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Vol. 53, Issue 1, 1-24, March 2001

Evolving Concepts in G Protein-Coupled Receptor Endocytosis: The Role in Receptor Desensitization and Signaling

Stephen S. G. Ferguson1

The John P. Robarts Research Institute and Departments of Physiology, Pharmacology and Toxicology, and Medicine, University of Western Ontario, London, Ontario, Canada

Abstract
I. Introduction
II. G Protein-Coupled Receptor Desensitization
    A. Protein Kinase Phosphorylation
        1. Second Messenger-Dependent Protein Kinases.
        2. G Protein-Coupled Receptor Kinases.
            a. The G Protein-Coupled Receptor Kinase Family.
            b. Targeting and Regulation.
            c. Site of Action.
        3. Other Kinases.
    B. The Arrestins
        1. The Arrestin Family.
        2. Receptor Binding.
III. G Protein-Coupled Receptor Internalization
    A. Molecular Mechanisms Involved in G Protein-Coupled Receptor Endocytosis
        1. Role of G Protein-Coupled Receptor Kinase and beta -Arrestin Proteins.
        2. Clathrin and beta -Adaptin Interactions.
        3. beta -Arrestin Regulation.
        4. Alternative G Protein-Coupled Receptor Endocytic Pathways.
        5. Receptor Determinants for Endocytosis.
    B. Biological Role of G Protein-Coupled Receptor Internalization
        1. Endocytosis and G Protein-Coupled Receptor Desensitization.
        2. Endocytosis and G Protein-Coupled Receptor Resensitization.
        3. Endocytosis and G Protein-Coupled Receptor Signaling.
IV. Conclusions
Acknowledgments
References


    Abstract
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G protein-coupled receptors (GPCRs) are seven transmembrane proteins that form the largest single family of integral membrane receptors. GPCRs transduce information provided by extracellular stimuli into intracellular second messengers via their coupling to heterotrimeric G proteins and the subsequent regulation of a diverse variety of effector systems. Agonist activation of GPCRs also initiates processes that are involved in the feedback desensitization of GPCR responsiveness, the internalization of GPCRs, and the coupling of GPCRs to heterotrimeric G protein-independent signal transduction pathways. GPCR desensitization occurs as a consequence of G protein uncoupling in response to phosphorylation by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes the binding of beta -arrestins, which not only uncouple receptors from heterotrimeric G proteins but also target many GPCRs for internalization in clathrin-coated vesicles. beta -Arrestin-dependent endocytosis of GPCRs involves the direct interaction of the carboxyl-terminal tail domain of beta -arrestins with both beta -adaptin and clathrin. The focus of this review is the current and evolving understanding of the contribution of GRKs, beta -arrestins, and endocytosis to GPCR-specific patterns of desensitization and resensitization. In addition to their role as GPCR-specific endocytic adaptor proteins, beta -arrestins also serve as molecular scaffolds that foster the formation of alternative, heterotrimeric G protein-independent signal transduction complexes. Similar to what is observed for GPCR desensitization and resensitization, beta -arrestin-dependent GPCR internalization is involved in the intracellular compartmentalization of these protein complexes.


    I. Introduction
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G protein-coupled receptors (GPCRs2) constitute a superfamily of seven transmembrane spanning proteins that respond to a diverse array of sensory and chemical stimuli, such as light, odor, taste, pheromones, hormones, and neurotransmitters. GPCRs transduce the information provided by these stimuli into intracellular second messengers that are interpreted as meaningful signals by the cell. This process involves the coupling of agonist-activated GPCRs to a wide variety of effector systems via their interaction with heterotrimeric guanine nucleotide binding proteins (G proteins). The binding of agonist to a GPCR selects for a receptor conformation state that promotes the exchange of GDP for GTP on the G protein alpha -subunit and is presumed to allow the dissociation of the G protein Galpha - and Gbeta gamma -subunits (Neer, 1995; Surya et al., 1998). Subsequently, the activated Galpha - and Gbeta gamma -subunits positively and/or negatively regulate the activity of effector enzymes and ion channels (reviewed by Neer et al., 1995; Gautam et al., 1998). Agonist activation of a GPCR not only results in the G protein-dependent activation of effector systems, but also sets in place a series of molecular interactions that allows for: 1) feedback regulation of G protein coupling, 2) receptor endocytosis, and 3) signaling through G protein-independent signal transduction pathways (Lefkowitz, 1993; Ferguson et al., 1996a; Ferguson and Caron, 1998; Krupnick and Benovic, 1998; Hall et al., 1999; Luttrell et al., 1999a; Schoneberg et al., 1999). Thus, in the relatively few years since the cloning of the first GPCRs (Nathans and Hogness; 1983; Dixon et al., 1986), work done in a large number of laboratories has made it apparent that the functional activity of GPCRs extends far beyond the traditional model of: receptor right-arrow G protein right-arrow effector.

GPCR activity represents a coordinated balance between molecular mechanisms governing receptor signaling, desensitization, and resensitization. Receptor desensitization, the waning of GPCR responsiveness to agonist with time, represents an important physiological "feedback" mechanism that protects against both acute and chronic receptor overstimulation. GPCR desensitization also acts to filter information from multiple receptor inputs into an integrated and meaningful biological signal through second messenger protein kinase-dependent phosphorylation and inactivation of weaker receptor-mediated signals. However, GPCR desensitization can also significantly limit the therapeutic usefulness of many receptor agonists.

Three families of regulatory molecules are known to contribute to the GPCR desensitization process: second messenger-dependent protein kinases, G protein-coupled receptor kinases (GRKs) and arrestins (reviewed by Lefkowitz, 1993; Ferguson et al., 1996a; Ferguson and Caron, 1998; Krupnick and Benovic, 1998). As will be documented in the present review, it is now recognized that the same regulatory molecules that contribute to agonist-stimulated receptor desensitization (GRKs and beta -arrestins), initiate and regulate GPCR endocytosis, intracellular trafficking, and resensitization (e.g., Tsuga et al., 1994; Ferguson et al., 1995, 1996b; Zhang et al., 1997; Oakley et al., 1999).

