the ins and outs of g protein-coupled receptor trafficking

The ins and outs of G protein-coupledreceptor traffickingAdriano Marchese1, Catherine Chen1, You-Me Kim2 and Jeffrey L. Benovic1

1Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA2Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA

All cells are faced with the complexity of converting a

myriad of extracellular stimuli to intracellular signals

and appropriate biological responses. This process

involves a series of highly orchestrated molecular

events, many of which occur at the plasma-membrane

interface. Endocytosis is one such event, and functions

to transport numerous small molecules and protein

cargo. For G protein-coupled receptors (GPCRs), endo-

cytosis serves as a mechanism to regulate cell-surface-

receptor levels, thereby, contributing to the regulation

of hormonal responsiveness. The trafficking of GPCRs

through the endocytic pathway involves three main

steps: recruitment of receptors to discrete endocytic

sites, internalization and intracellular sorting. Many

recent studies have advanced our understanding of

these steps. Novel mechanistic insight into the recruit-

ment of GPCRs to sites of endocytosis has been gained

and the discovery of novel protein–protein interactions,

which define more clearly the processes of internaliz-

ation and endocytic sorting, have been characterized. In

addition, a role for ubiquitin in internalization and sort-

ing to the degradative pathway has emerged.

The best characterized pathway for G protein-coupledreceptor (GPCR) endocytosis occurs through clathrin-coated pits (CCPs; Fig. 1). GPCR concentration incoated-pits typically involves agonist-dependent phos-phorylation and recruitment of the adaptor proteinarrestin [1,2]. Arrestins are cytosolic proteins that areinvolved in the regulation of GPCR signaling. Visualarrestins include arrestin-1, which regulates phototrans-duction in retinal rod cells, and arrestin-4, which isexpressed in cone cells. Non-visual arrestins (termedarrestin-2 and -3 or b-arrestin-1 and -2) uncouple Gprotein-coupled receptor kinase (GRK)-phosphorylatedGPCRs from heterotrimeric G proteins (a process termeddesensitization) and also appear to be importantmediators of receptor trafficking and signaling [1,2].Non-visual arrestins serve to link GPCRs to CCPs viatheir interaction with receptors, clathrin and clathrin-associated protein (AP)-2 complexes. Although endocy-tosis through CCPs appears to be the predominantpathway used by most GPCRs, additional pathwayshave been suggested, such as those in which receptorspass through caveolae [3] or through other ill-defined

plasma-membrane microdomains. GPCRs that havebeen identified in caveolae include the b2-adrenergic,bradykinin, endothelin, M2-muscarinic, adenosine-A1

and cholecystokinin receptors [4]. Although agonist-dependent translocation into and out of these domainshave been observed, internalization of GPCRs throughcaveolae has not been conclusively demonstrated.Additional studies that use cholesterol-disrupting agents,dominant negative constructs, intracellular co-localizationand more stringent methods of caveolae isolation appearnecessary to address this issue.

Although non-clathrin, non-caveolar pathways are notwell defined, the M2-muscarinic acetylcholine receptor(M2AChR) internalizes through an arrestin-independentand partially dynamin-dependent pathway that appearsto be independent of clathrin and caveolae in somecells. Recently, this route has been shown to depend onADP-ribosylation factor 6 (ARF6) [5] – a small Gprotein involved in phospholipid metabolism, cyto-skeletal dynamics and intracellular vesicle transport.ARF6 has also been implicated in clathrin-independentendocytic trafficking [6], although not necessarily in allcell types [7]. Moreover, ARF6-dependency does notnecessarily implicate a clathrin-independent pathway ofGPCR internalization, as suggested by the ARF6-sensitivenature of b2-adrenergic receptor (b2AR) internalization [8]and luteinizing hormone/chorionic gonadotropin (LH/CG)receptor desensitization [9]. The fate of GPCRs afterARF6-dependent internalization might be similar to thatof receptors that internalize through CCPs becauseinternalized M2AChRs co-localize with markers for endo-somes derived from CCPs [5].

