G protein-coupled receptor endocytosis in ADP-ribosylation factor 6-depleted cells

The internalization of G protein-coupled receptors is regulated by several important proteins that act in concert to finely control this complex cellular process. Here, we have applied the RNA interference approach to demonstrate that ADP-ribosylation factor 6 (ARF6) is essential for the endocytosis of a broad variety of receptors. Reduction of endogenous expression of ARF6 in HEK 293 cells resulted in a correlated inhibition of the beta(2) -adrenergic receptor internalization previously characterized as being sequestered via the clathrin-coated vesicle pathway. Furthermore, other receptors internalizing via this endocytic route, namely the angiotensin type 1 receptor and the vasopressin type 2 receptor, were also impaired in their ability to be sequestered when levels of endogenous ARF6 in cells were reduced. Interestingly, endocytosis of the endothelin type B receptor, characterized as being internalized via the caveolae pathway, was also markedly inhibited in ARF6-depleted cells. In contrast, internalization of the vasoactive intestinal peptide receptor was unaffected by reduced levels of ARF6. Finally, internalization of the acetylcholine-muscarinic type 2 receptor via the non-clathrin-coated vesicle pathway was also inhibited in ARF6-depleted cells. Taken together, our results demonstrate that ARF6 proteins play an essential role in the internalization process of most G protein-coupled receptors regardless of the endocytic route being utilized. However, this phenomenon is not general. In some cases, another ARF isoform or other proteins may be essential to regulate the endocytic process.     

Decoupling of activation and effector binding underlies ARF6 priming of fast endocytic recycling

The small GTP-binding protein ADP-ribosylation factor 6 (ARF6) controls the endocytic recycling pathway of several plasma membrane receptors. We analyzed the localization and GDP/GTP cycle of GFP-tagged ARF6 by total internal reflection fluorescent microscopy. We found that ARF6-GFP associates with clathrin-coated pits (CCPs) at the plasma membrane in a GTP-dependent manner in a mechanism requiring the adaptor protein complex AP-2. In CCP, GTP-ARF6 mediates the recruitment of the ARF-binding domain of downstream effectors including JNK-interacting proteins 3 and 4 (JIP3 and JIP4) after the burst recruitment of the clathrin uncoating component auxilin. ARF6 does not contribute to receptor-mediated clathrin-dependent endocytosis. In contrast, we found that interaction of ARF6 and JIPs on endocytic vesicles is required for trafficking of the transferrin receptor in the fast, microtubule-dependent endocytic recycling pathway. Our findings unravel a novel mechanism of separation of ARF6 activation and effector function, ensuring that fast recycling may be determined at the level of receptor incorporation into CCPs.     

Desensitization of the luteinizing hormone/choriogonadotropin receptor in ovarian follicular membranes is inhibited by catalytically inactive ARNO(+)

We have investigated the participation of endogenous ADP-ribosylation factor (ARF) nucleotide-binding site opener (ARNO) in desensitization of the luteinizing hormone/choriogonadotropin (LH/CG) receptor, independent of receptor internalization, using a cell-free plasma membrane model. We recently showed that the addition of recombinant ARNO promotes binding of beta-arrestin1 to the third intracellular (3i) loop of the active LH/CG receptor, thereby reducing the ability of the receptor to activate the stimulatory G protein and signal to adenylyl cyclase. In the present report we determined whether ARNO is detectable in follicular membranes and whether the catalytically inactive E156K ARNO mutant, containing a mutation in the Sec7 domain, can act in a dominant negative manner to block LH/CG receptor desensitization. Results show that ARNO is readily detected in follicular membranes and that levels of membrane-associated ARNO increase with follicular maturation. The addition of catalytically inactive E156K ARNO blocks both the release of beta-arrestin1 from its membrane docking site, based on Western blot analysis, and development of LH/CG receptor desensitization. We also investigated whether a point mutation in the pleckstrin homology (PH) domain of ARNO (R280D), which blocks binding of phosphoinositides like phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate (PIP(2)) but not catalytic activity, disrupts LH/CG receptor desensitization. R280D ARNO neither promotes nor inhibits LH/CG receptor desensitization, consistent with a requirement of the PH domain of ARNO for its association with the plasma membrane. LH/CG receptor activation of ARNO is not mediated by activation of phosphatidylinositol 3-kinase (PI 3-kinase) or by G protein beta gamma subunits. Taken together, these results suggest that LH/CG receptor promotes beta-arrestin1 release from its membrane docking site to bind to the 3i loop of the LH/CG receptor via activation of membrane delimited endogenous ARNO. As ARNO activation is independent of PI 3-kinase and G beta gamma, our results are consistent with a role for PIP(2) in receptor-stimulated ARNO activation.     