In addition to signaling via heterotrimeric G proteins, it is now recognized that GPCRs act as scaffolds promoting the formation and compartmentalization of G protein-independent signal transduction complexes. A growing number of proteins have been identified that bind GPCRs and either couple GPCRs to G protein-independent signal transduction pathways or alter G protein specificity and agonist selectivity. The list of GPCR interacting proteins now includes: GRKs (Benovic et al., 1991), arrestins (Lohse et al., 1990a), calmodulin (Minakami et al., 1997; Thomas et al., 1999; Wang et al., 1999), calcyon (Lezcano et al., 2000), A kinase-anchoring protein (AKAP) (Fraser et al., 2000), ATRAP (Daviet et al., 1999), tubulin (Ciruela et al., 1999), receptor activity modulating proteins (McLatchie et al., 1998), Homer (Brakeman et al., 1997), Janus kinase 2 (Marrero et al., 1995), PDZ domain-containing proteins (e.g., NHERF, RGS12), (Hall et al., 1998; Snow et al., 1998) SH3 domain-containing adaptor molecules (e.g., Grb2, Nck, c-Src, and endophilin) (Oldenhof et al., 1998; Tang et al., 1999; Cao et al., 2000), and small G proteins (Mitchell et al., 1998). Although this list of GPCR-interacting proteins is expanding rapidly, the global impact of these protein interactions on GPCR signaling has yet to be precisely determined. The potential contribution of these interactions to GPCR signaling has been reviewed previously (Bockaert and Pin; 1999; Hall et al., 1999).

The present review will focus on our current, yet evolving, understanding of the molecular mechanism(s) involved in GPCR endocytosis, as well as the contribution of receptor endocytosis to the regulation of GPCR signaling. The identification of the molecular mechanisms underlying GPCR endocytosis has progressed rapidly in recent years. However, as new information becomes available regarding the multitude of potential molecular interactions between GPCRs, their regulatory proteins and the cellular endocytic machinery, it is becoming clear that GPCR endocytosis is regulated by a myriad of complex determinants. Although many of the molecular mechanisms first described for the beta 2-adrenergic receptor (beta 2AR) might apply equally well to other GPCRs, this is more likely to be an exception rather than the rule. Thus, it can be anticipated that the diversity in GPCR structure/function will lead to important differences in the intracellular trafficking patterns of distinct GPCR subtypes. Therefore, the challenge awaiting researchers in the field will be to understand the reasons for observed differences in GPCR subtype regulation. Consequently, this review will not only focus on well established paradigms of GPCR regulation, but will also discuss the role of GPCR endocytosis in shifting the traditional paradigms for GPCR regulation and signaling.


    II. G Protein-Coupled Receptor Desensitization
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The exposure of GPCRs to agonists often results in a rapid attenuation of receptor responsiveness. This process, termed desensitization, is the consequence of a combination of different mechanisms. These mechanisms include the uncoupling of the receptor from heterotrimeric G proteins in response to receptor phosphorylation (e.g., Bouvier et al., 1988; Hausdorff et al., 1989; Lohse et al., 1990a,b), the internalization of cell surface receptors to intracellular membranous compartments (e.g., Hermans et al., 1997; Trejo and Coughlin, 1998; Oakley et al., 1999; Anborgh et al., 2000), and the down-regulation of the total cellular complement of receptors due to reduced receptor mRNA and protein synthesis, as well as both the lysosomal and plasma membrane degradation of pre-existing receptors (e.g., Doss et al., 1981; Hadcock and Malbon 1988; Valiquette et al., 1990, 1995; Jockers et al., 1999; Pak et al., 1999). The time frames over which these processes occur range from seconds (phosphorylation) to minutes (endocytosis) and hours (down-regulation). The extent of receptor desensitization varies from complete termination of signaling, as observed in the visual and olfactory systems, to the attenuation of agonist potency and maximal responsiveness, such as observed for the beta 2AR (Pippig et al., 1995; Zhang et al., 1997; Sakmar, 1998). The extent of receptor desensitization is regulated by a number of factors that include receptor structure and cellular environment (Jockers et al., 1996; Aramori et al., 1997; Menard et al., 1997; Barlic et al., 1999). Since GPCR endocytic mechanisms are intimately linked to the molecular events that contribute to the desensitization of GPCR responsiveness, a clear understanding of these desensitization processes is required.

Traditionally, GPCR desensitization has been characterized by events that contribute to the uncoupling of receptors from their heterotrimeric G proteins. Thus, for the purpose of this review, the term desensitization refers solely to the uncoupling of GPCRs from G protein-mediated signaling pathways. However, GPCR signaling can also be terminated at the level of the heterotrimeric G protein. For example, a family of proteins, termed regulators of G protein signaling (RGS) act to increase the rate of hydrolysis of GTP bound to both Gi and Gq alpha -subunits, thereby dampening signaling via Gi- and Gq-regulated signaling pathways (reviewed by Dohlman and Thorner, 1997; Siderovski et al., 1999). The recent demonstration that RGS12 interacts with the carboxyl-terminal PDZ domain binding motif of the interleukin-8 receptor B (CXCR2) suggests that the regulation of G protein signaling by RGS proteins may also involve direct interactions with the receptor (Snow et al., 1998).

A. Protein Kinase Phosphorylation

The most rapid means by which GPCRs are uncoupled from heterotrimeric G proteins is through the covalent modification of the receptor as a consequence of phosphorylation by intracellular kinases. It is generally accepted that both second messenger-dependent protein kinases [e.g., cAMP-dependent protein kinase (PKA) and protein kinase C (PKC)] and GRKs phosphorylate serine and threonine residues within the intracellular loop and carboxyl-terminal tail domains of GPCRs (reviewed by Lefkowitz, 1993; Ferguson et al., 1996a; Ferguson and Caron, 1998; Krupnick and Benovic, 1998). GRK family members selectively phosphorylate agonist-activated receptors, thereby promoting the binding of cytosolic cofactor proteins called arrestins, which sterically uncouple the receptor from heterotrimeric G protein (Benovic et al., 1987; Lohse et al., 1990b, Pippig et al., 1993). In contrast, second messenger-dependent protein kinases not only phosphorylate agonist-activated GPCRs, but also indiscriminately phosphorylate receptors that have not been exposed to agonist (Hausdorff et al., 1989; Lohse et al., 1990a). Thus, agonist-independent phosphorylation is a property that has generally been ascribed only to second messenger-dependent protein kinases and not GRKs (Lefkowitz, 1993). Nevertheless, it is now recognized that GPCRs spontaneously isomerize to an activated conformation in the absence of agonist, which suggests that GRKs may also contribute to the regulation of basal GPCR activity (Pei et al., 1994; Rim and Oprian, 1995). Second messenger-dependent protein kinases are also thought to represent the predominant mechanisms by which GPCR desensitization is achieved at low agonist concentrations. However, in young hypertensive patients, elevated GRK2 protein levels are correlated with enhanced beta 2AR desensitization in response to low levels of circulating catecholamines (Gros et al., 1997). This observation demonstrates the need to re-evaluate the relative contributions of second messenger-dependent protein kinases and GRKs to receptor/G protein uncoupling at low agonist concentrations. Moreover, second messenger-dependent protein kinase and GRK activities may not be independent from each other since, in the olfactory system, inhibition of either kinase family results in the complete abolition of olfactory receptor desensitization (Schleicher et al., 1993; Boekhoff et al., 1994). Thus, the relative contributions and mechanisms by which second messenger-dependent protein kinases and GRKs regulate GPCR desensitization are not fully understood and may be more complex than originally envisaged. GRKs also contain amino-terminal RGS domains, suggesting that they may not only regulate GPCR signaling at the level of the receptor, but also regulate the activity of the G protein as well (Carman et al., 1999; Sallese et al., 2000). The ability of GRKs to serve as RGS-like proteins may account for the phosphorylation-independent desensitization of the parathyroid hormone receptor responsiveness in response to GRK protein overexpression (Dicker et al., 1999).

