hKOR mutant still underwent down-regulation (102). Previously, it was reported that agonist-induced down-regulation of the hKOR was partially reduced by the proteasome inhibitor MG132 or the lysosome inhibitor chloroquine and virtually abolished by the combination of the two. These data indicate that lysosomes as well as proteasomes are involved in agonist-induced down-regulation of the hKOR (102). Studies of MOR and DOR opioid receptors using 4 different proteasome inhibitors confirmed the essential role of the proteasome in MOR and DOR basal turnover and agonist-induced down-regulation (104). This role of ubiquitin-dependent degradation leading to basal turnover of cell surface receptors via the proteasomal pathway has also been described for metabotropic glutamate receptors (mGlurR1 and mGluR5) (50) and the human follitropin receptor (105). Other GPCRs, such as rhodopsin and components of GPCR signaling including transducin, rhodopsin G protein, and G protein-coupled receptor kinase 2, are degraded by the ubiquitin/proteasome pathway (106, 107).

More recently it has been recognized that ubiquitination of GPCRs is involved in ER-associated protein degradation (ERAD) (104). In most such cases, ubiquitination contributes to quality control (QC) exerted in the ER by clearing newly synthesized but misfolded receptors. During biogenesis of GPCRs and other transmembrane proteins at the ER, proteins are folded and adopt distinct conformations, which are guided by interactions with chaperones before export to the Golgi for further processing. Misfolded or incompletely folded receptors like DOR, MOR, calcium-sensing receptor, and thyrotropin-releasing hormone receptor are retrotranslocated to the cytosolic side of the ER membrane, conjugated with ubiquitin, and degraded by the proteasome (49, 108, 109). ER-proteasome regulation could be demonstrated in both heterologous and physiologically relevant cell types, suggesting that proteasomes may play an important role in QC during biosynthesis and functions to sort misfolded receptors to the ERAD pathway, ensuring membrane delivery of only properly folded receptors. The relevance of this process was revealed after the discovery of naturally occurring point mutations in rhodopsin, V2R, and gonadotropin hormone-releasing hormone receptors that cause misfolding and loss of cell surface expression and contribute to disease progression (110). Pharmacological agents that bind specifically to misfolded gonadotropin-releasing hormone receptor have been discovered and by inducing proper receptor folding they are able to rescue defects in sorting at the ER, thereby resulting in increased cell surface expression (111). In addition to ubiquitination, deubiquitination of GPCRs during biosynthesis regulates ER QC and increases receptor surface. The A_2A adenosine receptor (A2AR) binds directly to ubiquitin-specific protease 4 (USP4), a deubiquitinating enzyme, which results in enhanced cell surface expression of functionally active receptor (112).

GPCR Transubiquitination and Role of Ubiquitination on GPCR-associated Proteins

GPCRs are known to transactivate growth factor tyrosine kinase receptors, such as the epidermal growth factor receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, by releasing membrane-anchored ligands or through modulation of receptor cytoplasmic domains (113), leading to both Ras-dependent MAPK activation and stimulation of PI3K (114). Recently, it has been observed that signaling by GPCRs can promote transubiquitination of other GPCRs, which can change their cell and tissue responses. Transubiquitination was first described for the angiotensin-II type 1 receptor (AT₁R) in response to dopamine D₅ receptor (D₅R) activation (115). Dopamine and angiotensin-II have counterregulatory effects on cellular signal transduction, production of reactive oxygen species, renal sodium excretion, and blood pressure, where AT₁R signaling is prohypertensive and D₅R signaling is antihypertensive. Li et al found that activation of the D₅R decreased AT₁R protein abundance by promoting AT₁R degradation in proteasomes via an ubiquitin/proteasome pathway, suggesting a possible mechanism for the regulation of both receptors and therefore their counterregulatory effects.

GPCRs can form functional homodimers or heterodimers. The regulation and trafficking of dimerized GPCRs after activation of one receptor protomers are of considerable interest. A recent study showed that, depending on the activating ligand, internalized DOR and MOR heterodimers are either recycled or sorted to lysosomes for degradation (116). The stimulation with a DOR-specific agonist induces ubiquitination but not phosphorylation of MOR, causing degradation of both DOR and the nonstimulated MOR. Disruption of the heterodimers rescued MOR cell surface expression and increased the sensitivity to opiate agonists. These results underscore the potential importance of heterodimer-mediated GPCR transubiquitination on therapies that depend on trafficking of GPCRs. Further, in addition to transubiquitination between GPCRs, recent studies suggest that different receptors and antagonists can stimulate GPCR transubiquitination and degradation. For example, the orexin receptor OX₂, which participates in regulating the sleep/wake cycle, is ubiquitinated and degraded in response to signaling by the proinflammatory cytokine TNF-α (117). Similarly, the S1PR inhibitor FTY720 (also known as fingolimod) induces phosphorylation of the carboxy-terminal domain of S1P1 receptor at multiple sites, promoting receptor internalization, polyubiquitination, and degradation by proteasomes. The ubiquitin E3 ligase WWP2 was also identified as a critical requirement for polyubiquitination of the S1PR carboxy-terminal domain induced by FTY720 (48).