ARF6 activation by Galpha q signaling: Galpha q forms molecular complexes with ARNO and ARF6

G protein-coupled receptors (GPCRs) are widely expressed hepta-helical receptors with tightly regulated pleiotropic effects. ADP-Ribosylation Factor 6 (ARF6) plays an important role in GPCR trafficking and is the subject of intense research. However, the mechanisms underlying activation and regulation of ARF6 by GPCRs are poorly characterized. Here we report that Galpha(q) signaling leads to the activation of ARF6. Stimulation of the TPbeta receptor triggered ARF6 activation which was completely inhibited by the RGS domain of GRK2 known to specifically bind and sequester Galpha(q). Co-immunoprecipitation studies revealed that ARNO (a guanine nucleotide exchange factor for ARF6) and ARF6 formed complexes preferentially with activated Galpha(q) compared to non-activated Galpha(q). Formation of the Galpha(q) complexes with ARNO and ARF6 was detected early and was optimal after 30 min of receptor stimulation corresponding with the profile of ARF6 activation. Interestingly, binding experiments using purified proteins showed that Galpha(q) interacted directly with ARNO. Galpha(q)-dependent TPbeta receptor-mediated activation of ARF6 resulted in phosphoinositol-4,5-bisphosphate production which was potently inhibited by dominant negative mutants of ARNO and ARF6. Furthermore, our data show that the expression of ARNO and ARF6 promoted, whereas dominant negative mutants of these proteins inhibited the internalization of the TPbeta receptor. This further elucidates our previous data on the PLCbeta- and PKC-independent mechanism involved in Galpha(q)-mediated internalization of the TPbeta receptor. Taken altogether, our results support a novel model where activated Galpha(q) forms molecular complexes with ARNO and ARF6, possibly through a direct interaction with ARNO, leading to ARF6 activation.     

ARF6 regulates angiotensin II type 1 receptor endocytosis by controlling the recruitment of AP-2 and clathrin

We have previously shown that the ADP-ribosylation factor 6 (ARF6), a small GTP-binding protein, is important for the internalization of several G protein-coupled receptors. Here, we propose to elucidate the molecular steps controlled by ARF6 in the endocytic process of the angiotensin II type 1 receptor (ATR), a model receptor being internalized via the clathrin-coated vesicle pathway. In HEK 293 cells, angiotensin II stimulation leads to the formation of a complex including ARF6, the beta-subunit of AP-2 and the heavy chain of clathrin. In vitro experiments indicate that the interactions between ARF6 and the beta-subunit of AP-2 as well as with the heavy chain of clathrin are direct, and dependent upon the nature of the nucleotide bound to ARF6. beta2-adaptin binds to ARF6-GDP while clathrin preferentially interacts with ARF6 when loaded with GTP. These interactions have an important physiological consequence. Indeed, depletion of ARF6 prevents the agonist-dependent recruitment of beta2-adaptin and clathrin to the activated ATR. Interestingly, in these cells, the plasma membrane redistribution of either beta2-adaptin-GFP or betaarrestin 2-GFP, following Ang II stimulation, is altered. Both proteins are defective in clustering into large punctated structure at the plasma membrane compared to control conditions. Taken together, these results suggest that the cycling of ARF6 between its GDP-and GTP-bound states coordinates the recruitment of AP-2 and clathrin to activated receptors during the endocytic process.     

Regulation of G protein-coupled receptor endocytosis by ARF6 GTP-binding proteins.