Several GPCRs, including the thromboxane-A2b recep-tor and protease-activated receptor-1 (PAR1), exhibitconstitutive internalization (i.e. internalization in theabsence of stimulus). Mechanisms of constitutive intern-alization are not defined, but might be distinct from thosemechanisms mediating agonist-dependent internaliz-ation. For example, the thromboxane-A2b receptor under-goes agonist-induced internalization through CCPs thatis GRK-, arrestin- and dynamin-dependent [10], whereasits constitutive internalization follows a CCP pathwaythat does not involve GRKs or arrestins [11]. The motif(Tyr-Xaa-Xaa-Xaa-Ile) mediating constitutive internaliz-ation of the thromboxane-A2b receptor is similar to thatused by the transferrin receptor and does not contribute toagonist-promoted internalization [11]. Another differenceCorresponding author: Jeffrey L. Benovic ([email protected]).

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between constitutive and agonist-dependent internaliz-ation might lie with the fate of the receptors once they areinternalized. For example, agonist-dependent internaliz-ation of PAR1 leads to the degradative pathway, whereasconstitutive internalization enables receptor recyclingfrom intracellular pools [12].

Role of phosphorylation in GPCR trafficking

Many GPCRs are targeted for phosphorylation by GRKs,a family of serine/threonine protein kinases thatspecifically phosphorylate agonist-activated GPCRs [1,2].GRK-mediated receptor phosphorylation promotes thebinding of arrestins that, in turn, promote associationof the receptor with components of the endocyticmachinery. A classic example of this is the b2AR,which undergoes agonist-dependent phosphorylation byGRK2, binding of arrestin and subsequent endocytosis.Overexpression of wild-type arrestin enhances b2ARinternalization [13,14], whereas disruption of arrestinbinding using dominant negative [14], antisense [15],knockout [16] or RNA interference (RNAi) [17] strat-egies attenuates internalization.

In addition to its actions as a receptor kinase, GRKsmight interact with other proteins to mediate GPCRinternalization. For example, GRK2 interactions withGRK-interacting protein 1 (GIT1) [18] and phosphoinosi-tide 3-kinase (PI3K) p110-a and -g [19] have beenimplicated in receptor trafficking. GIT1 is an ARFGTPase-activating protein that inhibits the internaliz-ation of several GPCRs when overexpressed [20]. Althougha role for GIT1 interaction with GRK2 in GPCR traffickinghas not been directly established, this interaction providesa potential mechanism for targeting GIT1 to an activatedreceptor and for contributing to subsequent traffickingsteps. Inhibition of PI3K activity using wortmannin ordisruption of GRK2–PI3K association by overexpressionof the PIK domain of PI3Kg effectively inhibits agonist-promoted internalization of the b2AR [19]. These findings

suggest an important role for localized generation of D-3phosphoinositides in GPCR recruitment to CCPs.

Many receptors are also subject to phosphorylation bysecond-messenger-dependent kinases such as proteinkinase A (PKA) and protein kinase C (PKC). GPCRphosphorylation by PKA and PKC has been primarilyimplicated in desensitization, but there is also evidencethat phosphorylation by PKC contributes to receptortrafficking. For example, phorbol myristate acetate(PMA) treatment promotes the endocytosis of CXCchemokine receptor 4 (CXCR4) [21], parathyroid hormone1 [22], sst2A somatostatin [23], d-opioid [24], 5-HT2A [25],endothelial differentiation gene 1 (EDG1) [26] and FPA

prostanoid [27] receptors in several cell lines, whereasinhibition of PKC attenuates agonist-promoted internal-ization of the gastrin-releasing peptide [28], parathyroidhormone 1 [22], 5-HT2A [25] and FPA prostanoid [27]receptors. Although mechanistic insight is lacking, thesestudies suggest that PKC phosphorylation functions totarget some GPCRs to endocytic pathways.

