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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Circ Res. Author manuscript; available in PMC Oct 27, 2007.
Published in final edited form as:
PMCID: PMC1952537
NIHMSID: NIHMS22688

Seven-Transmembrane Receptors and Ubiquitination

Abstract

Regulation of protein function by post-translational modification plays an important role in many biological pathways. The most well known among such modifications is protein phosphorylation performed by highly specific protein kinases. In the past decade, however, covalent linkage of the low molecular weight protein ubiquitin to substrate proteins (protein ubiquitination) has proven to be yet another widely utilized mechanism of protein regulation playing a crucial role in virtually all aspects of cellular functions. This review highlights some of the recently discovered and provocative roles for ubiquitination in the regulation of the life cycle and signal transduction properties of seven-transmembrane receptors, which serve to integrate many biological functions and play fundamental roles in cardiovascular homeostasis.

Keywords: Seven-transmembrane receptors, GPCR, GRK, β-arrestin, 7TMR, internalization, degradation, ubiquitin, E3 ubiquitin ligase, ubiquitination, deubiquitination

Introduction

The super-family of seven-transmembrane receptors, 7TMRs, (also called G protein coupled receptors, GPCRs) is the largest plasma membrane receptor family including approximately 1000 members 1, 2. 7TMRs are ubiquitously expressed, respond to a wide spectrum of sensory and chemical stimuli, and regulate most physiological responses 3. Ligands acting at 7TMRs constitute widely used pharmacological agents accounting for more than 30% of marketed prescription drugs 4. Of great significance is the fact that within this superfamily are members, which mediate the effects of various hormones and neurotransmitters of fundamental importance for regulation of the cardiovascular system. Examples of 7TMRs expressed in the heart, which play prominent roles in cardiac function include those that respond to epinephrine and norepinephrine (α and β adrenergic receptors), acetylcholine (M2 muscarinic cholinergic receptor), angiotensin II (AT1 receptors), and endothelin-1 (ET1B receptors). In addition to these “classical” 7TMRs, several others with yet undefined functions are also expressed in cardiac tissues 5.

Members of the 7TMR superfamily share common structural and functional properties. Structurally they possess an extracellular amino terminus, seven membrane traversing hydrophobic alpha helices and a cytoplasmic carboxyl terminus; functionally they exert their agonist-stimulated signaling properties by coupling to the heterotrimeric G proteins, and as well as by binding to G protein-coupled receptor kinases (GRKs) and the β-arrestin proteins 6. The latter two groups of proteins, namely GRKs that phosphorylate the 7TMRs on serine and threonine residues, and β-arrestins that bind phosphorylated 7TMRs to competitively inhibit G protein coupling, were originally defined as negative regulators that lead to receptor “desensitization” 79. More recently, GRKs and β-arrestins have been shown to function as positive signal mediators independent of receptor-G protein coupling 10. In addition, GRKs and β-arrestins have also been shown to bind to many non-receptor proteins and play indispensable roles in cellular processes including receptor endocytosis and signaling (e.g. mitogen activated protein kinase signaling) 6,1113.

In humans, the G protein family is represented by 35 genes, 16 of which encode a subunits, five which encode β and fourteen which encode γ subunits. Functional G protein heterotrimers result from a combination of one of each subunit and act as guanine nucleotide on-off switches 14. On the other hand, the GRK and arrestin families are represented by seven and four members respectively 6,15. GRK1 and GRK7 are expressed in the retina and act as kinases for the visual receptors Rhodopsin and Cone opsin, respectively. Arrestin1 and arrestin4, also known as visual arrestins, bind and regulate the visual receptors. The other GRKs, namely, the cytosolic GRK2 (also known as βARK1), and GRK3 and membrane bound GRK5 and GRK6 are ubiquitously expressed. Translocation of GRK2 and GRK3 to the plasma membrane requires their association with G protein βγ subunits 15. Outside the visual system, GRK4 is the only member with a restricted tissue distribution, with its expression reported to be testis and brain specific. Perhaps most amazing, however, is the observation that there exist only two nonvisual arrestin isoforms, namely, β-arrestin1 and β-arrestin2 (also called arrestin2 and arrestin3, respectively), which bind to and regulate the vast repertoire of 7TMRs.

Signal transduction via cell surface receptors requires the presence of receptor protein at the plasma membrane. The plasma membrane expression of 7TMRs results from a balance between two pathways, one that delivers properly folded receptors to the cell surface and the second that removes the receptors by endocytosis (reviewed in 16), either temporarily (by internalization) or permanently (internalization followed by subsequent degradation in acidic endosomal vesicles, called lysosomes). Receptors are first internalized into early or sorting endosomes, which fuse with late endosomes that have internal membranes (Fig 1A). Late endosomes are also called multivesicular bodies (MVBs) 17. In some cases, the internalized cargo can be directed from the early endosomes to recycling endosomes to deliver the receptors back to the cell surface. Lysosomal delivery of receptors is believed to involve the fusion of MVBs with lysosomes. For many 7TMRs, chronic treatment with agonist results in a significant decrease in the cellular receptor levels by a process termed down-regulation, primarily ascribed to lysosomal receptor degradation (Fig 1A). Importantly some of the key molecular mechanisms governing receptor degradation have been only recently discovered and are attributed to post-translational receptor modification by the protein tag, ubiquitin (Fig 1B, C and D).