1. Second Messenger-Dependent Protein Kinases. The second messenger-dependent protein kinases, PKA and PKC, are phosphotransferases that catalyze the transfer of the gamma -phosphate group of ATP to serine and threonine residues contained within specific amino acid consensus sequences of proteins. Second messenger-dependent protein kinase are activated in response to GPCR-stimulated increases in intracellular second messengers such as cAMP, Ca2+, and diacyglycerol and participate in GPCR signaling by mediating the phosphorylation of downstream target proteins. However, these kinases also feedback phosphorylate GPCRs at phosphorylation consensus sites within their intracellular loops and carboxyl-terminal tail domains. For example, beta 2AR-activated PKA activity leads to receptor desensitization in response to PKA-mediated phosphorylation of at least one of two PKA consensus sites within the receptor (Bouvier et al., 1988; Yuan et al., 1994; Moffet et al., 1996). One site is found within the G protein-binding domain of the third intracellular loop of the beta 2AR and the other site is found within the proximal region of the beta 2AR carboxyl-terminal tail (Bouvier et al., 1988; Yuan et al., 1994; Moffett et al., 1996). It is proposed that covalent modification of the beta 2AR at only the PKA site within the third intracellular loop domain contributes to receptor/G protein coupling (Yuan et al., 1994). However, phosphorylation of the PKA site within the beta 2AR carboxyl-terminal tail occurs subsequent to the depalmitoylation of cysteine residue 341, suggesting that this site contributes to the agonist-dependent desensitization of beta 2AR responsiveness (Moffet et al., 1996). Two recent studies suggest that PKA-mediated phosphorylation of the beta 2AR involves the direct and constitutive association of the AKAP (AKAP79/150 and AKAP250) with the receptor (Fraser et al., 2000; Lin et al., 2000). PKC activation leads to the phosphorylation and desensitization of many Gi- and Gq-linked GPCRs (e.g., Diviani et al., 1997; Liang et al., 1998; Tang et al., 1998). Nonetheless, second messenger-dependent protein kinase-mediated mechanisms of receptor desensitization have received less attention than GRK-mediated mechanisms of receptor desensitization.

2. G Protein-Coupled Receptor Kinases. a. The G Protein-Coupled Receptor Kinase Family. The GRK family of kinases is comprised of seven family members that share significant sequence homology (reviewed by Premont et al., 1995; Stoffel et al., 1997) (Table 1). Each of the GRKs share a similar functional organization with a central catalytic domain, an amino-terminal domain that is thought to be important for substrate recognition and that contains an RGS-like domain, and a carboxyl-terminal domain that contributes to the plasma membrane targeting of the kinase (Fig. 1). The members of the GRK family can be subdivided into three groups based on sequence and functional homology: 1) GRK1 (rhodopsin kinase) (Shichi and Somers, 1978) and GRK7 (a new candidate cone opsin kinase) (Weiss et al., 1998); 2) GRK2 (beta -adrenergic receptor kinase 1, beta ARK1) (Benovic et al., 1986) and GRK3 (beta -adrenergic receptor kinase 2, beta ARK2) (Benovic et al., 1991); and 3) GRK4 (Premont et al., 1994; Sallese et al., 1994), GRK5 (Kunapuli and Benovic, 1993), and GRK6 (Benovic and Gomez, 1993).


                              
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TABLE 1
Characteristics of GRK family members


                              
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TABLE 2
Characteristics of arrestin family members



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Fig. 1.   Schematic representation of the domain architecture for GRK1-GRK7. The amino-terminal GPCR-binding domain of GRK1-GRK7 contains a conserved RGS domain (Carman et al., 1999). The plasma membrane targeting of each of the GRKs is mediated by distinct mechanisms that involves their carboxyl-terminal domains. GRK1 and GRK7 are farnesylated at CAAX motifs in their carboxyl termini (Inglese et al., 1992). The carboxyl-terminal domains of GRK2 and GRK3 contain a beta gamma -subunit binding domain that exhibits sequence homology to a pleckstrin homology domain (Pitcher et al., 1992; Touhara et al., 1994). The GRK5 carboxyl-terminal domain contains a stretch of 46 basic amino acids that mediate plasma membrane phospholipid interactions (Kunapuli et al., 1994; Premont et al., 1994). GRK4 and GRK6 are palmitoylated at cysteine residues (Stoffel et al., 1994, 1998). Figure adapted from Stoffel et al., 1997.

b. Targeting and Regulation. In unstimulated cells, GRK1-3 are localized to the cytosol and translocate to bind their substrates in response to the agonist activation of their plasma membrane-bound receptor targets. For GRK1, the light-activated association of the kinase with the plasma membrane is facilitated by the post-translational farnesylation of its carboxyl-terminal CAAX motif (Inglese et al., 1992). The activity of GRK1, but not GRK2-6, can be regulated by the calcium sensor protein recoverin (Iacovelli et al., 1999). Although GRK2 and GRK3 are not isoprenylated, the plasma membrane translocation of these kinases is regulated in part by their association with the beta gamma -subunit of heterotrimeric G proteins (Pitcher et al., 1992; Boekhoff et al., 1994). The association of GRK2 and GRK3 with G protein beta gamma -subunits is mediated by a 125 amino acid beta gamma -subunit-binding domain in the carboxyl termini of the kinases that bears striking sequence homology with pleckstrin homology domains (Koch et al., 1993; Touhara et al., 1994) (Fig. 1). Membrane translocation of endogenous GRK2 can be impaired by the overexpression of the carboxyl-terminal beta gamma -binding domain of GRK2, which presumably acts to sequester free G protein beta gamma -subunits (Koch et al., 1993). The expression of the beta gamma -binding domain has been used to block GRK-mediated desensitization in both in vitro cell culture systems and in vivo using transgenic mice (Koch et al., 1993, 1995; Dicker et al., 1999). The targeting of GRK2 and GRK3 to the plasma membrane is also influenced by phosphatidylinositol 4,5-bisphosphate binding to the carboxyl-terminal pleckstrin homology domain of the kinases (Pitcher et al., 1995a). Recently, it was demonstrated that mitogen-activated protein kinase (MAPK) phosphorylation of the GRK2 carboxyl-terminal domain decreased the efficacy of the kinase toward GPCR substrates (Pitcher et al., 1999; Elorza et al., 2000). In contrast, GRK2 activity and plasma membrane translocation are potentiated in response to serine phosphorylation by both PKC and tyrosine phosphorylation by c-Src (Chuang et al., 1995; Winstel et al., 1996; Sarnago et al., 1999). Consequently, GRK2 activity seems to be regulated by a complex series of protein phosphorylation events.