Role of arrestins in GPCR trafficking

A role for arrestins in GPCR trafficking was first suggestedby the finding that overexpression of arrestin-2 or -3promoted internalization of a mutant b2AR impaired inagonist-induced phosphorylation and internalization [14].Involvement of arrestins in receptor endocytosis was alsodemonstrated by the inhibitory action of various mutantarrestins on b2AR internalization [14]. Moreover, anti-sense [15], knockout [16] and RNAi [17] strategies haveprovided definitive evidence of a direct role for arrestins inb2AR internalization. In addition, arrestins have now beenimplicated in the trafficking of a wide variety of GPCRs(Table 1).

A possible role for arrestins in regulating receptorrecycling has been suggested by spatially and tem-porally distinct patterns of arrestin-receptor associ-ation among various GPCRs. Activation of a widerange of GPCRs can promote the rapid translocation of

Fig. 1. Pathways of G protein-coupled receptor (GPCR) internalization. GPCR internalization occurs through two mechanistically distinct endocytic pathways: agonist-

dependent and agonist-independent. Upon agonist (orange oval) binding in the agonist-dependent pathway, receptors are phosphorylated by a G protein-coupled receptor

kinase (GRK) leading to the recruitment of arrestins. Arrestins serve as adaptor proteins by linking receptors to components of the transport machinery, such as clathrin,

adaptor protein AP-2 and phosphoinositides, leading to recruitment to clathrin-coated pits and subsequent internalization. Additional interactions with GRKs [e.g. GRK-

interacting protein (GIT)] and arrestins [e.g. ADP-ribosylation factor 6 (ARF6), ARF nucleotide-binding site opener (ARNO) and murine double minute 2 (MDM2)] might also

play a role in internalization. AP2 might also bind to some receptors directly circumventing a need for arrestin in internalization. For the agonist-independent pathway, little

is known about the molecular mechanisms mediating internalization. The mechanism by which GPCRs are recruited to clathrin-coated pits, or whether another route is

involved, is not known. However, studies using the thromboxane-A2b receptor suggest that GRKs and arrestins are not required.

Ti BS

GRKP

P

GIT

Agonist-dependent Agonist-independent

Clathrin-coated pitClathrinAP2Mdm2Ubiquitin

ARF6ARNOPIP2/3

Arrestin

ArrestinP

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expressed arrestin-2-GFP and arrestin-3-GFP chimerasfrom the cytosol to the plasma membrane. However,following activation of the a1bAR, b2AR, dopamine D1a,endothelin ETa and m-opioid receptors (class A recep-tors), translocated arrestins are confined to theperiphery of cells and do not travel into endocyticvesicles with receptors [29,30]. By contrast, stimulation

of the angiotensin AT1a, neurotensin-NT1, TRH, sub-stance-P and vasopressin-V2 receptors (class B recep-tors) triggers redistribution of arrestins intointracellular vesicular compartments where they co-localize with internalized receptors [29,30]. Interest-ingly, class B receptors have stretches of serine andthreonine residues, the phosphorylation of which hasbeen proposed to mediate high-affinity arrestin bindingand prolonged receptor–arrestin association in endo-cytic vesicles [31]. This prolonged association witharrestin might regulate the kinetics of receptorrecycling because both angiotensin-AT1a and vasopres-sin-V2 receptors traffic to intracellular vesicles withbound arrestins and recycle very slowly [32,33].Similarly, prolonged association of arrestin-2 with theadenosine-A2b receptor appears to slow the rate ofreceptor recycling [34]. Interestingly, recent evidencesuggests that the prolonged association of arrestin-3with GPCRs might correlate with the ubiquitinationstatus of the arrestin [35].

Although there are numerous examples for the regu-latory role of non-visual arrestins in GPCR trafficking, thespecificity of arrestin-2 and -3 in these processes has notbeen extensively investigated. In rat basophilic leukemia(RBL)-2H3 cells endogenous arrestin-3, but not arrestin-2,is rapidly recruited to CCPs upon stimulation of the b2ARand M1AChR, suggesting a possible selectivity forarrestin-3 by these receptors [36]. In addition, b2ARinternalization was markedly decreased in arrestin-3knockout cells but was minimally affected in cells lackingarrestin-2, again implying a preferential role for arrestin-3in b2AR internalization [16]. In type-1a metabotropicglutamate receptors (mGluR1a), overexpression of GRKswith arrestin-2, but not arrestin-3, promotes agonist-stimulated receptor internalization, providing an exampleof arrestin-2 specificity in this process [37].