Figure 1Figure 1Figure 1
7TMR trafficking and Ubiquitination

Protein modifications and their roles

The post-translational modification of proteins after ribosomal translation is a highly effective method found within nature to finely tune protein activity in a highly sophisticated manner. Among the post-translational modifications that involve addition of a functional group to amino acid side chains, glycosylation (the addition of oligosaccharides to the side chain amide of asparagine residues), palmitoylation (the covalent attachment of the fatty acid palmitate on cysteine residues) and phosphorylation (the addition of phosphate groups to serine, threonine or tyrosine residues) have been previously described to play roles in the regulation of 7TMR biology. An elaborate discussion of these modifications is beyond the scope of this review. Readers are directed to several recent excellent reviews on these modifications 1824. Likely, the most-studied post-translational 7TMR modification is receptor phosphorylation on serine and threonine residues by second messenger kinases and by the GRKs. As will be discussed below, GRK mediated 7TMR phosphorylation plays an important role in both β-arrestin binding and receptor ubiquitination.

Ubiquitination

Ubiquitination (also referred to as ubiquitylation or ubiquitinylation) is a post-translational modification involving the covalent addition of a small protein, ubiquitin, to the lysine side chains of substrate proteins (Fig 1B). Ubiquitin is a highly conserved low molecular weight protein of 76 amino acid residues found in all eukaryotic cells. Ubiquitination was originally discovered as an ATP-dependent process that resulted in the degradation of modified proteins in a cell free system and was later found to be the primary mechanism for degrading majority of short-lived proteins in eukaryotic cells 2529.

Three enzymatic activities acting in concert are required for ubiquitination (Fig 1B). These enzymes were named E1 or ubiquitin activating enzyme, E2 or ubiquitin carrier protein and E3 or ubiquitin protein ligase. E1 carries out the ATP-dependent activation of the carboxyl-terminal glycine residue of ubiquitin, by the formation of ubiquitin adenylate, followed by the transfer of activated ubiquitin to a thiol site of E1 thereby forming a thiolester linkage 27. Activated ubiquitin is then transferred to a thiol site of E2 by transacylation and is then directly conjugated to lysine residues in the substrate proteins or to an E3 enzyme, which can modify the substrate on lysine residues 27. Ubiquitin has seven lysines, at positions 6, 11, 27, 29, 33, 48 and 63 of its primary amino acid sequence. After ubiquitin conjugation to a substrate, subsequent addition occurs on a lysine (generally at lysine-48) of the previously attached ubiquitin. When this process occurs repeatedly a polyubiquitin chain is formed. Polyubiquitinated substrate proteins, are recognized for degradation by the multisubunit protease complex in the cell known as the 26S proteasome 30. A chain of 4 ubiquitins conjugated at lysine-48 in the ubiquitin moieties on a substrate is sufficient for recognition and degradation by the proteasomes 31. Besides tryptic and chymotryptic enzymatic activities, proteasomes also have associated deubiquitinating enzymes, which cleave the ubiquitin chains prior to shredding of the substrate protein within the proteasomal core 32. Thus, ubiquitin itself is not degraded but rather recycled for subsequent rounds of activity.

According to a recent genomic annotation, humans express a single E1, approximately 60 E2s and nearly 400 E3s 33. This distribution strongly suggests that the specificity of substrate ubiquitination is primarily governed by E3-substrate interactions. E3 ubiquitin ligases are traditionally categorized in to two main groups, namely, HECT (Homologous to E6AP C Terminus) E3s and RING (Really Interesting New Gene) E3s based upon their catalytic domains 34. However, this classification does not include some members such as E3 alpha, a zinc finger containing ligase that recognizes specific destabilizing motifs at the amino termini of substrate proteins and some newly identified E3 ubiquitin ligases, such as the F-box, U-box and PHD (plant homeodomain) E3s. Whereas HECT E3s accept ubiquitin from E2s and then ligate the ubiquitin on to substrates, RING E3s are believed to function by facilitating ubiquitin transfer from the E2 enzyme on to substrate lysines. The U-box proteins 35 act like RING E3s and mediate polyubiquitination, but they lack the metal chelating residues that are present in the RING domains. PHD E3s have a specialized form of zinc finger motif 36. U-box proteins are also referred to as E4 ligases since they accelerate polyubiquitination of ubiquitinated proteins.

Although originally described as a signal for the proteasomal degradation of substrate proteins, ubiquitin and polyubiquitin chains have recently been shown to have non-canonical functions in diverse cellular pathways 37. Thus, monoubiquitination (one ubiquitin moiety attached) of yeast cell surface receptors 38 and multi-mono or poly ubiquitination 39, 40 of mammalian growth factor receptors have been shown to function as internalization and lysosomal sorting signals respectively. In addition, polyubiquitin chain containing lysine-63 linkage between ubiquitin moieties has been shown to act as a trigger for the activation of a kinase cascade leading to NF B activity, as well as to play an important role in DNA repair 41. In general, ubiquitination involving a lysine-48 linkage is believed to signal degradation via the proteasomal route whereas a lysine-63 linkage is associated with nonproteasomal pathways. Additionally, most endocytic roles of ubiquitination are believed to be mediated by monoubiquitination. Recent work, however, indicates that rapid clathrin-dependent internalization is promoted by polyubiquitin more efficiently than monoubiquitin 42,43.

Recently, eleven ubiquitin-like proteins (UBLs) (reviewed in 44) have been discovered that are covalently attached to a variety of target proteins in a highly regulated manner which is very reminiscent of ubiquitination. The UBLs all share with ubiquitin a “ubiquitin superfold” (β-grasp fold) despite very little similarity to ubiquitin at the level of primary sequence. Another twist to the general modality of ubiquitination is recent work indicating that protein ubiquitination can occur on a substrate protein’s amino terminus 45 or cysteine residues 46 rather than on the normal sites of modification, namely, the epsilon amino group of lysine residues.

A major distinction between ubiquitination and other post-translational modifications is that it results in the addition of a new tertiary structure to the modified protein. Further variation in the chain linkage between ubiquitin moieties lends additional architectural diversity that favors particular protein-protein interactions. Moreover, the downstream fate of ubiquitinated substrates is mediated by proteins with ubiquitin binding domains, which play an important role in recognition mechanisms in both proteasomal and nonproteasomal pathways 47.