The function of G protein-coupled receptors is regulated by a broad variety of membrane-bound and intracellular proteins. These act in concert to activate signaling pathways that will lead to the desensitization of activated receptors and, for most receptor types, their trafficking to intracellular compartments. This review focuses mainly on the endocytic pathways used by a G protein-coupled receptor and on the proteins that play an essential role in the regulation of the internalization process, most specifically the ADP-ribosylation factors. This family of proteins has been shown to be important for vesicle trafficking between different cellular membranes. The latest findings regarding the molecular mechanisms that regulate internalization of an agonist-stimulated receptor are presented here. Finally, a perspective on how ARF6 proteins might regulate the internalization process is also proposed.     

Properties of secretin receptor internalization differ from those of the beta(2)-adrenergic receptor.

The endocytic pathway of the secretin receptor, a class II GPCR, is unknown. Some class I G protein-coupled receptors (GPCRs), such as the beta(2)-adrenergic receptor (beta(2)-AR), internalize in clathrin-coated vesicles and this process is mediated by G protein-coupled receptor kinases (GRKs), beta-arrestin, and dynamin. However, other class I GPCRs, for example, the angiotensin II type 1A receptor (AT(1A)R), exhibit different internalization properties than the beta(2)-AR. The secretin receptor, a class II GPCR, is a GRK substrate, suggesting that like the beta(2)-AR, it may internalize via a beta-arrestin and dynamin directed process. In this paper we characterize the internalization of a wild-type and carboxyl-terminal (COOH-terminal) truncated secretin receptor using flow cytometry and fluorescence imaging, and compare the properties of secretin receptor internalization to that of the beta(2)-AR. In HEK 293 cells, sequestration of both the wild-type and COOH-terminal truncated secretin receptors was unaffected by GRK phosphorylation, whereas inhibition of cAMP-dependent protein kinase mediated phosphorylation markedly decreased sequestration. Addition of secretin to cells resulted in a rapid translocation of beta-arrestin to plasma membrane localized receptors; however, secretin receptor internalization was not reduced by expression of dominant negative beta-arrestin. Thus, like the AT(1A)R, secretin receptor internalization is not inhibited by reagents that interfere with clathrin-coated vesicle-mediated internalization and in accordance with these results, we show that secretin and AT(1A) receptors colocalize in endocytic vesicles. This study demonstrates that the ability of secretin receptor to undergo GRK phosphorylation and beta-arrestin binding is not sufficient to facilitate or mediate its internalization. These results suggest that other receptors may undergo endocytosis by mechanisms used by the secretin and AT(1A) receptors and that kinases other than GRKs may play a greater role in GPCR endocytosis than previously appreciated.     

N-terminal tyrosine modulation of the endocytic adaptor function of the beta-arrestins

The highly homologous beta-arrestin1 and -2 adaptor proteins play important roles in the function of G protein-coupled receptors. Either beta-arrestin variant can function as a molecular chaperone for clathrin-mediated receptor internalization. This role depends primarily upon two distinct, contiguous C-terminal beta-arrestin motifs recognizing clathrin and the beta-adaptin subunit of AP2. However, a molecular basis is lacking to explain the different endocytic efficacies of the two beta-arrestin isoforms and the observation that beta-arrestin N-terminal substitution mutants can act as dominant negative inhibitors of receptor endocytosis. Despite the near identity of the beta-arrestins throughout their N termini, sequence variability is present at a small number of residues and includes tyrosine to phenylalanine substitutions. Here we show that corresponding N-terminal (Y/F)VTL sequences in beta-arrestin1 and -2 differentially regulate mu-adaptin binding. Our results indicate that the beta-arrestin1 Tyr-54 lessens the interaction with mu-adaptin and moreover is a Src phosphorylation site. A gain of endocytic function is obtained with the beta-arrestin1 Y54F substitution, which improves both the beta-arrestin1 interaction with mu-adaptin and the ability to enhance beta2-adrenergic receptor internalization. These data indicate that beta-arrestin2 utilizes mu-adaptin as an endocytic partner, and that the inability of beta-arrestin1 to sustain a similar degree of interaction with mu-adaptin may result from coordination of Tyr-54 by neighboring residues or its modification by Src kinase. Additionally, these naturally occurring variations in beta-arrestins may also differentially regulate the composition of the signaling complexes organized on the receptor.     