Molecular mechanisms underlying arrestin-promoted

GPCR internalization

Initial molecular insight into the mechanism of arrestin-promoted receptor internalization was provided by thefinding that non-visual arrestins directly interact withclathrin [13]. The clathrin-binding domain in arrestin-2and -3 has been localized to a C-terminal clathrin box (Leu-f-Xaa-f-[Asp/Glu], where f represents an aliphaticresidue) that is common to a large number of clathrin-binding proteins. Deletion of the clathrin box completelyabrogates clathrin binding and reveals an essential role forarrestin–clathrin interaction in arrestin-promoted traf-ficking of the b2AR [38]. Crystallographic analysis ofclathrin in complex with an arrestin-3 clathrin-box peptidedemonstrates that the aliphatic residues in arrestin fit intotwo hydrophobic-binding pockets of the clathrin terminaldomain, whereas the C-terminal acidic residue is flankedby two basic residues in clathrin [39].

Targeting of non-visual arrestins into CCPs alsoinvolves interaction with the b subunit of the adaptorprotein AP-2. AP-2 functions to promote clathrin-coatassembly and receptor recruitment to CCPs. Arrestininteraction with b2-adaptin was first identified by yeasttwo-hybrid and co-immunoprecipitation analyses [40], and

Table 1. Role of arrestins in GPCR internalization

GPCRa Arrestins

testedb

Methodc Assayd Refse

Adenosine A2b 2,3 AS ELISA, IF [2]

a1a-AR 2 DN IF [1]

a1b-AR 2 DN IF [1]

a2a-AR 2,3 OE ELISA, IF [2]

a2b-AR 2,3 OE ELISA, IF [2]

a2c-AR 3 OE ELISA, IF [2]

b2-AR 2,3 OE, DN LB [14]

b2-AR 2,3 AS ELISA, LB [15]

b2-AR 2,3 KO LB [16]

Angiotensin A1a 2 DN LB, IF [1]

Angiotensin A1a 2,3 KO LB [16]

Bradykinin B2 2 DN LB [66]

CCR5-chemokine 2,3 OE, DN FC [2]

CXCR1-chemokine 2,3 OE, DN IF [67]

CXCR4-chemokine 2,3 DN ELISA, IF [2]

CXCR4-chemokine 2,3 OE, DN FC [2]

CRLR 2 DN FC [68]

Dopamine D2 2,3 OE LB, IF [2]

Endothelin ETa 2,3 OE, DN LB [2]

Endothelin ETb 2,3 OE, DN LB [2]

FSH 2,3 OE, DN LB [2]

LH/CG 2,3 OE, DN LB [1]

m1-AChR 2 DN LB [2]

m2-AChR 2 OE LB [69]

m3-AChR 2 DN LB [2]

m4-AChR 2 DN LB [2]

mGluR1a 2 DN ELISA [70]

mGluR1a 2 OE FC [37]

mGluR1b 2 DN ELISA [71]

mGluR1c 2 DN ELISA [71]

k-Opioid 2 DN LB [2]

m-Opioid 2 OE, DN FC [72]

m-Opioid 2 OE WB [2]

ORLR1 3 OE LB [73]

PAF 2,3 OE, DN FC, IF [74]

PAR2 2 OE, DN IF [2]

PGE2 EP4 2 OE ELISA, IF [2]

PGE2 EP4 2 DN ELISA [2]

Somatostatin SST3 2 OE, DN LB [2]

Substance P NK1 2 DN IF [2]

Substance P NK3 2 DN IF [2]

Thromboxane TXA2b 2,3 OE, DN ELISA [11]