The discussion thus far focused on the following 3 modes of GPCR ubiquitination: 1) constitutive receptor ubiquitination; 2) rapid, direct agonist-induced GPCR ubiquitination followed by proteasomal degradation; and 3) transubiquitination. In addition to these modes of regulation, ubiquitin modification of GPCR-associated proteins and GPCR-initiated signaling pathways provides another mechanism by which ubiquitination regulates GPCR trafficking and function. As noted above, internalization of mammalian GPCRs does not require ubiquitin posttranslational modification, in contrast to the paradigm in yeast. Shenoy and coworkers discovered that agonist-stimulated B2AR internalization required polyubiquitination of β-arrestin2 and MDM2, an E3 ubiquitin ligase that mediates β-arrestin2 ubiquitination (45). This ubiquitination appeared crucial for the proper function of arrestins (44, 118–120). β-Arrestins regulate the fate of the receptor by acting on the balance between receptor recycling to the plasma membrane or its lysosomal degradation. Interestingly, β-arrestin is able to recruit ubiquitin ligases and promote receptor ubiquitination, acting as adaptor proteins. In this case, β-arrestin2 interacts with the ubiquitin ligase Nedd4, which promotes B2AR ubiquitination at endosomes, leading to its lysosomal targeting (44). Ubiquitin ligases of the Need4 family harbor WW domains that can interact with specific proline-rich motifs. Surprisingly, no such PPxY motif has been identified in β-arrestin, and polyproline regions are not involved in Nedd4 interactions. In addition, Nedd4 recruitment to the B2AR was not affected by mutations in Nedd4 WW domains, indicating that the interaction between Nedd4 and β-arrestin involves noncanonical binding (121). Another notable feature of β-arrestin ubiquitination is the distinct kinetics that correspond to a particular receptor type (122). Although stimulation of the B2AR leads to transient β-arrestin ubiquitination, stimulation of the AT1aR angiotensin receptor results in a relatively sustained β-arrestin ubiquitination (123). An additional layer of complexity emerges from observations that β-arrestin interacts with deubiquitinase enzymes that regulate their ubiquitination status as well as receptor ubiquitination. In fact, USP33 (a deubiquitinase enzyme) was first identified as a β-arrestin interactant (120), suggesting that β-arrestin2 could be involved in USP33 recruitment to the B2AR. However, Berthouze and coworkers demonstrated that USP33 is transferred from agonist-activated B2AR to β-arrestin2, triggering its deubiquitination and dissociation from the receptor, once internalized (124). These data suggest that the association and dissociation of receptor from β-arrestin may coordinate the ubiquitin conjugation and deconjugating activities toward B2AR to tune the balance between receptor degradation and recycling.

Several recent studies have demonstrated that GPCR-associated proteins and β-arrestin are also regulated by ubiquitination. The number of these proteins seems to be relatively limited, but the effects of ubiquitination are diverse. Both yeast and mammalian stimulatory G protein α-subunits and the effectors they activate are dynamically ubiquitinated and degraded by the 26S proteasome (125–130). Other G protein subunits, such as the olfactory (Gαo), inhibitory (Gαi), Gt, and rhodopsin βγ, are degraded by proteasomal-dependent pathways. Ubiquitin ligases involved in G protein ubiquitination have yet to be identified with the exception of Gαi₃, which is regulated by the RING domain E3 ligase GIPN (131). Other signal transduction proteins, including regulator of G protein signaling proteins and G protein-coupled receptor kinase 2, are also ubiquitinated, although the significance of this process is undefined (107, 132). Immediate downstream effector molecules of the GPCR pathway have also been shown to be regulated by ubiquitination. The most representative example is the inositol 1,4,5-trisphosphate receptor, Ins(1,4,5)P₃R, that is ubiquitinated on activation of phospholipase C-linked receptors (133). Initial studies showed that Ins(1,4,5)P₃R levels were reduced by chronic activation of certain phospholipase C-linked GPCRs such as muscarinic acetylcholine receptors. This down-regulation was due to increased receptor degradation, and the receptors were processed by the ubiquitin proteasome pathway. The ubiquitination and degradation of Ins(1,4,5)P₃Rs represent the first example of an intracellular protein being processed by the ERAD pathway following GPCR activation.