Ubiquitination of Cell surface receptors

Initial clues that single transmembrane cell surface receptors were ubiquitinated came from microsequencing studies of isolated receptor proteins performed almost twenty years ago. The covalent attachment of ubiquitin was first reported in 1986 for the lymphocyte homing receptor 48 and the growth factor receptor, PDGFR (platelet-derived growth factor receptor) 49. These findings were followed by the identification of the growth hormone receptor 50 as a ubiquitin substrate although in each case the cause or consequence of such modification was unknown. These initial studies were followed by reports demonstrating ligand-induced ubiquitination of the PDGFR 51 and the T cell antigen receptor 52. Currently a host of growth factor receptors is known to be ubiquitinated and their degradation is suggested to occur through an ubiquitin-dependent pathway. Since several excellent reviews on growth factor receptor regulation and ubiquitination are available 5355, only some of the most salient features which are applicable to the 7TMRs will be presented in this review.

The yeast system has been used not only as a means to decipher the molecular mechanisms of ubiquitination and proteasomal degradation of cytosolic proteins, but has also been used to understand the role of ubiquitination in the regulation of membrane proteins 56. An initial hint that ubiquitination regulates the degradation of cell surface receptors was provided by studies of Ste6, a yeast peptide transporter. As such, Ste6 displays a slow turnover rate in yeast strains defective in ubiquitin conjugation and ubiquitinated forms of Ste6 were found to accumulate in endocytosis deficient strains 56. Internalization and vacuolar degradation of yeast Ste2, a seven transmembrane receptor for pheromone alpha factor were found to be regulated via monoubiquitination (Fig 1C) 57. For the yeast Ste3 (a factor receptor, also a 7TMR), although monoubiquitination will suffice, polyubiquitination enhances the rate of receptor endocytosis 58. Thus, until recently the characterization of ubiquitin-dependent yeast pheromone receptor internalization served as the prototype of 7TMR endocytosis. As will be described below, however, a more specialized bimodal ubiquitin-dependent regulation (in which receptor degradation is controlled by one ubiquitin-dependent process and receptor internalization by another) has recently been discovered for the mammalian 7TMRs.

Agonist-dependent ubiquitination of 7TMRs: lysosomal degradation

Agonist-stimulated ubiquitination of mammalian 7TMRs, at both endogenous and exogenous expression levels, was first reported for the human β2 adrenergic receptor (AR) 59. Receptor ubiquitination was detectable with both in vitro and cellular assays as the appearance of high molecular weight protein bands with altered electrophoretic mobility that positively reacted to ubiquitin antibodies. Moreover, ubiquitination was abolished in a mutant β2AR, in which all of the lysine residues had been mutated to arginines (β2AR-0K) (Fig 1D and 59). Interestingly, unlike the yeast Ste2 protein, which does not undergo pheromone-dependent internalization in the absence of ubiquitination (Fig 1C, and 57), the mutant β2AR-0K receptor was internalized from the cell membrane with the same kinetics as the wild type receptor, in response to an acute (minutes) dose of agonist. However, with chronic (24 hours) agonist treatment, the mutant β2AR failed to undergo lysosomal degradation. Thus, for a prototypic mammalian 7TMR, ubiquitination is not required as an internalization signal but rather functions to direct the internalized receptors to appropriate degradative compartments. The exact site(s) of ubiquitination or details of a degradation motif in the β2AR remain to be elucidated. Another very striking feature of the mammalian system is that receptor internalization is also dependent on the agonist-induced ubiquitination of the adaptor protein β-arrestin, as will be discussed in the following section.

An important feature common to both Ste2 and the β2AR is the dependence of receptor ubiquitination on the prior phosphorylation of the receptors’ cytoplasmic domains. Serine phosphorylation of Ste2 occurs within a motif (SINNDAKSS) and thereby triggers the ubiquitination of lysine in the receptor’s carboxyl tail which is important for mediating both constitutive and ligand-induced endocytosis 60. A GRK homolog is absent in yeast. Thus, Ste2 phosphorylation is reported to be due to the activity of yeast casein kinase I, since mutant yeast strains defective in this kinase are unable to internalize α-factor and both phosphorylation and ubiquitination of Ste2 are impaired in the mutant strains. For the β2AR, mutation of all the phosphorylation sites in the carboxyl tail results in impairment of receptor ubiquitination. Notably, β-arrestin has been shown to function as a required adaptor in the process of receptor ubiquitination (see below) and hence this defect in the mutant β2AR may also be due to the absence of β-arrestin binding, since β-arrestin is recruited only to phosphorylated β2ARs 59,61.

Agonist-stimulated ubiquitination has also been reported for the 7TMR, CXCR4, a chemokine receptor that serves as the coreceptor for HIV 62. Unlike the β2AR modification, CXCR4 is modified with a single ubiquitin. However, similar to the β2AR, a CXCR4 mutant defective in ubiquitination was internalized at a normal rate, but was not degraded in lysosomes, confirming that for at least some (and perhaps all) mammalian 7TMRs, obstructing receptor ubiquitination does not lead to retention of receptors at the plasma membrane.

Yet another receptor that is ubiquitinated upon agonist stimulation is the V2 vasopressin receptor 63. Vasopressin has an essential role in the maintenance of total body water acting in the kidney to increase sodium and therefore water absorption. Vasopressin and its analogues are used clinically to treat diabetes insipidus. In the case of the vasopressin receptor, polyubiquitination occurs at a single lysine (lysine 268) in the third intracellular loop of the receptor. Abrogation of ubiquitination, by mutation of lysine-268 to arginine does not affect agonist-induced receptor internalization, but substantially increases receptor half-life 63.