TRH 2 OE, DN LB [2]

aAbbreviations: GPCR, G protein-coupled receptors shown to undergo arrestin-

mediated internalization; CRLR, calcitonin receptor-like receptor; CXCR, CXC

chemokine receptor; FSH, follicle stimulating hormone; LH/CG, luteinizing hormo-

ne/choriogonadotropin; mGluR, metabotropic glutamate receptor; NK1, neurokinin

1; ORLR1, opioid receptor-like receptor 1; PAF, platelet activating factor; PAR2,

protease-activated receptor 2; PGE2, prostaglandin E2; TRH, thyrotropin releasing

hormone.bArrestins tested: 2, arrestin-2; 3, arrestin-3.cMethods used to test effects of arrestins: AS, arrestin-specific antisense RNA

expression; DN, overexpression of specific dominant negative arrestins; KO,

knockout of specific arrestin gene; OE, overexpression of specific arrestin.dAssays used to determine internalization of receptors: ELISA, enzyme-linked

immunosorbent assay; FC, flow cytometry; IF, immuno-fluorescent microscopy; LB,

radioactive ligand binding assay; WB, western blotting analysis of surface-

biotinylated receptors.eMultiple references are cited for a particular GPCR if they demonstrate the effect of

arrestins using different methods or assays.




involves ionic and hydrophobic interactions between theC-terminal domain of non-visual arrestins and theb2-adaptin appendage domain [38,41]. Additional insightinto this interaction was provided by the discovery that thebinding of arrestin to b2-adaptin and clathrin is enhancedby conformational changes in arrestin that are probablyinduced by receptor binding [38]. Interestingly, thearrestin-binding surface on b2-adaptin partially overlapswith the binding surfaces for many other b-adaptin-binding proteins, including clathrin, AP180, eps15 andepsin [42]. Moreover, because arrestin and b-adaptinbinding residues in the clathrin terminal domain alsooverlap [39], the spatial and temporal regulation ofarrestin, b2-adaptin and clathrin interaction could provecrucial in mediating GPCR endocytosis.

Molecular interactions of arrestin that are implicated inGPCR trafficking are not limited to protein–proteininteractions. Like many other proteins involved inclathrin-mediated endocytosis, non-visual arrestins bindphosphoinositides with high affinity [43]. Interestingly, amutant arrestin that lacks phosphoinositide bindingretains its ability to bind b2AR and clathrin, but doesnot promote b2AR internalization [43]. This suggests thatphosphoinositide binding plays a crucial role in delivery ofGPCR–arrestin complexes to CCPs. Interestingly, someGPCR-containing CCPs might be distinct from CCPscontaining constitutively internalized cargo such as thetransferrin receptor [44]. Thus, a plausible model forarrestin-promoted trafficking of GPCRs involves a confor-mational change in the arrestin induced by receptorbinding that promotes subsequent association of thearrestin–receptor complex with CCPs via arrestin inter-action with clathrin, b2-adaptin and phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P2].

The interaction of non-visual arrestins with CCPsraises intriguing questions concerning whether recep-tor–arrestin complexes promote de novo formation ofCCPs. Previously, it was reported that arrestin itself doesnot mediate clathrin-lattice assembly [13], whereas morerecent studies demonstrate that receptor-arrestin com-plexes accumulate in pre-existing CCPs rather thannucleating new pits [45,46]. However, under some con-ditions, recruitment of receptor–arrestin complexes topre-existing CCPs appears to increase CCP number andclustering [45]. These changes also occur, albeit with areduced magnitude, in the presence of a mutant arrestin-3that is impaired in clathrin or b2-adaptin interaction.These findings suggest that a functional GPCR–arrestin-3complex might affect the dynamics of CCP formation [45].