Balanced Ubiquitination and Deubiquitination Determine GPCR Fate

GPCR ubiquitination is dynamic and can be reversed. Similar to phosphorylation and dephosphorylation, which are carried out by large families of kinases and phosphatases, ubiquitination is counteracted by deubiquitinating enzymes (DUBs) that deconjugate ubiquitin-modified proteins and rescue the substrate from proteasomal degradation. There are some 90 DUBs in the human proteome. DUBs can be divided into 5 different classes: ubiquitin C-terminal hydrolases, USPs, Machado-Joseph disease protein domain proteases, ovarian tumor proteases, and JAMM motif proteases. All DUBs are cysteine proteases, with the exception of JAMM motif proteases, which are metalloproteases (134). DUBs can display specificity at multiple levels, thus distinguishing between the many ubiquitin-like molecules, isopeptides (using an ϵ-amino group), and linear peptides (using an α-amino group), and between different types of ubiquitin linkage and chain structure (135). Ligand-induced deubiquitinases have been shown only for a few GPCRs. Adenosine A2AR receptors are deubiquitinated by USP4 (112); the β2-AR undergoes increased agonist-stimulated ubiquitination, lysosomal trafficking, and degradation after knockdown of USPs 20 and 33 (124). PAR1 is tightly regulated in a spatiotemporal manner through the actions of ubiquitin ligases and DUBs (86). Similarly, the balance between ubiquitination and deubiquitination of Frizzled-4, a 7-transmembrane receptor for Wnt ligands, is also important for regulating surface expression and cellular responsiveness (136). Constitutive ubiquitination of Frizzled-4 promotes internalization and lysosomal degradation, whereas deubiquitination mediated by USP8 leads to recycling and increased surface expression that occurs in a Wnt-independent manner (136). DUBs are remarkably specific. Overexpression of USP4, which promotes cell surface targeting of the A2AR (112), does not affect B2AR ubiquitination (124). Thus, receptor deubiquitination involves specific USP isoforms. Berthouze and coworkers proposed that fast recycling from early endosomes mainly involves dephosphorylation of nonubiquitinated receptors, whereas slow recycling of ubiquitinated receptors from deeper subcellular compartments is regulated by DUBs and protein phosphatases (124). Thus, the DUBs switch the receptor's fate from lysosomal/proteasomal degradation to recycling and enhanced cellular resensitization (Figure 4).

Figure 4.

A, Ubiquitination is a reversible process. DUBs deconjugate ubiquitin-modified proteins and rescue the substrate from proteasomal degradation. B, The balance between GPCR ubiquitination and deubiquitination regulates their surface expression and cellular responsiveness. Greater receptor ubiquitination results in fewer cell surface receptors by virtue of degradation and down-regulation. Conversely, with more DUBs, the final number of receptors that are ubiquitinated is diminished, with less attendant degradation, consequently more receptor recycling, and greater numbers of membrane-delimited GPCRs.

Other GPCRs regulated by ubiquitination and deubiquitination include the CXCR4. CXCR4 undergoes CXCL12-induced ubiquitination, resulting in lysosomal degradation (137). Overexpression of USP14 provoked deubiquitination of CXCR4, and therefore, its escape from degradation (138). Furthermore, the deubiquitinating enzyme USP8 has been shown to participate indirectly in CXCR4 regulation by modulating the dynamics of signaling endosomes (139). CXCR7 binds CXCL12 with high affinity. Canals and coworkers showed that in the basal state, CXCR7 is ubiquitinated and the Lys residues on its carboxy-terminus are the site of ubiquitination (140). They observed that receptor activation by CXCL12 results in reversible deubiquitination because subsequent removal of the chemokine partially restored the ubiquitinated receptor to levels detected in the basal state. These results contrast to what has been described for CXCR4 and highlight the differences that could potentially underlie distinct functions and/or patterns of expression of the 2 CXCL12-binding receptors (140). Thus, the same ligand can exert dissimilar effects on different receptors. In the same manner, the activation of a single GPCR by 2 or more distinct ligands can elicit distinct responses. This phenomenon is known as “biased agonism” (141) and knowledge of the molecular basis for biased agonism has important implications on drug development in pharmacology. Our recent work revealed that ubiquitination is important in determining differential GPCR regulation induced by biased agonists. The PTHR regulates bone growth and mineral ion balance and is responsive to distinct activating [PTH(1–84), PTH(1–34)] and nonactivating [PTH(7–84), PTH(7–34)] ligands. Stimulation with the activating ligand PTH(1–34) elicited transient PTHR ubiquitination (62). The receptor is internalized and then recycled following deubiquitination. In contrast, nonactivating PTH(7–34) triggered continuous PTHR ubiquitination and receptor degradation. The different trafficking behaviors exhibited by the PTHR appear to be regulated by the deubiquitinating enzyme USP2. PTH(1–34) increase USP2 levels, favoring the balance toward rapid deubiquitination and recycling of the receptor. However, PTH(7–34) does not increase or activate USP2. The slow deubiquitination induced by PTH(7–34) leads to an accumulation of ubiquitin tags on the PTHR and subsequent receptor degradation by the proteasomal pathway (62). These findings indicate that specific PTHR ligands distinctly modulate the activity of the ubiquitination machinery, resulting in differential receptor ubiquitination and trafficking and provide a novel paradigm for ligand biased GPCR ubiquitination.