In the absence of GPCR activation by agonist, GRK4, GRK5, and GRK6 all exhibit substantial membrane localization. Both GRK4 and GRK6 are palmitoylated on carboxyl-terminal cysteine residues (Stoffel et al., 1994; Premont et al., 1996; Stoffel et al., 1998) (Fig. 1). The palmitoylation of these kinases seems essential for their plasma membrane localization since only the palmitoylated form of these kinases is isolated from membrane fractions (Stoffel et al., 1994; Premont et al., 1996; Stoffel et al., 1998). Moreover, palmitoylated GRK6 is 10-fold more active at phosphorylating beta 2AR than the nonpalmitoylated GRK6 (Stoffel et al., 1998). Since protein palmitoylation is a reversible post-translational protein modification, dynamic regulation of the palmitoylation state of GRK4 and GRK6 may have important effects on the functional activity of these kinases.

GRK5 association with the plasma membrane is thought to be mediated by the electrostatic interaction between 46 highly basic amino acid residues contained within the carboxyl terminus of the kinase and plasma membrane phospholipids (Kunapuli et al., 1994; Premont et al., 1994) (Fig. 1). The activity of the GRK5 enzyme is not only influenced by autophosphorylation of serine and threonine residues in the carboxyl terminus of the kinase, but also by the binding of membrane phospholipids (Kunapuli et al., 1994). Unlike GRK2, PKC-mediated phosphorylation reduces GRK5 activity (Chuang et al., 1996). In addition, calmodulin associates directly with the amino-terminal domain of GRK5 and not only reduces the ability of the kinase to bind both receptor and phospholipids, but also inhibits the activity of the kinase by stimulating autophosphorylation of serine and threonine residues that are distinct from those involved in the activation of the kinase (Pronin and Benovic, 1997; Pronin et al., 1997; Iacovelli et al., 1999). Since the activation of PKC, Ca2+-calmodulin, and/or phospholipid metabolism is stimulated by some, but not all GPCRs, it is likely that GPCR subtype differences in the stimulation of GRK5 activity will be observed.

c. Site of Action. GRKs phosphorylate GPCRs at both serine and threonine residues localized within either the third intracellular loop or carboxyl-terminal tail domains. Some GPCRs, for example the alpha 2-adrenergic receptor (alpha 2AR) and m2 muscarinic acetylcholine receptor (mAChR), have short carboxyl-terminal tails containing relatively few serine and threonine residues, but have enlarged third intracellular loop domains containing multiple serine and threonine residues. In contrast, receptors such as rhodopsin and the beta 2AR have relatively short third intracellular loops but have long carboxyl-terminal tails containing several serine and threonine residues. Mutation of all of the serine and threonine residues within either the carboxyl-terminal tail of the beta 2AR or the third intracellular loop of the m2 mAChR abolishes GRK-mediated phosphorylation of these receptors (Bouvier et al., 1988; Nakata et al., 1994). Although no distinct GRK phosphorylation consensus motifs have been identified, localization of acidic amino acid residues proximal to the site of phosphorylation seems to favor GRK2-mediated phosphorylation (Onorato et al., 1991; Chen et al., 1993).

The stoichiometry of GRK phosphorylation differs, depending upon the GPCR studied. Nonetheless, whereas GRKs phosphorylate receptors on many sites in vitro, it is thought that, at least in the case of rhodopsin, receptor desensitization requires only the initial phosphorylation event (Ohguro et al., 1993). In addition, high-affinity binding of arrestins to rhodopsin and beta 2AR requires GRK phosphorylation to a stoichiometry of only 2 mol of phosphate per mole of receptor in vitro (Gurevich et al., 1995). However, mutation of the primary GRK-phosphorylated residues on the beta 2AR identified in vitro (Fredericks et al., 1996) did not prevent GRK-mediated beta 2AR desensitization in cells (Seibold et al., 1998). Thus, the primary sites of GRK phosphorylation identified in vitro may not necessarily represent the GRK-phosphorylated residues in vivo. Alternatively, the phosphorylation of secondary GRK phosphorylation sites may compensate for the loss of the primary site for GRK-mediated phosphorylation. It is also becoming apparent that the GRK-mediated phosphorylation of clusters of serine and threonine residues in the carboxyl-terminal tails of some receptors may regulate the stability of receptor/arrestin complexes (Oakley et al., 1999). Thus, it is likely that difference in the stoichiometry of GRK-mediated phosphorylation of GPCR subtypes may underlie observed differences in the intracellular trafficking and signaling of desensitized receptors (see Section III.B.).

3. Other Kinases. In addition to serving as substrates for PKA, PKC, and GRK phosphorylation, GPCRs have been shown to serve as substrates for phosphorylation by other protein kinases. Casein kinase 1a-mediated phosphorylation of the third intracellular loop domain of the m3 mAChR occurs in response to agonist activation of the receptor (Tobin et al., 1997; Budd et al., 2000). Moreover, casein kinase 1a-mediated phosphorylation of the m3 mAChR was blocked by either the expression of a catalytically inactive casein kinase 1a mutant or a peptide corresponding to the third intracellular loop domain of the m3 mAChR (Budd et al., 2000). Nonetheless, the functional consequence of casein kinase 1a receptor phosphorylation remains to be fully elucidated (Budd et al., 2000). The presence of casein kinase phosphorylation consensus motifs within the intracellular loop and carboxyl-terminal tail domains of many GPCRs makes these observations particularly intriguing.

There is evidence that tyrosine kinase-mediated GPCR phosphorylation may influence the activity of some GPCRs. Mutagenesis of tyrosine residues in the carboxyl-terminal tail of the µ opioid receptor reduced the agonist stimulated down-regulation of the receptor (Pak et al., 1999). This effect could be mimicked using the tyrosine kinase inhibitor genistein (Pak et al., 1999). Insulin-stimulated tyrosine phosphorylation of the beta 2AR has also been reported (Valiquette et al., 1995). However, whereas carboxyl-terminal tyrosine residues contributed to beta 2AR down-regulation, these residues were not the substrates for insulin-promoted tyrosine phosphorylation (Valiquette et al., 1990, 1995). Tyrosine phosphorylation of the bradykinin B2 receptor in response to agonist can be prevented by genistein treatment and seems to contribute to receptor signaling leading to arachidonic acid release (Jong et al., 1993). The identity of the tyrosine kinase(s) mediating the phosphorylation of these receptors is unknown.