ARNO through its coiled-coil domain regulates endocytosis at the apical surface of polarized epithelial cells

ARNO is a guanine-nucleotide exchange protein for the ARF family of GTPases. Here we show that in polarized epithelial cells, ARNO is localized exclusively to the apical plasma membrane, where it regulates endocytosis. Expression of ARNO stimulates apical endocytosis of the polymeric immunoglobulin receptor, and coexpression of ARF6 with ARNO leads to a synergistic stimulation of apical endocytosis. Expression of a dominant negative ARF6 mutant, ARF6-T27N, antagonizes this stimulatory effect. Deletion of the N-terminal coiled-coil (CC) domain of ARNO causes the mutant ARNO to localize to both the apical and basolateral plasma membranes. Expression of the CC domain alone abolishes ARNO-induced apical endocytosis as well as co-localization of IgA-receptor complexes with ARNO and clathrin. These results suggest that the CC domain contributes to the specificity of apical localization of ARNO through association with components of the apical plasma membrane. We conclude that ARNO acts together with ARF6 to regulate apical endocytosis.     

EFA6 regulates endosomal trafficking and affects early endosomes in polarized MDCK cells

The small-GTPase family of ADP ribosylation factors (ARFs) recruit coat proteins to promote vesicle budding. ARFs are activated by an association with sec7-containing exchange factors which load them with GTP. In epithelial cells, the small GTPase ARF6 operates within the endocytic system and has been shown to associate with ARNO to promote apical endocytosis and early to late endosomal trafficking. EFA6 has been shown to stimulate tight-junction formation and maintenance. Here, we show that in polarized epithelial MDCK cells, EFA6 is localized to early endosomes, causes their dramatic enlargement, and promotes basolateral targeting of IgA, which is normally targeted to the apical PM. These results suggest that the physiological function of ARF6 within the endocytic system is regulated by the exchange factor it associates with.     

A Highlights from MBoC Selection: Unregulated ARF6 Activation in Epithelial Cysts Generates Hyperactive Signaling Endosomes and Disrupts Morphogenesis

Tumor development in glandular tissues is associated with structural alterations in the hollow ducts and spherical structures that comprise such tissues. We describe a signaling axis involving sustained activation of the GTP-binding protein, ARF6, that provokes dramatic changes in the organization of epithelial cysts, reminiscent of tumorigenic glandular phenotypes. In reconstituted basement membrane cultures of renal epithelial cysts, enhanced ARF6 activation induces the formation of cell-filled glandular structures with multiple lumens and disassembled cadherin-based cell–cell contacts. All of these alterations are accompanied by growth factor receptor internalization into signaling endosomes and reversed by blocking ARF6 activation or receptor endocytosis. Receptor localization in signaling endosomes results in hyperactive extracellular signal-regulated kinase signaling leading to Bcl-2 stabilization and aberrant cysts. Similarly, formation of hyperproliferative and disorganized mammary acini induced by chronic stimulation of colony-stimulating factor 1 receptor is coupled to endogenous ARF6 activation and constitutive receptor internalization and is reversed by ARF6 inhibition. These findings identify a previously unrecognized link between ARF6-regulated receptor internalization and events that drive dramatic alterations in cyst morphogenesis providing new mechanistic insight into the molecular processes that can promote epithelial glandular disruption.     

ARF6-Dependent Regulation of P2Y Receptor Traffic and Function in Human Platelets

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.     

Unraveling G protein-coupled receptor endocytosis pathways using real-time monitoring of agonist-promoted interaction between beta-arrestins and AP-2

The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves beta-arrestin and clathrin-coated pits. However, both beta-arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that beta-arrestin binding to the beta2 subunit of the clathrin adaptor AP-2 (beta2-adaptin) is needed for the beta-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted beta-arrestin 1 and 2 interaction with beta2-adaptin, indicating a beta-arrestin- and clathrin-dependent endocytic process. Detailed analyses of beta-arrestin interactions with both the receptor and beta2-adaptin also allowed us to demonstrate that recruitment of beta-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between beta-arrestins and beta2-adaptin. However, both receptors recruited beta-arrestins upon agonist stimulation, suggesting a beta-arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based beta-arrestin/beta2-adaptin interaction assay represents a novel biosensor to assess receptor activation.     