Agonist-induced ubiquitination of the protease activated receptor 2 (PAR2), a receptor activated by cleavage of part of its extracellular domain has been recently reported 64. Importantly, PAR2 is expressed not only on platelets, but also on endothelial cells, and myocytes where it mediates both vascular contractility and proliferation, suggesting that it helps to regulate vascular homeostasis. A PAR2 mutant with no lysine acceptor sites was not ubiquitinated and was retained in early endosomes following internalization, thus preventing lysosomal trafficking and degradation. Another 7TMR, the neurokinin-1 receptor, whose spectrum of biological activities includes sensory transmission in the nervous system and contraction/relaxation of peripheral smooth muscles, is also ubiquitinated in response to chronic stimulation with the agonist substance P 65. Indeed, ubiquitination defective NK1R undergoes internalization but not degradation. Moreover, lysine mutants of the NK1R recycled to the plasma membrane with kinetics similar to those observed in their wild type counterparts suggesting that receptors lacking ubiquitin tags preferentially traffic through recycling vesicles. Ligand-stimulated lysosomal degradation of the platelet-activating factor (PAF) receptor is reported to be ubiquitin-dependent, although receptor ubiquitination itself was not completely agonist dependent 66. PAFR is a 7TMR that mediates responses to PAF, a unique phospholipid mediator, which possesses potent proinflammatory, smooth muscle contractile and hypotensive activities, and is involved in various inflammatory disease states including allergic asthma, atherosclerosis, and psoriasis.

As summarized above, several well-studied 7TMRs undergo agonist-dependent ubiquitination, and in each case, the modification is dispensable for receptor endocytosis into early endosomal vesicles but is requisite for subsequent steps of intracellular receptor trafficking involving proper receptor sorting to degradative compartments or lysosomes. Importantly, however, exceptions to this phenomenon do exist. While the human β2AR is regulated by agonist induced ubiquitination, the β1AR is resistant both to ubiquitination and degradation as demonstrated in heterologous cells 67. Moreover, both the wild type and lysine mutants of murine delta opioid receptor traffic to and are degraded in lysosomes suggesting a ubiquitin-independent pathway for this receptor 68. However, its lysosomal trafficking requires components of the endocytic machinery such as Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and VPS4 (see later section on accessory proteins) that play important roles in sorting ubiquitinated endocytic cargo 69.

Agonist-independent ubiquitination of 7TMRs: Proteasomal degradation

At present, ubiquitin-dependent degradation leading to basal turnover of cell surface receptors via the proteasomal pathway has been described for two 7TMRs, namely, the metabotropic glutamate receptors (mGluR1 and mGluR5) 70 and the human follitropin receptor (FSHR) (Fig 2) 71. Additionally, over the past several years, it has been reported that a handful of 7TMRs become ubiquitinated in the endoplasmic reticulum (ER) and degraded by the proteasomal pathway (Fig 2). In most such cases, ubiquitination was found to promote quality control exerted in the ER by clearing newly synthesized but misfolded receptors. As such, misfolded or incompletely folded human delta opioid receptor (δOR) is retrotranslocated to the cytosolic side of the ER membrane via the Sec61 translocon complex, where it is deglycosylated, conjugated by ubiquitin and degraded by the 26S proteasome 72. In addition, the calcium-sensing receptor, (CaR; a 7TMR that functions to control serum Ca2+ levels) and the thyrotropin releasing hormone receptor (TRHR) (a 7TMR that controls thyrotropin secretion from the anterior pituitary gland) also undergo ubiquitination and proteasomal degradation when misfolded receptor proteins accumulate in the ER 73 74. For some of these receptors, the ER-proteasome regulation could be demonstrated in both heterologous and physiologically relevant cell types, indicating that proteasomes may play an important role post-ER synthesis, ensuring membrane delivery of only properly folded receptors. Moreover, the efficient coupling of ER quality control 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 7577, luteinizing hormone receptors 78,79 and V2 vasopressin receptors 80, respectively.

Figure 2
7TMR regulatory pathways and proteasomal degradation

7TMR internalization: role of β-arrestin ubiquitination

As mentioned above, internalization of mammalian 7TMRs does not require a ubiquitin tag attached to the receptor itself, a situation that differs from the yeast 7TMR Ste2, in which eliminating receptor ubiquitination prevents receptor internalization. Internalization of mammalian 7TMRs, however, depends on the ubiquitination of the adaptor protein β-arrestin. In response to β2AR stimulation, β-arrestin undergoes rapid ubiquitination as demonstrated by Western blot 59 as well as real time BRET (bioluminescence resonance energy transfer) assays 81. Interestingly, in yeast two-hybrid screens using β-arrestin (1 or 2) as bait, Mdm2 (a RING domain containg E3 ubiquitin ligase) was identified as a unique β-arrestin-binding partner 59. Quite unexpectedly, in mouse embryonic fibroblasts that are Mdm2-null, β2AR is ubiquitinated in response to agonist, but β-arrestin is not. In the same cells, the β2AR was internalized poorly in response to acute agonist challenge, but its degradation in response to chronic agonist treatment was almost equivalent to that in Mdm2 containing cell types. Moreover, expression of Mdm2 in the original Mdm2-null cells results in recovery of both β-arrestin ubiquitination and rapid receptor internalization. The inhibition of β2AR internalization observed in Mdm2 null cells is also reproduced in heterologous cells such as HEK-293 in the presence of Mdm2 specific siRNA (Shenoy lab unpublished data). Moreover, an Mdm2 deletion mutant that lacks the catalytic domain, but retains β-arrestin-binding also inhibits receptor internalization and β-arrestin modification. Thus, quite remarkably, Mdm2 dependent β-arrestin ubiquitination is required for rapid internalization of the β2AR into clathrin-coated vesicles. It is conceivable that ubiquitinated β-arrestin binds or attracts certain endocytic components that also serve as ubiquitin receptors. The exact mechanisms by which ubiquitin-dependent processes mediate β-arrestin-dependent endocytosis, however, remain to be elucidated.