Non-visual arrestin interactions with additional proteins

Non-visual arrestin interaction with several additionalproteins also appears to contribute to GPCR trafficking.Non-visual arrestins have been demonstrated to bind ARFnucleotide-binding site opener (ARNO), an ARF guanine-nucleotide-exchange factor and the GDP-bound form ofARF6 [8]. The involvement of ARF6 and ARNO in GPCRregulation was first suggested by the fact that activation ofARF6 by ARNO promotes the release of membrane-boundarrestin-2 and the subsequent desensitization of LH/CGreceptor in ovarian follicles [47]. The regulatory role of

ARF6 in GPCR trafficking has also been suggested by thefinding that GTP binding-defective and hydrolysis-deficient ARF mutants and GIT1 inhibit b2AR internaliz-ation, whereas the ARF activator ARNO enhancesinternalization [8]. The ability of arrestins to regulateARF6 activity might serve as a general mechanism inGPCR trafficking.

N-ethylmaleimide sensitive factor (NSF) is anothernon-visual arrestin-interacting protein that, potentially,plays a role in intracellular trafficking of GPCRs. NSF isan ATPase that binds to soluble NSF-attachment protein(SNAP) and helps to promote disassembly of the SNAPreceptor (SNARE) complexes after vesicle fusion. NSFinteraction with arrestin-2 was first identified in yeasttwo-hybrid screening and confirmed by co-immuno-precipitation [48]. Arrestin-2 preferentially binds theATP-bound form of NSF and overexpression of NSFenhances b2AR internalization [48]. Although thephysiological consequences of arrestin–NSF interactionhave not been completely defined, arrestin interactionwith proteins involved in intracellular trafficking andvesicle fusion suggests that arrestins might contributeto post-endocytic events.

Murine double minute 2 (MDM2), a RING-finger E3ubiquitin ligase, has also recently been identified as anon-visual arrestin-interacting protein, thus implicat-ing a role for ubiquitin in arrestin function [49].Interestingly, ubiquitination of arrestin-3 by the E3ubiquitin ligase MDM2 appears to regulate internaliz-ation of the b2AR [49].

Additional clathrin-dependent pathways of

internalization

Several studies have suggested an arrestin-independentpathway in which GPCRs are internalized through CCPs[50,51]. Studies examining the trafficking of PAR1 inmouse embryonic fibroblasts (MEFs) derived from wild-type, and arrestin-2 and -3 knockout mice have revealedthat arrestins are not required for PAR1 internalization,but arrestins do play a role in the desensitization of PAR1[50]. This finding raises the intriguing possibility that anadaptor other than arrestin fulfils the role of linking someGPCRs to the internalization machinery. Indeed, studieswith the chemokine receptor CXCR2 suggest that such anadaptor might be AP-2 [51]. AP-2 binds to arrestins, but itcan also bind to tyrosine-based signals and di-leucinemotifs located within the cytoplasmic domains of cargomolecules, raising the possibility of direct associationbetween AP-2 and GPCRs containing similar motifs.CXCR2 has two di-leucine motifs within its C-terminaltail and a mutant receptor lacking these motifs retainsnormal binding to arrestin but fails to associate with AP-2and internalize upon agonist activation [51]. Thus, CXCR2is another example of a GPCR for which arrestins functionin desensitization but might not contribute to internaliz-ation. Whether AP-2 or other factors serve as adaptors tolink PAR1 or other GPCRs to the internalization machin-ery remains to be determined. Future studies using thearrestin knockout MEFs and employing gene-knockdownstrategies should help define the precise role that arrestinsand other factors play in the internalization of GPCRs.




Post-internalization trafficking

Once plasma membrane proteins are internalized intoendocytic vesicles, they are subject to one of two sortingfates (Fig. 2). One fate is the recycling pathway thatrestores them to the plasma membrane resulting infunctional resensitization of receptor-mediated signaling.Another fate is the degradative pathway during whichreceptors are transported to lysosomes and proteolyzedleading to long-term attenuation of signaling, a processknown as down-regulation. In general, GPCRs can bedivided into two groups: those that readily enter a recylingpathway and those that readily enter a degradationpathway. There are receptors that efficiently recycle,such as the b2AR and neurokinin-1 receptor, and thosethat are rapidly degraded such as PAR1 [50], d-opioidreceptor [52] and CXCR4 [53]. The sorting decisionappears to occur in an early endosomal compartment,where recycling receptors are segregated from receptorsdestined for degradation [54]. Details about the molecularmechanisms mediating the sorting of GPCRs are only nowbeginning to emerge.