Agonist-stimulated beta-adrenergic receptor internalization requires dynamic cytoskeletal actin turnover

Stimulation of beta-adrenergic receptors (betaARs) leads to sequential recruitment of beta-arrestin, AP-2 adaptor protein, clathrin, and dynamin to the receptor complex, resulting in endocytosis. Whether a dynamic actin cytoskeleton is required for betaAR endocytosis is not known. In this study, we have used beta(1)- and beta(2) ARs, two ubiquitously expressed members of the betaAR family, to comprehensively evaluate the requirement of the actin cytoskeleton in receptor internalization. The integrity of the actin cytoskeleton was manipulated with the agent latrunculin B (LB) and mutants of cofilin to depolymerize actin filaments. Treatment of cells with LB resulted in dose-dependent depolymerization of the cortical actin cytoskeleton that was associated with significant attenuation in internalization of beta(2)ARs, beta(1)ARs, and mutants of beta(1)ARs that internalize via either clathrin- or caveolin-dependent pathways. Importantly, LB treatment did not inhibit beta-arrestin translocation or dynamin recruitment to the agonist-stimulated receptor. To unequivocally demonstrate the requirement of the actin cytoskeleton for beta(2)AR endocytosis, we used an actin-binding protein cofilin that biochemically depolymerizes and severs actin filaments. Isoproterenol-mediated internalization of beta(2)AR was completely blocked in the presence of wild type cofilin, which could be rescued by a mutant of cofilin that mimics a constitutive phosphorylated state and leads to normal agonist-stimulated beta(2)AR endocytosis. Finally, treatment with jasplakinolide, an inhibitor of actin turnover, resulted in dose-dependent inhibition of beta(2)AR internalization, suggesting that turnover of actin filaments at the receptor complex is required for endocytosis. Taken together, these data demonstrate that intact and functional dynamic actin cytoskeleton is required for normal betaAR internalization.     

The role of beta-arrestins in the formyl peptide receptor-like 1 internalization and signaling

The N-formyl peptide receptor-like 1 (FPRL1) is a G protein-coupled receptor (GPCR) that transmits intracellular signals in response to a variety of agonists, many of them being clearly implicated in human pathology. beta-arrestins are adaptor proteins that uncouple GPCRs from G protein and regulate receptor internalization. They can also function as signal transducers through the scaffolding of signaling molecules, such as components of the extracellular signal-regulated kinase (ERK) cascade. We investigated the role of beta-arrestins in ligand-induced FPRL1 internalization and signaling. In HEK293 cells expressing FPRL1, fluorescence microscopy revealed that agonist-stimulated FPRL1 remained co-localized with beta-arrestins during endocytosis. Internalization of FPRL1, expressed in a mouse embryonic fibroblast (MEF) cell line lacking endogenous beta-arrestins, was highly compromised. This distinguishes FPRL1 from the prototypical formyl peptide receptor FPR that is efficiently internalized in the absence of beta-arrestins. In both HEK293 and MEF cells, FPRL1-mediated ERK1/2 activation was a rapid and transient event. The kinetics and extent of ERK1/2 activation were not significantly modified by beta-arrestin overexpression. The pattern of FPRL1-mediated ERK1/2 activation was similar whether cells express or not beta-arrestins. Furthermore, treatment of the FPRL1 expressing cells with pertussis toxin inhibited ERK1/2 activation in MEF and in HEK293 cells. These results led us to conclude that activation of ERK1/2 mediated by FPRL1 occurs primarily through G protein signaling. Since beta-arrestin-mediated signaling has been observed essentially for receptors coupled to G proteins other than G(i), this may be a characteristic of G(i) protein-coupled chemoattractant receptors.     