B. The Arrestins

GRK-mediated phosphorylation of either rhodopsin or the beta 2AR was not sufficient to promote the full inactivation of these GPCRs; full inactivation required an additional component or "arresting agent". The identification of an arresting protein was first made in rod outer segments where a 48-kDa protein, now called visual arrestin, was demonstrated to bind light-activated rhodopsin (Pfister et al., 1985). Subsequently, a visual arrestin-like protein, beta -arrestin1, was identified as a cofactor required for GRK2-mediated beta 2AR desensitization in vitro (Benovic et al., 1987). The cloning of beta -arrestin1 revealed 59% sequence homology with visual arrestin (Lohse et al., 1990a). The role of arrestins in regulating the desensitization of GPCRs has been demonstrated in intact cells (Pippig et al., 1995; Zhang et al., 1997), in the Drosophila photosystem in vivo (Dolph et al., 1993), and in mice either lacking visual arrestin or beta -arrestin2 (Xu et al., 1997; Bohn et al., 1999). The mechanism(s) by which arrestins contribute to GPCR desensitization involves both the physical uncoupling of GPCRs from heterotrimeric G proteins (visual arrestins and beta -arrestins) and the targeting of GPCRs for endocytosis (beta -arrestins) (see Sections II.B.2. and III.B.1.).

1. The Arrestin Family. To date, four arrestin family members have been identified (Table 1). The members of the arrestin family can be divided into two groups based on sequence homology, function, and tissue distribution: 1) visual arrestin (S antigen) (Shinohara et al., 1987; Yamaki et al., 1987) and cone arrestin (X-arrestin or C-arrestin) (Murakami et al., 1993; Craft et al., 1994) and 2) beta -arrestins (beta -arrestin1 and beta -arrestin2) (Lohse et al., 1990a; Attramadal et al., 1992). Visual arrestin is a major protein constituent of rod outer segments and is localized primarily to the retina with low expression in the pineal gland (Smith et al., 1994). C-Arrestin is highly enriched in both retina and pineal gland, but is localized primarily within cone photoreceptors in the retina (Craft et al., 1994). The beta -arrestins are ubiquitously expressed outside the retina, but are predominantly localized in neuronal tissues and in the spleen (Attramadal et al., 1992). In the rat central nervous system beta -arrestin2 is more abundant than beta -arrestin1 (Attramadal et al., 1992). The evaluation of beta -arrestin1 and beta -arrestin2 protein expression in the brain reveals extensive, heterogenous neuronal labeling (Attramadal et al., 1992). beta -Arrestin protein is found in several neuronal pathways and immunoelectron microscopy reveals that beta -arrestins are concentrated at neuronal synapses along with GRKs (Arriza et al., 1992; Attramadal et al., 1992). Thus, these proteins are ideally localized to modulate neuronal function. A third family of arrestin proteins might exist, since partial cDNA clones for D- and E-arrestin have been reported (Craft et al., 1994). However, although the mRNAs for D- and E-arrestin are expressed in a broad range of tissues, there is still question whether full-length D- and E-arrestin proteins truly exist (Craft et al., 1994).

Alternative splice variants have been identified for visual arrestin, beta -arrestin1, and beta -arrestin2. Bovine visual arrestin is expressed as a 404 amino acid residue protein, as well as two polypetide variants, one for which the last 35 amino acid residues are replaced by an alanine residue (p44) and another that lacks residues 338-345 encoded by exon 13 (Yamaki et al., 1987, 1990; Smith et al., 1994). The p44 visual arrestin variant is specifically localized to the rod outer segment and is severalfold more potent an inhibitor of rhodopsin signal transduction than the long form (Palczewski et al., 1994). Thus, the carboxyl-terminal domain of visual arrestin does not seem to be important for binding to rhodopsin. This observation has been confirmed using beta -arrestin truncation mutants (Gurevich, 1998). However, it is the beta -arrestin carboxyl-terminal domain that distinguishes these arrestin isoforms from visual arrestins (Fig. 2, A and B). Similar to visual arrestin, at least two alternatively spliced forms of beta -arrestin1 and beta -arrestin2 are expressed (Parruti et al., 1993b; Sterne-Marr et al., 1993). The variant form of beta -arrestin1 involves the insertion of eight amino acid residues between amino acids 333 and 334, and the variant form of beta -arrestin2 involves the insertion of 11 amino acid residues between amino acids 361-362 (Parruti et al., 1993b; Sterne-Marr et al., 1993). Although the existence of alternatively spliced variant beta -arrestin isoforms increases the potential number of functionally distinct beta -arrestins, there are no reported differences in the functional activity of the beta -arrestin variants. However, considering the limited number of arrestins and the preponderance of GPCR subtypes, it is likely that receptor specificity is governed by discrete differences in receptor structure and tissue-specific arrestin protein expression patterns rather than by a multitude of different arrestin isoforms.



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Fig. 2.   Molecular architecture of arrestins. Panel A, the arrestin regulatory domains identified by the solution of the visual arrestin crystal structure (Hirsch et al., 1999) and mutagenesis studies (Gurevich et al., 1995). R1, amino terminal regulatory domain (residues 1-24); A, receptor activation domain (residues 24-180); P, phosphate sensor domain (residues 163-182); S, secondary receptor-binding domain (residues 180-330); and R2, carboxyl-terminal regulatory domain (residues 330-404). Underlined is the proline-rich region conserved in mammalian beta -arrestin1 and beta -arrestin2 but not visual arrestins (Luttrell et al., 1999). The arrow points to serine residue 412, which is phosphorylated by MAPK (Lin et al., 1999). The black box highlights the clathrin- and beta -adaptin-binding domains that are conserved among nonvisual arrestins. Panel B, sequence alignment of visual and nonvisual arrestins from different species. The alignment highlights the functional differences between visual versus nonvisual arrestins that arise as the consequence of the clathrin (bold) and beta 2-adaptin (bold and asterisked) binding domains among nonvisual arrestins (Krupnick et al. 1997; Laporte et al., 2000).

