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 -subunit and is presumed to allow the dissociation of the G protein G- and G-subunits (Neer, 1995; Surya et al., 1998). Subsequently, the activated G- and G-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  G protein  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 -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 ₂-adrenergic receptor (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 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 G_i and G_q -subunits, thereby dampening signaling via G_i- and G_q-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).

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 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 -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, 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 ₂AR and the other site is found within the proximal region of the ₂AR carboxyl-terminal tail (Bouvier et al., 1988; Yuan et al., 1994; Moffett et al., 1996). It is proposed that covalent modification of the 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 ₂AR carboxyl-terminal tail occurs subsequent to the depalmitoylation of cysteine residue 341, suggesting that this site contributes to the agonist-dependent desensitization of ₂AR responsiveness (Moffet et al., 1996). Two recent studies suggest that PKA-mediated phosphorylation of the 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 G_i- and G_q-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 (-adrenergic receptor kinase 1, ARK1) (Benovic et al., 1986) and GRK3 (-adrenergic receptor kinase 2, 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).

View larger version (15K): [in this window] [in a new window]   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 -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.

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 ₂-adrenergic receptor (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 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 ₂AR or the third intracellular loop of the m2 mAChR abolishes GRK-mediated phosphorylation of these receptors (Bouvier et al., 1988

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.

GRK-mediated phosphorylation of either rhodopsin or the 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, -arrestin1, was identified as a cofactor required for GRK2-mediated ₂AR desensitization in vitro (Benovic et al., 1987). The cloning of -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 -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 -arrestins) and the targeting of GPCRs for endocytosis (-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) -arrestins (-arrestin1 and -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 -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 -arrestin2 is more abundant than -arrestin1 (Attramadal et al., 1992). The evaluation of -arrestin1 and -arrestin2 protein expression in the brain reveals extensive, heterogenous neuronal labeling (Attramadal et al., 1992). -Arrestin protein is found in several neuronal pathways and immunoelectron microscopy reveals that -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).

View larger version (29K): [in this window] [in a new window]   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 -arrestin1 and -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 -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 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 -arrestin binding to the 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 _2AAR) or the carboxyl-terminal tails of receptors (e.g., rhodopsin and 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) -arrestin can be coimmunoprecipitated with the third intracellular loop domains of the m3 mAChR, _2AAR, and 5-hydroxytryptamine2A receptor (Wu et al., 1997; Gelber et al., 1999); and 3) the ₂AR carboxyl-terminal tail is not absolutely required for -arrestin binding (Ferguson et al., 1996b). Gurevich et al. (1995) examined the ability of several arrestins to bind to various functional forms of rhodopsin, 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 -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).

	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 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 -adrenergic agonist treatment resulted in a loss of -adrenergic receptor recognition sites on the surface of frog erythrocytes (Chuang and Costa, 1979). Subsequently, cell surface versus internalized ₂AR binding sites were discriminated from one another either by differential sedimentation on a sucrose gradient or by using hydrophobic and hydrophilic -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 ₂AR were accessible to hydrophobic, but not hydrophilic, adrenergic ligands (Staehelin and Simons, 1982). More recently, the subcellular redistribution of cell surface 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 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 (t_1/2 = 90 min) when compared with the A3 adenosine receptor (t_1/2 = 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 -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 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 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.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Molecular mechanisms involved in the GRK- and -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 -arrestin proteins to the receptor. -arrestins, via their association with the 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 2-adaptins, -arrestins also bind clathrin (Goodman et al., 1996). The GPCR is subsequently internalized via clathrin-coated vesicles. AP-2, AP-2 heterotetrameric adaptor complex; Arr, -arrestin; H, hormone; P, phosphate group.

2. Clathrin and -Adaptin Interactions. The first indication that -arrestins specifically target GPCRs for endocytosis via clathrin-coated vesicles came from experiments testing the effects of -arrestin and dynamin dominant-negative mutants on the internalization of the ₂AR and AT_1AR (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 ₂AR internalization and -arrestin-stimulated AT_1AR internalization (Zhang et al., 1996). Furthermore, Goodman et al. (1996) demonstrated that both ₂ARs and -arrestins were colocalized with clathrin in clathrin-coated pits. The idea that -arrestins specifically target GPCRs for endocytosis via clathrin coated vesicles has been corroborated by recent studies showing that -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). -Arrestins bind to both the clathrin heavy chain and the 2-adaptin subunit of the heterotetromeric AP-2 adaptor complex (Goodman et al., 1997; Laporte et al., 1999, 2000).