Increased GTP-binding to dynamin II does not stimulate receptor-mediated endocytosis

Regarding the molecular mechanism of dynamin in receptor-mediated endocytosis, GTPase activity of dynamin has been thought to have a critical role in endocytic vesicle internalization. However, a recent report suggested that GTP-binding to dynamin itself activates the dynamin to recruit molecular machinery necessary for endocytosis. In this study, to investigate the role of GTP binding to dynamin II, we generated two mutant dynamin II constructs: G38V and K44E. G38V, its GTP binding site might be mainly occupied by GTP caused by reduced GTPase activity, and K44E mutant, its GTP binding site might be vacant, caused by its decreased affinity for GTP and GDP. From the analysis of the ratio of GTP vs GDP bound to dynamin, we confirmed these properties. To test the effect of these mutant dynamins on endocytosis, we performed flow cytometry and confocal immunofluorescence analysis and found that these two mutants have inhibitory effect on transferrin-induced endocytosis. Whereas fluorescent transferrin was completely internalized in wild-type (WT) dynamin II expressing cells, no intracellular accumulation of fluorescent transferrin was found in the cells overexpressing K44E and G38V mutant. Interestingly, the amount of GTP bound to K44E was increased when endocytosis was induced than that bound to WT. The present results suggested that the GTPase activity of dynamin II is required for formation of endocytic vesicle and GTP-binding to dynamin II per se is not sufficient for stimulating endocytosis.     

Importance of constitutive activity and arrestin-independent mechanisms for intracellular trafficking of the ghrelin receptor

The ghrelin receptor (GhrelinR) and its related orphan GPR39 each display constitutive signaling, but only GhrelinRs undergo basal internalization. Here we investigate these differences by considering the roles of the C tail receptor domains for constitutive internalization and activity. Furthermore the interaction between phosphorylated receptors and beta-arrestin adaptor proteins has been examined. Replacement of the FLAG-tagged GhrelinR C tail with the equivalent GPR39 domain (GhR-39 chimera) preserved G(q) signaling. However in contrast to the GhrelinR, GhR-39 receptors exhibited no basal and substantially decreased agonist-induced internalization in transiently transfected HEK293 cells. Internalized GhrelinR and GhR-39 were predominantly localized to recycling compartments, identified with transferrin and the monomeric G proteins Rab5 and Rab11. Both the inverse agonist [d-Arg(1), d-Phe(5), d-Trp(7,9), Leu(11)] substance P and a naturally occurring mutant GhrelinR (A204E) with eliminated constitutive activity inhibited basal GhrelinR internalization. Surprisingly, we found that noninternalizing GPR39 was highly phosphorylated and that basal and agonist-induced phosphorylation of the GhR-39 chimera was elevated compared with GhrelinRs. Moreover, basal GhrelinR endocytosis occurred without significant phosphorylation, and it was not prevented by cotransfection of a dominant-negative beta-arrestin1(319-418) fragment or by expression in beta-arrestin1/2 double-knockout mouse embryonic fibroblasts. In contrast, agonist-stimulated GhrelinRs recruited the clathrin adaptor green fluorescent protein-tagged beta-arrestin2 to endosomes, coincident with increased receptor phosphorylation. Thus, GhrelinR internalization to recycling compartments depends on C-terminal motifs and constitutive activity, but the high levels of GPR39 phosphorylation, and of the GhR-39 chimera, are not sufficient to drive endocytosis. In addition, basal GhrelinR internalization occurs independently of beta-arrestins.     

beta-Arrestin/AP-2 interaction in G protein-coupled receptor internalization: identification of a beta-arrestin binging site in beta 2-adaptin.