2. Receptor Binding. Arrestins preferentially bind to agonist-activated and GRK-phosphorylated GPCRs as opposed to second messenger protein kinase-phosphorylated or nonphosphorylated receptors (Lohse et al., 1990a, 1992). In vitro, the affinity of beta -arrestin binding to the beta 2AR is increased 10- to 30-fold by GRK phosphorylation (Lohse et al., 1992), and this selectivity is even more pronounced for visual arrestin binding to rhodopsin (Gurevich et al., 1995). GRK phosphorylation occurs within either the third intracellular loop domain (e.g., m2 mAChR and alpha 2AAR) or the carboxyl-terminal tails of receptors (e.g., rhodopsin and beta 2AR) (Bouvier et al., 1988; Nakata et al., 1994; Eason et al., 1995; Brannock et al., 1999). Thus, to bind and interdict the signaling of multiple distinct GPCR subtypes, arrestins must exhibit the capacity to recognize and bind multiple receptor domains and conformations. This idea is supported by the following observations: 1) the interaction of visual arrestin with rhodopsin can be blocked by synthetic peptides representing the first and third intracellular loops of rhodopsin (Krupnick et al., 1994); 2) beta -arrestin can be coimmunoprecipitated with the third intracellular loop domains of the m3 mAChR, alpha 2AAR, and 5-hydroxytryptamine2A receptor (Wu et al., 1997; Gelber et al., 1999); and 3) the beta 2AR carboxyl-terminal tail is not absolutely required for beta -arrestin binding (Ferguson et al., 1996b). Gurevich et al. (1995) examined the ability of several arrestins to bind to various functional forms of rhodopsin, beta 2AR, and m2 mAChR. Although each of the arrestin isoforms demonstrated preference for binding to the GRK-phosphorylated agonist-activated form of the receptors, there was also substantial binding to phosphorylated nonactivated receptors, as well as agonist-activated nonphosphorylated receptors (Gurevich et al., 1995). This suggests that, depending on the GPCR isoform studied, agonist-independent beta -arrestin association may be observed (Anborgh et al., 2000). The clear exception is visual arrestin, which binds selectively to only GRK-phosphorylated and light-activated rhodopsin (Gurevich et al., 1995). In addition, the site of phosphorylation within the rhodopsin carboxyl-terminal tail appears critical for visual arrestin-dependent quenching of rhodopsin activity (Brannock et al., 1999).

The recent solution of the crystal structure for arrestin, together with mutagenesis studies, has provided further insight into the molecular events involved in arrestin binding to phosphorylated light-activated rhodopsin (Gurevich et al., 1995; Granzin et al., 1998; Vishnivetskiy et al., 1999; Hirsch et al., 1999). With respect to receptor binding, mutagenesis studies revealed that the molecular structure of visual arrestin can be divided into three functional and two regulatory domains (Gurevich et al., 1995). The functional domains include: a receptor activation recognition domain (amino acid residues 24-180), a secondary receptor binding domain (amino acids 180-330), and a phosphate sensor domain (amino acid residues 163-182). The regulatory domains are comprised of an amino-terminal regulatory domain (amino acid residues 1-24) and a carboxyl-terminal regulatory domain (amino acid residues 330-404) (Fig. 2A). The solution of the visual arrestin crystal structure is consistent with these observations and reveals that visual arrestin is comprised of two major functional domains that are each constructed from a seven-stranded beta  sandwich (Granzin et al., 1998; Hirsch et al., 1999). The two domains, the N domain (amino acid residues 8-180) and C domain (amino acid residues 188-362), respectively, comprise the activation recognition and secondary receptor binding domains originally identified by mutational analysis (Fig. 2A). The carboxyl-terminal tail of visual arrestin (amino acids 372-404) is connected to the C domain by a flexible linker, and the carboxyl-terminal tail forms various interactions with parts of the arrestin N and C domains to regulate their structure. The phosphate sensor domain, identified by mutagenesis, constitutes a polar core that in the basal state is embedded between the N and C domains and forms the fulcrum of the arrestin molecule. It is likely that this core structure is highly conserved among arrestin isoforms. Residues from both the amino-terminal and carboxyl-terminal regulatory domains (Asp-30 and Arg-382) also contribute to the polar core of visual arrestin. It is predicted from both mutagenesis studies and the crystal structure for visual arrestin that the interaction of the carboxyl-terminal tail with the polar core stabilizes the basal state structure of visual arrestin (Gurevich et al., 1995; Hirsch et al., 1999). However, in response to receptor binding, the phosphorylated receptor tail invades the polar core, thereby disrupting polar residues and releasing the arrestin carboxyl-terminal tail. This leads to the reorientation of the N and C domains along the fulcrum formed by the polar core facilitating the formation of a receptor-arrestin complex (Hirsch et al., 1999). This model is consistent with the observation that the p44 visual arrestin isoform exhibits greater affinity for rhodopsin and that the mutation of polar residues within the polar core of the visual arrestin protein results in arrestin mutants able to bind nonphosphorylated rhodopsin (Palczewski et al., 1994; Vishnivetskiy et al., 1999). Thus, it can be concluded that the conformation of free arrestin has evolved to resist agonist- and phosphorylation-independent interactions with receptors. This may be further ensured by the formation of arrestin oligomers (Schubert et al., 1999).


    III. G Protein-Coupled Receptor Internalization
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An important aspect of GPCR activity and regulation is the internalization or sequestration of agonist-activated receptors into the intracellular membrane compartments of the cell. GPCR internalization has become the subject of intensive investigation over the past several years (reviewed by Sterne-Marr and Benovic, 1995; Ferguson et al., 1996a; Bohm et al., 1997a; Ferguson and Caron, 1998; Krupnick and Benovic, 1998). Consequently, a large volume of data has accumulated regarding the mechanisms regulating the endocytosis of a wide variety of different GPCRs. These studies have revealed GPCR domains involved in receptor endocytosis, some of the molecular intermediates that regulate GPCR endocytosis, and the potential for GPCRs to internalize by multiple endocytic mechanisms. In addition, whereas the molecular mechanism(s) involved in the initiation of GPCR endocytosis are best characterized for the beta 2AR, recent studies using other GPCRs have revealed important diversity in the patterns of GPCR endocytosis and intracellular trafficking. Therefore, the following sections will review the current understanding of the mechanism(s) involved in the initiation and regulation of GPCR endocytosis, how differences in GPCR structure affect the formation of endocytic complexes, and how these complexes contribute to distinct GPCR signaling and intracellular trafficking patterns.

The concept that GPCRs are lost from the cell surface following agonist activation originated from the observation that beta -adrenergic agonist treatment resulted in a loss of beta -adrenergic receptor recognition sites on the surface of frog erythrocytes (Chuang and Costa, 1979). Subsequently, cell surface versus internalized beta 2AR binding sites were discriminated from one another either by differential sedimentation on a sucrose gradient or by using hydrophobic and hydrophilic beta -adrenergic ligands (Harden et al., 1980; Staehelin and Simons, 1982). Internalized receptors were found in a "light vesicular" fraction, whereas cell surface receptors were found in a "heavy vesicular" plasma membrane fraction (Harden et al., 1980). Similarly, internalized beta 2AR were accessible to hydrophobic, but not hydrophilic, adrenergic ligands (Staehelin and Simons, 1982). More recently, the subcellular redistribution of cell surface beta 2AR in response to agonist activation was demonstrated by immunocytochemical staining of epitope-tagged receptors (von Zastrow and Kobilka, 1992), as well as in real time in living cells using a green fluorescent protein (GFP)-tagged beta 2AR (Barak et al., 1997a). Similar experiments have now been performed for several GPCRs (e.g., Tarasova et al., 1997; Schulein et al., 1998; Barlic et al., 1999; Bevan et al., 1999; Doherty et al., 1999; Drmota et al., 1999; Liu et al., 1999; Bremmes et al., 2000). The rate at which GPCRs internalize seems to be receptor specific. For example, the A1 adenosine receptor internalizes quite slowly (t1/2 = 90 min) when compared with the A3 adenosine receptor (t1/2 = 19 min) (Ferguson et al., 2000). These kinetic differences suggest that GPCR internalization can be mediated by multiple endocytic mechanisms and/or that structural heterogeneity between receptor subtypes modulates their relative affinities to bind endocytic adaptor proteins.