Another interesting feature of β-arrestin ubiquitination is the distinct kinetics which correspond to a particular receptor type 82 (Fig 3A). Whereas stimulation of the β2AR leads to transient β-arrestin ubiquitination, stimulation of the AT1aR results in a relatively sustained β-arrestin ubiquitination. Importantly, the β2AR and the AT1aR can be classified into discrete groups, denoted ‘class A’ and ‘class B’ respectively, based upon the pattern of recruitment of green fluorescent protein tagged β-arrestin in response to agonist. Whereas class A receptors (including the β2AR, α1bAR, μ opioid receptor, endothelin A receptor, and dopamine D1A receptor) recruit β-arrestins only to the plasma membrane, class B receptors (including the V2 vasopressin, Angiotensin AT1a, thyrotropin releasing hormone, neurotensin 1, and neurokinin NK1- receptors) form stable complexes with the β-arrestin proteins in endocytic vesicles 83. Thus, while class A receptors release β-arrestins upon internalization, class B receptors remain stably associated with β-arrestins even after internalization. Interestingly, these patterns also correlate with the kinetics and stability of β-arrestin-ubiquitination patterns: transient β-arrestin-ubiquitination parallels transient binding of β-arrestin and β2AR at the cell membrane (class A pattern), whereas stable β-arrestin ubiquitination corresponds to stable receptor-β-arrestin complexes localized on endosomes (class B pattern) 82. The dissociation of β-arrestin from the internalizing receptor most likely occurs due to deubiquitination events since a β-arrestin2-Ub chimeric protein remains stably bound to the class A β2AR during internalization 82. Interestingly, induction of sustained β-arrestin ubiquitination upon 7TMR stimulation occurs at specific lysine acceptor sites. For the AT1aR dependent β-arrestin modification, stable ubiquitin attachment occurs primarily at lysines 11 and 12 in rat β-arrestin2 84. Mutation of these lysines to arginines leads to reversal of AngII stimulated β-arrestin ubiquitination from a sustained to a transient pattern, with a corresponding reversal of AT1aR-β-arrestin binding (i.e. from stable endosome localized complexes to transiently associated complexes seen only at the plasma membrane). Quite unexpectedly, however, the same β-arrestin mutant was stably ubiquitinated and was found to associate tightly with two other activated class B receptors, the V2R and NK1R that like the AT1aR are capable of β-arrestin recruitment to endosomes. These provocative findings suggest that different 7TMRs are capable of inducing sustained β-arrestin ubiquitination at distinct sites. Whether the nature of the target lysines in the β-arrestin proteins (a total of 35 in β-arrestin1 and 31 in rat β-arrestin2) susceptible to ubiquitination is a function of differences in the conformation of β-arrestin when bound to a particular receptor, or whether it reflects the activity of differing E3 ubiquitin ligases remains to be determined. Thus far, Mdm2 is the only E3 ligase that has been demonstrated to bind β-arrestins in a variety of cell types and assay systems. On the other hand, β-arrestins have been shown to interact with many non-receptor partners and are multifunctional adaptor proteins; as such, it is certainly possible that they will be found capable of binding several members of the ubiquitination machinery.

Figure 3Figure 3
β-arrestin’s role in endocytosis, signaling and receptor ubiquitination

An important effect of 7TMR activity that is intertwined with receptor trafficking is the formation and localization of signaling complexes termed “signalosomes” 13,61. Activation of 7TMRs, besides leading to β-arrestin recruitment, also invokes the formation of β-arrestin-dependent signaling complexes, in which β-arrestin, bound to activated receptor is able to scaffold various components of a kinase activation complex. At present, the best-studied example of this is that of β-arrestin-dependent stimulation of extracellular signal related kinase (ERK), a mitogen activated protein kinase (MAPK) which controls many cellular functions including cellular growth, proliferation, motility and shape. For many ‘class B’ receptors, such β-arrestin-receptor signalosomes containing activated ERK are compartmentalized to perinuclear endosomes (Fig 3A). Sustained ubiquitination of β-arrestin is required both to form stable endocytic complexes with the receptor as well to engage active ERK on the signalosomes (Fig 3A and 84). Infact, mutation of lysines 11 and 12 in β-arrestin (1 or 2), (mentioned above) leads to an impairment of AngII-induced sustained β-arrestin ubiquitination, with subsequent impairment of the endosome-localized AT1aR-β-arrestin complexes and the β-arrestin-mediated ERK scaffolding, suggesting a crucial role for β-arrestin ubiquitination in this process 84. Interestingly, polyubiquitination of TRAF6, an adaptor that binds the tumor necrosis factor receptor (TNFR), has been shown to activate the first kinase, TGFβ1-activating kinase (TAK1) of the kinase cascade involved in NF-κB signaling 41. Whether β-arrestin ubiquitination actually triggers the MAP kinase cascade in response to 7TMR stimulation is a provocative question that remains to be addressed.

Notably, a β-arrestin homolog is not expressed in yeast and other lesser eukaryotes. However, recent studies have indicated the presence of arrestin-like proteins in the model fungus, Aspergillus nidulans 85. Activation of PalH, a 7TMR expressed by this model fungus leads to a pH signaling pathway involving phosphorylation and ubiquitination of the arrestin-like protein, PalF. PalF ubiquitination is suggested to play a crucial role in guiding the internalization of the receptor as well as in downstream signaling mechanisms. This analogy between mammalian β-arrestin and PalF suggests possible evolutionary conservation in the role of β-arrestin ubiquitination to mediate 7TMR internalization and signal transduction.