Sorting signals

Recent studies have revealed that the cytoplasmicdomains of plasma-membrane proteins carry the necess-ary sorting signals that dictate the route that is takenonce in an endosomal compartment. Analysis ofchimeras between PAR1 – a receptor readily targetedto lysosomes – and the neurokinin-1 receptor – arecycling receptor – have revealed that the sortingsignals reside in the C-terminal tails of these receptors[55]. Indeed, work with the b2AR has revealed that aC-terminal PDZ (PSD-95, Dlg, ZO-1 homology)-bindingdomain regulates endosomal sorting [56]. In particular,

recycling of the b2AR appears to be regulated byinteraction of ezrin-radixin-moesin (EBP50, also knownas NHERF) with the PDZ-binding domain of the b2ARand the cortical actin cytoskeleton [56]. Phosphoryl-ation of a serine residue within this domain by GRK5disrupts this interaction and results in receptor sortingto a degradative pathway [56]. Interestingly, inter-action of this same C-terminal region with NSF alsofunctions to regulate b2AR recycling [57]. Thus, acommon region in the b2AR might control recycling bymultiple mechanisms that could be tightly regulated ina temporal and spatial manner. This example serves tounderscore the complexity with which GPCRs aresorted within the endocytic system.

For receptors that are rapidly degraded, it appears thatthe C-terminal tail mediates interaction with distinctproteins that are important for lysosomal targeting. Forexample, PAR1 associates with sorting nexin 1 anddisruption of this interaction attenuates agonist-promoteddegradation [58]. Not surprisingly, the interaction ismediated by the C-terminal tail of PAR1 and might bespecific for receptors that are preferentially sorted to thedegradative pathway [58]. Interestingly, sorting nexin 1also regulates degradation of the epidermal growth factor(EGF) receptor and might play a general role in vesiculartrafficking [59]. For the d-opioid receptor, binding to aprotein referred to as GPCR-associated sorting protein(GASP) appears to be crucial for targeting the receptor tolysosomes [60]. GASP binds to the C-terminal tail of thed-opioid receptor but not to the closely related m-opioidreceptor, a receptor that efficiently recycles [60]. There-fore, GASP might preferentially bind to receptors that arereadily targeted to lysosomes, although the precisemolecular determinants are not known. It is interesting

Fig. 2. Pathways of endocytic sorting of G protein-coupled receptors (GPCRs). Once endosomes are formed, recycling receptors (recycling pathway) are readily segregated

from receptors destined for lysosomes (degradation pathway). Although poorly understood, the sorting decision might be regulated by specific and distinct protein inter-

actions or modification by ubiquitin. Receptors that enter the recycling pathway are dephosphorylated in part owing to the low acidic environment of endosomes and traffic

back to the cell surface resulting in functional resensitization. Several proteins that might be involved in this process including arrestin, N-ethylmaleimide sensitive factor

(NSF) and ezrin-radixin-moesin (EBP50) have been identified, although precise molecular details remain to be determined. Receptors that enter the degradation pathway

traffic to lysosomes where they are proteolyzed, leading to a loss in the total cellular complement of the receptor – a process known as down-regulation. GPCR-associated

sorting protein (GASP) and sorting nexin 1 are two proteins that appear to dictate the entry of the d-opioid and thrombin (PAR1) receptors, respectively, into the degra-

dation pathway. Agonist dependent modification of the b2-adrenergic receptor?? (b2AR) and CXC chemokine receptor 4 (CXCR4) by ubiquitin dictates the entry of these two

receptors into the degradation pathway. Although poorly understood, ubiquitin acts as a sorting signal in endosomes.