A. Molecular Mechanisms Involved in G Protein-Coupled Receptor Endocytosis

1. Role of G Protein-Coupled Receptor Kinase and beta -Arrestin Proteins. Sibley et al. (1986) were the first to suggest that receptor phosphorylation might be involved in GPCR endocytosis. However, when this hypothesis was tested, using beta 2AR mutants lacking sites for both second messenger-dependent protein kinase- and GRK-mediated receptor phosphorylation, no significant differences were observed between the internalization of wild-type and mutant beta 2ARs (Bouvier et al., 1988; Hausdorff et al., 1989). A similar result was obtained when permeablized A431 cells were treated with PKA and GRK inhibitors (Lohse et al., 1990b). This led to a commonly held view that receptor phosphorylation did not contribute to GPCR endocytosis.

Despite the fact that phosphorylation was originally not considered to play a role in the internalization of the beta 2AR, there was growing evidence that phosphorylation might be involved in the endocytosis of other GPCRs, such as the m2 mAChR (Moro et al., 1993). Early experiments demonstrated that the internalization of the m2 mAChR was reduced by the mutation of serine and threonine residues within the third intracellular loop domain of the receptor (Moro et al., 1993). Subsequently, Tsuga et al. (1994) demonstrated that the overexpression of wild-type GRK2 enhanced both the rate and maximal extent of m2 mAChR internalization, whereas a dominant-negative GRK2 mutant (K220W) impaired both the phosphorylation and the internalization of the receptor in COS7 cells. However, whereas catalytically inactive GRK2 dominant-negative mutants blocked m2 mAChR in COS7 cells, they had no effect on m2 mAChR internalization in BHK-21 and HEK 293 cells (Tsuga et al., 1994; Pals-Rylaarsdam et al., 1995). Thus, these experiments provided the first indication that differences in the cellular context in which dominant-negative mutants are used could result in discordant observations. Subsequently, it was determined that GRK2 protein expression levels vary from cell type to cell type, with lowest levels of GRK2 protein expression found in COS7 cells and substantially higher levels found in cells derived from the hematopoetic system (e.g., COS7 < HEK 293 cells < RBL-2H3 cells) (Aramori et al., 1997; Menard et al., 1997; Barlic et al., 1999). Therefore, the effectiveness of dominant-negative proteins may be dependent on the level of GRK protein expressed in the particular cell line utilized for experimentation.

The observation that GRK2 phosphorylation played a role in the internalization of the m2 mAChR, but that beta 2AR mutants lacking sites for GRK2 phosphorylation internalized normally, led to speculation that GRK2-dependent internalization was peculiar to Gi-coupled receptors (Tsuga et al., 1994). However, a role for GRK-mediated phosphorylation in the internalization of the Gs-coupled beta 2AR was eventually demonstrated using an internalization-defective beta 2AR-Y326A mutant (Ferguson et al., 1995). The beta 2AR-Y326A mutant was not only internalization-defective, but also did not serve as a substrate for GRK-mediated phosphorylation. The overexpression of GRK2 not only promoted the internalization of this receptor mutant, but re-established GRK-mediated phosphorylation of the mutant receptor. The GRK expression-dependent rescue of beta 2AR-Y326A internalization required intact sites for GRK2 phosphorylation, indicating that receptor phosphorylation rather than GRK2 association per se was required to allow beta 2AR internalization in HEK 293 cells. Wild-type beta 2AR phosphorylation and internalization were reduced by the overexpression of a catalytically inactive GRK2 mutant. The role of GRK2-mediated phosphorylation in facilitating GPCR internalization has now been confirmed for multiple other receptors, e.g., the AT1AR (Smith et al., 1998), endothelin A receptor (Bremmes et al., 2000), D2 dopamine receptor (Itokawa et al., 1996), follitropin receptor (Lazari et al., 1999), and monocyte chemoattractant protein-1 receptors (Franci et al., 1996) CCR-5 (Aramori et al., 1997) and CXCR1 (Barlic et al., 1999).

The contribution of other GRK family members to GPCR internalization has not been the subject of intense investigation and their role in promoting GPCR internalization remains less clear. For example, the agonist-promoted internalization of the beta 2AR-Y326A mutant was facilitated by GRK2, GRK3, GRK5, and GRK6, but not by GRK4, which increased receptor internalization in the absence of agonist (Menard et al., 1996). The GRK-mediated phosphorylation of the follitropin receptor was blocked by the expression of dominant-negative mutants of both GRK2 and GRK6 (Lazari et al., 1999). However, the internalization of the follitropin receptor was blocked only by the dominant-negative GRK2 mutant (Lazari et al., 1999). The role of different GRK isoforms in facilitating the internalization of mAChR subtypes seemed to be even more complicated (Tsuga et al., 1998a). The internalization of human m2-m5, but not m1 mAChR, was increased by GRK2 overexpression (Tsuga et al., 1998a). However, the ability of GRK4, GRK5, and GRK6 to promote the internalization of mAChR subtypes differed for each receptor subtype tested and the cell line used (Tsuga et al., 1998a).

Depending on the GPCR studied, GRK-mediated phosphorylation is not absolutely required for internalization (Bouvier et al., 1988; Hausdorff et al., 1989; Ferguson et al., 1995). Rather, it seems that phosphorylation stabilizes a conformation state required to promote the interaction of GPCRs with some other cellular element that directly promotes the internalization of the receptor. Consequently, beta 2AR mutants lacking sites for GRK-mediated phosphorylation must be able to interact with an endocytic adaptor protein, even in the absence of phosphorylation. In fact, GRK-phosphorylation increases the affinity of the beta 2AR to bind beta -arrestins, which in addition to uncoupling receptors from heterotrimeric G proteins, act as endocytic adaptor proteins targeting GPCRs for internalization via clathrin-coated vesicles (Ferguson et al., 1996b; Zhang et al., 1996) (Fig. 3). When overexpressed, both beta -arrestin1 and beta -arrestin2 ameliorate the internalization defect of the beta 2AR-Y326A even in the absence of GRK-mediated phosphorylation (Ferguson et al., 1996b). Furthermore, beta -arrestins facilitate the endocytosis of beta 2ARs lacking either carboxyl-terminal tails or putative sites for GRK phosphorylation. Thus, depending on the level of beta -arrestin protein expression and beta -arrestin binding affinity, GRK-mediated phosphorylation may be dispensable for some GPCR subtypes (Menard et al., 1997). However, for most receptors, including the beta 2AR, a synergistic relationship exists between GRK-mediated phosphorylation and beta -arrestin binding (Ferguson et al., 1996b).