β-arrestin: An E3 ubiquitin ligase adaptor

Both β2AR and V2R ubiquitination are not detectable in β-arrestin1/2 double nullmouse embryonic fibroblasts, but are induced only when these cells are transfected with the exogenous β-arrestin2 isoform suggesting that at least for these 7TMRs, β-arrestin2 likely functions as a required E3 ligase adaptor (Fig 3B, 59,63). The identity of the specific E3 ligase recruited, however, remains to be determined. Although originally discovered as a protein that binds 7TMRs, β-arrestin has more recently been shown to be recruited to various types of cell surface receptors including those for growth factors 6. In fact, β-arrestin1 escorts the E3 ligase Mdm2 to bind the insulin like growth factor-1 receptor, leading to receptor ubiquitination and downregulation 86. A similar adaptor role for Kurtz, the Drosophila non-visual arrestin, has been demonstrated in the recruitment of the E3 ligase Deltex to ubiquitinate the Notch receptor 87 (Fig 3B). Although a requirement for β-arrestin has not yet been documented for other 7TMRs which are ubiquitinated, such a role seems likely.

E3 ligases implicated in 7TMR regulation

Because of the hierarchical nature of the ubiquitination cascade (one E1 binds dozens of E2s and each E2 can communicate with hundreds of E3s), the net specificity of substrate recognition as well as the timing and nature of the modification(s) is dependent upon the E3 ligases in the cell. Thus, malfunctioning E3 ligases can result in drastic changes in cellular substrate protein levels and lead to various biological consequences. As example, a mutation in the ‘PY’ binding motif in the epithelial sodium channel (ENaC) expressed in the kidney prevents binding of the ENaC cognate E3 ligase Nedd4 88. The result is increased levels of the ENaC at the membrane, leading to excessive reabsorption of Na+ and H2O and consequently, a severe form of an early onset type of familial hypertension referred to as Liddle’s syndrome. Because several such examples of E3 related diseases exist, the identification of the specific E3 ligases that ubiquitinate corresponding 7TMRs is an important step toward understanding and exploiting these regulatory mechanisms that dictate optimal receptor expression at the cell surface.

Ubiquitination of the chemokine receptor CXCR4 is carried out by the HECT domain-containing E3 ligase, AIP4 (Atrophin interacting protein 4) 89. Coexpression of a HECT domain mutant of AIP4, but not of the related HECT E3s, namely Nedd4 and Nedd4-2, results in stabilization and retention of agonist activated CXCR4 in endosomes. On the other hand, PAR2 ubiquitination and lysosomal sorting is carried out by the RING domain containing E3 ligase, c-Cbl, the E3 ligase which is also known to ubiquitinate the EGF and the PDGF receptors 64. Interestingly, PAR2 activation leads to cSrc dependent tyrosine phosphorylation and the subsequent recruitment of c-Cbl to the receptor at the cell surface. Ligand-induced degradation of PAFR was also found to be inhibited by a dominant negative Cbl mutant (NCbl) 66. Ubiquitination and degradation of mGluRs is mediated by yet another RING domain containing E3 ubiquitin ligase, Siah1A (seven in absentia homolog 1A) that can directly bind to the carboxyl terminal domains of mGluR1a and mGluR5 70. Finally, dorfin (double RING finger protein) was identified as a yeast two-hybrid partner for the CaR and was shown to mediate ubiquitination and ER associated degradation (ERAD) of CaR thus assuring CaR quality control during biosynthesis 73. In total, the above examples indicate that many representative E3 ligases can participate in 7TMR regulation. Although examples of 7TMRs paired with their respective E3 ligases provide some clues as to the regulatory mechanisms involved in controlling receptor degradation, the further identification and characterization of E3 ligases for many more 7TMRs will be necessary in order to obtain a more complete appreciation of the intricacies of ubiquitination in 7TMR regulation.

Ubiquitination of 7TMR regulatory proteins and downstream effectors

Several 7TMR-associated proteins other than β-arrestin (as described above) are also regulated by ubiquitination. Both yeast and mammalian stimulatory G protein alpha subunit (Gαs) proteins are ubiquitinated and degraded by the 26S proteasomes (Fig 2) 9095. Interestingly, β2AR-stimulation leads to downregulation of mammalian Gas protein via the proteasomal pathway 95 suggesting that the regulated degradation of G protein is yet another mechanism for fine tuning receptor signal transduction (Fig 2). In addition, proteasomal degradation has been reported for the olfactory (Gαo) 96, and inhibitory (Gαi) G protein subunits 97 as well as for both the Gt 98 and βγ subunits of retinal G protein transducin 92, although whether these processes are choreographed by 7TMR activation is not known. With the exception of G alpha i3, which is regulated by the RING domain E3 ligase GIPN 97, the identification of E3 ligases involved in G protein ubiquitination remains to be determined.

In addition to leading to both β2AR and β-arrestin ubiquitination, activation of the β2AR has remarkably also been demonstrated to induce the ubiquitination and proteasomal degradation of G protein coupled receptor kinase 2 (GRK2) (Fig 2 and 99). Additional studies have also shown that β-arrestin recruitment to activated β2ARs facilitates both c-Src and MAPK mediated phosphorylation of GRK2 on tyrosine and serine/threonine residues respectively, and subsequent GRK2 degradation 100,101. Moreover, β-arrestin bound Mdm2 mediates the ubiquitination and degradation of GRK2 102. Importantly, altered levels of GRK2 have been noted in various human diseases including rheumatoid arthritis, multiple sclerosis, congestive heart failure, and hypertension. Future studies should illuminate the role played by the proteasomal pathway, as evoked by a 7TMR, in affecting GRK2 levels in these and other pathologic states.