Ti BS

GASPSorting nexin 1

EBP50NSFArrestinRecycling

endosome Earlyendosome

Lysosome

Recycling pathway

Degradation pathway

pH

P

P

Ub

Ub




to note that GASP also binds strongly to the C-terminaltail of the b2AR [60], a receptor that is efficiently recycled.Defining how GASP regulates the preferential targeting ofcertain GPCRs to lysosomes awaits further analysis.Moreover, it will be interesting to determine whethersorting nexin 1 and GASP will also regulate the sorting ofother GPCRs that are efficiently targeted to lysosomes.

Role of ubiquitination in GPCR sorting

It has been known for several years that mono-ubiquitina-tion serves as a signal for internalization and subsequenttargeting of the yeast a-mating factor receptor fordegradation in the vacuole, the yeast equilvalent of thelysosome [61]. Recently, it has been shown that ubiquiti-nation of CXCR4 [53] and the b2AR [49] also functions as atargeting signal for lysosomal degradation. Receptormutants that fail to become ubiquitinated upon agonistactivation internalize normally, but are not degraded.Exactly how the ubiquitin moiety serves as a sorting signalremains to be elucidated, however, recent developmentsshed some light on this process. It appears that endosomalcomponents of the trafficking machinery are involved inthe ubiquitin-dependent sorting of cargo into invaginatingdomains of endosomes [62]. Some of these componentsbind ubiquitin and, therefore, might serve as receptors forubiquitinated cargo targeted for degradation [62].Whether these components are involved in the sorting ofubiquitinated mammalian GPCRs to a degradative path-way remains to be determined.

The attachment of ubiquitin to substrate proteins iscarried out by an ATP-dependent mechanism that iscatalyzed by the sequential activity of three enzymes:ubiquitin-activating enzymes (E1), ubiquitin-conjugating(UBC) enzymes (E2) and ubiquitin ligases (E3) [63]. Thereare two major classes of E3s that mediate the ubiquitina-tion of cell signaling receptors, and both function bybinding either directly or indirectly to specific substrateproteins to catalyze the covalent attachment of ubiquitinto a lysine residue of the protein substrate [63]. Presently,the E3 ubiquitin ligases that mediate the ubiquitination ofmammalian GPCRs are not known. However, studiesperformed in arrestin knockout MEFs offer insight intohow an E3 enzyme mediates GPCR ubiquitination. InMEFs lacking arrestin-2 and -3, ubiquitination of theb2AR is abrogated, which suggests a role for arrestin inb2AR ubiquitination [49]. The ability of arrestin-3 to bindto the E3 ubiquitin ligase MDM2 suggests that arrestinsmight act as adaptors to recruit E3s to activated GPCRs,however, MDM2 does not appear to be the ligase thatubiquitinates the b2AR [49]. Thus, the mechanismsinvolved in targeting ubiquitin ligases to GPCRs remainunestablished. Moreover, because ubiquitination at mul-tiple steps in the endocytic pathway appears necessary forproper trafficking of cell-signaling receptors [64], it will beinteresting to determine whether ubiquitination plays arole in lysosomal targeting of GPCRs that are not directlyubiquitinated [65].

Future directions

Although many recent studies have shed light on themolecular mechanisms mediating GPCR internalization

and sorting, many questions remain. Are there additionaladaptor molecules that serve to recruit GPCRs to CCPs? Ifso, what is the molecular composition of these CCPs, andare they functionally distinct from other CCPs? How broada role does ubiquitination play in sorting GPCRs to thedegradative pathway? What are the components of thetransport machinery mediating ubiquitin-dependent sort-ing and are these components different from those used tosort GPCRs that are not ubiquitinated? If the recent pastserves as any indication, many new and exciting dis-coveries await concerning the GPCR-trafficking field.

AcknowledgementsWe thank Raymond Penn and James Keen for their suggestions. Thiswork was supported by grants from the National Institutes of Health(J.L.B. and C.C.) and by postdoctoral fellowships from the CanadianInstitutes of Health Research (A.M.) and the American HeartAssociation (A.M.).

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