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Fig. 3.   Molecular mechanisms involved in the GRK- and beta -arrestin-dependent desensitization and internalization of GPCRs. GPCR activation leads to GRK-dependent phosphorylation of intracellular serine and threonine residues that facilitate the translocation and binding of beta -arrestin proteins to the receptor. beta -arrestins, via their association with the beta 2-adaptin subunit of the AP-2 heterotetrameric adaptor complex, target GPCRs to clathrin-coated pits (Ferguson et al., 1996b; Zhang et al., 1996; Barak et al., 1997b; Laporte et al., 1999). In addition to their association with beta 2-adaptins, beta -arrestins also bind clathrin (Goodman et al., 1996). The GPCR is subsequently internalized via clathrin-coated vesicles. AP-2, AP-2 heterotetrameric adaptor complex; beta Arr, beta -arrestin; H, hormone; P, phosphate group.

As mentioned previously, the relationship between GRK-mediated phosphorylation and beta -arrestin binding is likely different for each GPCR subtype. This may explain the myriad of related, but dissimilar, observations that have been reported in the literature regarding the relative importance of GRKs and beta -arrestins in the internalization of different GPCR subtypes. For example, the internalization of two chemokine receptors, CCR-5 and CXCR1, in HEK 293 cells requires the overexpression of both GRK and beta -arrestin proteins (Aramori et al., 1997; Barlic et al., 1999). In contrast, whereas the internalization of the m2 mAChR seems dependent on GRK-mediated phosphorylation, the internalization of this receptor subtype does not seem to require beta -arrestin, depending on the cellular system in which the receptor is expressed (Tsuga et al., 1994; Schlador and Nathanson, 1997; Vogler et al., 1999; Werbonat et al., 2000). There are also examples of receptors that do not serve as substrates for GRK and beta -arrestin proteins and do not internalize in response to agonist activation (Jockers et al., 1996). Thus, differences in GPCR structure-activity relationships likely play an equally important role in regulating differences in the patterns of GPCR endocytosis as they do for regulating differences in agonist and G protein coupling specificity.

In addition to apparent differences in GPCR structure-activity relationships with regard to GRK and beta -arrestin binding, GPCR endocytosis is also regulated by both the context of agonist activation and the cellular milieu in which a receptor is expressed. There are several excellent examples that illustrate this point. 1) Etorphine, but not morphine, stimulates µ-opioid receptor phosphorylation and internalization in HEK 293 cells (Zhang et al., 1998). However, GRK2, but not beta -arrestin overexpression, allows both phosphorylation and internalization of the µ-opioid receptor in response to morphine. This observation indicates that different receptor agonists stabilize distinct receptor conformations that are able to discriminate between G protein coupling and GRK phosphorylation. This may explain why some peptide receptor antagonists are observed to stimulate receptor internalization (Roettger et al., 1997; Bhowmick et al., 1998). 2) The extent of agonist-promoted beta 2AR internalization was different, depending on the cell line in which it was tested (Menard et al., 1997). For example, the maximal extent of beta 2AR internalization in different cell lines correlates nicely with the levels of GRK and beta -arrestin protein expression (Menard et al., 1997). It is possible that GPCR structure may have evolved to match the levels of GRK and beta -arrestin protein expression in the cells in which they are normally expressed in vivo. For example, CXCR1 is effectively internalized in neutrophil-like RBL-2H3 cells, but does not internalize in HEK 293 cells (Barlic et al., 1999). The difference between these cell lines is that RBL-2H3 cells express substantially high levels of GRK2 and beta -arrestin2 protein than HEK 293 cells (Barlic et al., 1999). 3) The endocytosis of GPCR subtypes differs depending on the complement of beta -arrestin protein isoforms expressed by a particular cell. It was recently reported that there are striking differences in the ability of different arrestin isoforms to bind different GPCRs at the plasma membrane (Oakley et al., 2000). Therefore, the ability of a specific GPCR to internalize in a particular cell type may be dictated by the complement of beta -arrestin isoforms expressed in the cell. Consequently, the endogenous complement of GRK and beta -arrestin proteins expressed in immortalized cell culture systems, such as HEK 293 cells, may not accurately reflect protein expression levels that will be observed in the diverse physiological environments in which a particular GPCR subtype may be expressed such as in hematopoetic, cardiac or neuronal cells. Therefore, potentially important differences in the physiological regulation of different GPCR subtypes that occur in vivo will require the examination of endocytosis patterns under conditions of varied GRK and beta -arrestin protein expression levels.

2. Clathrin and beta -Adaptin Interactions. The first indication that beta -arrestins specifically target GPCRs for endocytosis via clathrin-coated vesicles came from experiments testing the effects of beta -arrestin and dynamin dominant-negative mutants on the internalization of the beta 2AR and AT1AR (Zhang et al., 1996). Dynamin is a large GTPase that is involved in the pinching off of clathrin-coated vesicles from the plasma membrane (Damke et al., 1994). The expression of a dynamin mutant (K44A) lacking GTPase activity effectively blocked both beta 2AR internalization and beta -arrestin-stimulated AT1AR internalization (Zhang et al., 1996). Furthermore, Goodman et al. (1996) demonstrated that both beta 2ARs and beta -arrestins were colocalized with clathrin in clathrin-coated pits. The idea that beta -arrestins specifically target GPCRs for endocytosis via clathrin coated vesicles has been corroborated by recent studies showing that beta -arrestins interact directly with components of the endocytic machinery involved in the formation of clathrin-coated pits (Goodman et al., 1996; Laporte et al., 1999, 2000). beta -Arrestins bind to both the clathrin heavy chain and the beta 2-adaptin subunit of the heterotetromeric AP-2 adaptor complex (Goodman et al., 1997; Laporte et al., 1999, 2000).

beta -Arrestins bind with high affinity and stoichiometry to purified clathrin in vitro (Goodman et al., 1996). beta -Arrestin2 binds clathrin with approximately 6-fold higher affinity than beta -arrestin1 (Goodman et al., 1996). Visual arrestin, while structurally related to the beta -arrestins, does not promote beta 2AR internalization and does not bind to clathrin (Goodman et al., 1996). The clathrin beta -arrestin binding domain is localized to residues 89-100 of the amino-terminal globular region in the terminal domain of the clathrin heavy chain that lies at the distal end of each clathrin triskelion (Goodman et al., 1997). The beta -arrestin domain involved in clathrin binding is localized to amino acid residues 373-377 in the carboxyl terminus of beta -arrestin2 (Krupnick et al., 1997) (Fig. 2). Mutation of the residues within this region of beta -arrestin2 substantially reduced clathrin cage binding without altering binding to phosp