Thus far, very few immediate downstream effector molecules of the 7TMR pathway have been shown to be regulated by ubiquitination. The most interesting example described to date is that of the inositol 1,4,5-trisphosphate receptors, Ins(1,4,5)P3R (Fig 2) that are ubiquitinated upon activation of phospholipase-C-linked receptors 103. Ins(1,4,5)P3Rs are found in the ER membrane and regulate Ca2+ flow from the ER to the cytosol, and their downregulation could be a protective effect to control the levels of Ca2+ in the cytosol. Elevation of both Ca2+and inositol 1,4,5-trisphosphate in the cell provoke ubiquitination and downregulation of Ins(1,4,5)P3Rs via the proteasomal pathway104,105.

Role of accessory proteins in sorting ubiquitinated 7TMRs to degradative pathways

The intracellular sorting of internalized 7TMRs en route to lysosomes might be expected to be directed solely by mono- or poly- ubiquitin tags. As such, ubiquitin by itself carries sufficient information to target membrane cargo for lysosomal degradation. Moreover, substitution of the C-terminal domains of cell surface receptors with a ubiquitin moiety is sufficient to initiate the endocytic process 106. The mechanism by which this is achieved has become evident from recent work describing the discovery of ‘ubiquitin receptors’ as trafficking compartment-associated proteins which carry one or more ubiquitin interaction domains 47.

CXCR4 trafficking and sorting to lysosomes was recently shown to involve Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate), a protein that colocalizes with ubiquitinated proteins on clathrin-coated endosomal microdomains and prevents the recycling of internalized cargo 89,107. Specifically, the E3 ligase AIP4, in addition to being recruited to ubiquitinate CXCR4 was also capable of ubiquitinating Hrs on endosomes. Further, this Hrs ubiquitination is also CXCR4 dependent. It should be noted that Hrs functions not only to recruit ubiquitinated receptors but also to recruit downstream ‘mono-Ub receptors’ such as the ESCRT (endosomal sorting complexes required for transport) complexes 108. The role of the ESCRT network in mediating transport of ubiquitinated cargo has been well documented for the model cargo protein carboxypeptidase S in yeast and for the mammalian EGFR 109, but its significance in 7TMR degradative pathways has not yet been established. It has also been proposed that several sorting proteins similar to Hrs may act to functionally increase the efficiency and improve the specificity of the sorting process.

VPS4, a member of the AAA protein family (ATPases associated with diverse cellular activities) regulates the transport of proteins out of a prevacuolar endosomal compartment 110. Interestingly, an ATPase defective mutant was shown to completely block CXCR4 degradation leading to the accumulation of ubiquitinated CXCR4 89. It is proposed that Hrs and VPS4 complex assembly is stimulated by CXCR4 activation, and that the activity of VPS4 is necessary for the proper delivery of ubiquitinated CXCR4 to late endosomal compartments. Interestingly, another AAA-ATPase, the p97/valosin-containing protein, which functions to chaperone misfolded proteins along the ERAD pathway, is reported to bind both the CaR and the E3 ligase Dorfin in HEK-293 cells. As such, the process by which ubiquitinated 7TMRs are both recognized and targeted is likely very complex, and involves the participation of many members of the endocytic machinery. Future work should shed some light on the general and specific details of the exact sorting itinerary of ubiquitinated 7TMRs.

7TMR regulation and the role of proteasomes

While the trafficking of ubiquitinated yeast receptors to the vacuole (the yeast equivalent of the lysosome) does not involve the proteasomal machinery, the trafficking of several mammalian receptors does involve a component of the proteasomal pathway. In the case of ubiquitinated misfolded 7TMRs that are directed to the ERAD pathway, the role of the proteasomal pathway is not surprising due to the known coupling of the ERAD and proteasomal pathways. However, an issue that remains perplexing is that several mammalian cell surface receptors seem to require proteasomal activity for endocytosis to occur. In fact, proteasomal inhibitors actually prevent the degradation of these receptors, which are well documented to occur in lysosomes. Interestingly, in the case of the single transmembrane growth hormone receptor (GHR), endocytosis occurs in the absence of ubiquitination but does require intact proteasomal activity 111. Similarly, a functional proteasome ensures the optimal endocytosis and subsequent lysosomal degradation of the Interleukin 2 receptor/ligand complex 112. Studies also indicate that agonist induced human k-opioid receptor degradation can be reduced by both lysosomal and proteasomal inhibitors 113. Moreover proteasomal inhibitors have been shown to retard the downregulation of both μ and δ opioid receptors 114. Receptor ubiquitination has been demonstrated for the latter two opioid receptors, albeit it is agonist independent. Both lysosomal and proteasomal inhibitors completely block agonist-induced degradation of the PAFR 66. Proteasomal inhibition blocks both internalization and degradation of the β2AR 59. While the biochemical inhibition of proteasomes provides strong evidence of a role for proteasomal activity in directing lysosomal degradation of internalized receptors, it is very likely that the effect is indirect. It is known that during endocytic travel, 7TMRs are complexed with a multitude of intracellular partners, including kinases, phosphatases, adaptors and cytoskeletal proteins. It is tempting to speculate that some of these accessory molecules need to be subjected to regulated degradation by the proteasome for optimal lysosomal 7TMR trafficking to occur. As such, obstruction of accessory molecule degradation in the proteasome would indirectly result in altered 7TMR trafficking and lysosomal degradation. Another interesting hypothesis is that both proteasomes and lysosomes cooperate to degrade membrane proteins. In this scenario, 7TMR intracellular domains are cleared by the proteasomes, while the remaining portion of the 7TMR is subsequently degraded in the lumen of the lysosome.