Ubiquitination, GPCRs, and Disease

Dysregulated GPCR trafficking and the deregulation of ubiquitin pathways (142), as well as defective endocytosis, are associated with several human diseases, including many tumor types (143). GPR55 in mouse skin, for example, drives tumor development and is up-regulated in human squamous cell carcinomas (144). In this context, recent studies show that CXCR7 expression increases tumor formation and metastasis for some cancers (145, 146). The data also suggest that an important function of CXCR7 is to prevent degradation of CXCR4 (147). Therefore, high expression of CXCR7 in tumor cells may contribute to disproportionate signaling through CXCR4, a landmark of the pathophysiology of WHIM syndrome, a rare congenital immunodeficiency disorder that is also associated with tumor growth and metastasis (148). The ubiquitination state of CXCR7 under these pathophysiological conditions could help explain this scenario; however, it remains to be explored. Additionally, disrupted trafficking of CXCR4 and PAR1 within the endosomal-lysosomal system contributes to increased surface expression of receptors in breast cancer cells and cancer progression (149, 150). Tumor cells display aberrant PAR1 trafficking, which causes persistent signaling and cellular invasion. These defects appear to be specific to breast cancer cells, because PAR1 in normal human mammary epithelial cells displays appropriate trafficking and signal termination. It remains to be determined if the ubiquitin pathway is the mechanism responsible for defective PAR1 trafficking in breast carcinoma (149).

Conversely, the efficient coupling of ER QC and proteasomal activity may well be necessary to avert inherited diseases, such as retinitis pigmentosa, male pseudohermaphroditism, and nephrogenic diabetes insipidus, that result from ER retention of mutated rhodopsin (151, 152), luteinizing hormone receptors (153, 154), and V2R receptors (155).

It is increasingly apparent that ubiquitination is crucial to cell integrity. This awareness has informed the development of ubiquitin-proteasome pathway inhibitors as potential therapeutic agents, culminating in the recent approval for patients with refractory multiple myeloma of bortezomib, a potent proteasome inhibitor that blocks the degradation of ubiquitinated proteins (156). Moreover, proteasomal inhibitors have anti-inflammatory effects and as such their short-term use is proposed to exert beneficial actions in pathologic conditions associated with acute inflammation, including myocardial infarction or stroke (157–160). Thus, it is also likely that inhibitors of either ubiquitinating or DUBs regulating various cardiovascular proteins may have similar therapeutic utility. These studies suggest that the development of pharmacological agents capable of directly or indirectly inducing ubiquitination and down-regulation of targeted GPCRs could be applied in particular disease scenarios.

Conclusion and Future Directions

The ubiquitin system has integral roles in most cellular events and is therefore an important therapeutic target, for example, for the development of treatments against cancer and neurodegenerative disorders, as well as for inflammatory and infectious diseases (161, 103). In an interesting way, it revealed an unanticipated role for ubiquitination in regulating mammalian GPCR longevity and lysosomal sorting. Similarly astonishing is the function that modification by ubiquitin plays in the modulation of GPCR regulators such as GRKs, MAPKs, and β-arrestins. Although the ubiquitin posttranslational modification is carried out by the addition of the same ubiquitin chemical tag, this modification is capable of mediating discrete and very different molecular consequences. Our understanding of the regulation of GPCR signaling and trafficking by ubiquitination and deubiquitination is extraordinarily limited. Thus, studies are needed to explore new functions of ubiquitin in GPCR signaling. This field is an emerging area at the nexus of endocrinology and pharmacology that has important implications in drug development with fewer adverse side effects.