Driving in reverse: deubiquitination and 7TMR regulation

Importantly, ubiquitin attachment to substrate proteins is a reversible process. Ubiquitin itself is not degraded, but rather specialized enzymes called deubiquitinating enzymes remove the Ub moieties from modified proteins 115. A “housekeeping” deubiquitinating activity is normally associated with the 26S proteasomes 116. These enzymes are categorized as isopeptidases or Ub-C-terminal hydrolases (UCHs), and have molecular masses of less than 50 kDa. A second group of deubiquitinating enzymes called Ubiquitin Specific Proteases (USP) have molecular weights of > 100 kDa. Three additional groups of deubiquitinating enzymes are recognized based on their catalytic domain structures. These include otubain proteases (OTU), Machado-Joseph disease protease (MJD) and the JAMM metalloproteases that require Zn2+ for activity 115. In Saccharomyces cerevisiae, 16 USPs and 1 UCH (Yuh1p) have been identified in the genome. According to a recent genomic inventory, the human genome has 58 USP, 4 UCH, 5MJD, 14 OTU and 14 JAMM-containing genes 115. The high sequence diversity displayed by many USPs at the carboxyl and amino termini likely provides a basis for a breadth of substrate recognition and specificity.

The yeast deubiquitinases Doa4 and UBP3 are linked to vacuolar protein sorting and pheromone signaling, respectively 117,118. Faf (Fat facets, Drosophila compound eye) is a deubiquitinase that has been linked to endocytosis 119 acting on the substrate Lqf (liquid facets), an ortholog of the well-characterized clathrin adaptor, Epsin. Deubiquitinating enzymes have also been shown to play important roles in EGFR down regulation via the lysosomes 120. However, any role of deubiquitination in mammalian 7TMR trafficking remains to be determined. Recently, the deubiquitinating enzyme USP4 was shown to prevent the ubiquitination and proteasomal degradation of newly synthesized intracellular A2a adenosine receptors via the ERAD pathway (Fig 2 121). Moreover, USP4 expression facilitated robust functional expression of the A2a receptor at the plasma membrane, suggesting that deubiquitination can help cell-surface targeting of membrane proteins. Future studies should reveal if deubiquitinating enzymes play a role in the sorting mechanisms for ubiquitinated 7TMRs.

Impact of ubiquitination on cardiovascular functions

As detailed above, ubiquitination affects many aspects of 7TMR biology, including the regulation of cellular trafficking and downstream signaling events central to cardiovascular homeostasis. Indeed, cell surface receptor expression levels play a pivotal role in the regulation of cardiac tissue hormonal responsiveness. Interestingly, animal models of heart failure display high circulating levels of catecholamines but concomitantly decreased β-AR stimulation. Such decreased receptor stimulation results from both increased levels of uncoupled receptors (desensitization) and decreases in receptor density due to receptor downregulation 122. It is very tempting to speculate that dysregulation of a ubiquitination-mediated pathway could be a major factor in such pathophysiological settings. Further, since ubiquitination can direct the proteasomal degradation of additional components of 7TMR signaling, including GRK2, G proteins, MAPKs and the Ins(1,4,5)P3R, the role of ubiquitination in cardiac physiology is probably more intricate than currently appreciated. Examples of disease-causing alleles of a 7TMR or other downstream signaling components that are defective in ubiquitination and/or ubiquitin-mediated degradation have not yet been identified, but certainly pose exciting possibilities for future research.

Finally, the emerging role of the ubiquitin-proteasome system in maintaining control of cellular cardiac protein levels 123125, suggests that ubiquitination plays a broader role in cardiac biology, particularly in familial cardiomyopathies. Moreover, a few studies have also demonstrated that proteasomal inhibitors have anti-inflammatory effects and as such their short-term use is proposed to have beneficial effects in pathologic conditions associated with acute inflammation including myocardial infarction or stroke 126129. It is also likely that inhibitors of either ubiquitinating or deubiquitinating enzymes regulating various cardiovascular proteins, could have similar therapeutic uses.

Conclusions

Recent studies have uncovered an unanticipated role for ubiquitination in regulating mammalian 7TMR longevity and lysosomal sorting. Equally surprising and fascinating is the role that modification by ubiquitin plays in the modulation of receptor regulators such as GRKs and β-arrestins. Further, it appears that in addition to affecting multiple loci, namely, 7TMR, G protein, GRK, β-arrestin and Ins(1,4,5)P3 receptor in 7TMR pathways, ubiquitination is also capable of mediating discrete effects. As such, ubiquitination of 7TMRs targets them for lysosomal degradation, ubiquitination of GRKs targets them for proteasomal destruction, and ubiquitination of β-arrestin functions as a signal for rapid 7TMR endocytosis and facilitates the formation and intracellular targeting of receptor signalosomes. Thus, although all these processes are induced by an agonist and carried out by the addition of the same molecular tag ubiquitin, they result in very different molecular consequences.

At present, the activity of several different E3 ubiquitin ligases has been identified in the context of 7TMR ubiquitination. The present model, in which nonredundant E3 ligase activities have been demonstrated for different 7TMRs, suggests that the diversity of E3 ligases encoded in the genome likely helps to increase the specificity of the process. Alternatively, recruitment of a particular enzymatic activity may be prompted by the specific conformation of the activated receptor itself. However, further dissection of each molecular step, as well as the identification of novel members of both the ubiquitination and endocytic machinery, will be necessary to fully appreciate and understand the roles of this ubiquitous modification in the regulation of 7TMR biology.

Acknowledgments

I thank Drs. Robert J. Lefkowitz and Matthew T. Drake for critically reviewing this manuscript. This work was supported by National Institutes of Health grant HL080525 and American Heart Association grant 0530014N.

Footnotes

None

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