beta-Arrestins, proteins involved in the turn-off of G protein-coupled receptor (GPCR) activation, bind to the beta(2)-adaptin subunit of the clathrin adaptor AP-2. The interaction of beta(2)-adaptin with beta-arrestin involves critical arginine residues in the C-terminal domain of beta-arrestin and plays an important role in initiating clathrin-mediated endocytosis of the beta(2)-adrenergic receptor (beta(2)AR) (Laporte, S. A., Oakley, R. H., Holt, J. A., Barak, L. S., and Caron, M. G. (2000) J. Biol. Chem. 275, 23120--23126). However, the beta-arrestin-binding site in beta(2)-adaptin has not been identified, and little is known about the role of beta-arrestin/AP-2 interaction in the endocytosis of other GPCRs. Using in vitro binding assays, we have identified two glutamate residues (Glu-849 and Glu-902) in beta(2)-adaptin that are important in beta-arrestin binding. These residues are located in the platform subdomain of the C terminus of beta(2)-adaptin, where accessory/adapter endocytic proteins for other classes of receptors interact, distinct from the main site where clathrin interacts. The functional significance of the beta-arrestin/AP-2/clathrin complex in the endocytosis of GPCRs such as the beta(2)AR and vasopressin type II receptor was evaluated using mutant constructs of the beta(2)-adaptin C terminus containing either the clathrin and the beta-arrestin binding domains or the beta-arrestin-binding domain alone. When expressed in human embryonic kidney 293 cells, both constructs acted as dominant negatives inhibiting the agonist-induced internalization of the beta(2)AR and the vasopressin type II receptor. In addition, although the beta(2)-adaptin construct containing both the clathrin and beta-arrestin binding domains was able to block the endocytosis of transferrin receptors, a beta(2)-adaptin construct capable of associating with beta-arrestin but lacking its high affinity clathrin interaction did not interfere with transferrin receptor endocytosis. These results suggest that the interaction of beta-arrestin with beta(2)-adaptin represents a selective endocytic trigger for several members of the GPCR family.     

HCMV-encoded chemokine receptor US28 employs multiple routes for internalization

The human cytomegalovirus-encoded G protein-coupled receptor homologue US28 binds inflammatory chemokines and sequesters them from the environment of infected cells. Low surface deposition and endocytosis are dependent on constitutive C-terminal phosphorylation, suggesting a requirement for beta-arrestin binding in receptor internalization. In this report, a US28-dependent redistribution of beta-arrestin into vesicular structures occurred, although internalization of US28 was independent of beta-arrestin. Internalization of US28 was dynamin-dependent, and US28 partially partitioned into the detergent-resistant membrane fraction. Endocytosis was diminished by cholesterol depletion, yet sucrose inhibition was even stronger. The relevance of the clathrin-coated pit pathway was supported by colocalization of beta(2)-adaptin and US28 in endocytic compartments. Exchange of the C-terminal dileucine endocytosis motif inhibited rapid endocytosis, indicating a direct interaction of US28 with the AP-2 adaptor complex. We suggest that the arrestin-independent, dynamin-dependent internalization of US28 reveals a differential sorting of beta-arrestins and the virally encoded chemokine receptor homologue.     

Palmitoylation of the V2 vasopressin receptor carboxyl tail enhances beta-arrestin recruitment leading to efficient receptor endocytosis and ERK1/2 activation

A large number of G protein-coupled receptors are palmitoylated on cysteine residues located in their carboxyl tail, but the general role of this post-translational modification remains poorly understood. Here we show that preventing palmitoylation of the V2 vasopressin receptor, by site-directed mutagenesis of cysteines 341 and 342, significantly delayed and decreased both agonist-promoted receptor endocytosis and mitogen-activated protein kinase activation. Pharmacological blockade of receptor endocytosis is without effect on the vasopressin-stimulated mitogen-activated protein kinase activity, excluding the possibility that the reduced kinase activation mediated by the palmitoylation-less mutant could result from altered receptor endocytosis. In contrast, two dominant negative mutants of beta-arrestin which inhibit receptor endocytosis also attenuated vasopressin-stimulated mitogen-activated protein kinase activity, suggesting that the scaffolding protein, beta-arrestin, represents the common link among receptor palmitoylation, endocytosis, and kinase activation. Coimmunoprecipitation and bioluminescence resonance energy transfer experiments confirmed that inhibiting receptor palmitoylation considerably reduced the vasopressin-stimulated recruitment of beta-arrestin to the receptor. Interestingly, the changes in beta-arrestin recruitment kinetics were similar to those observed for vasopressin-stimulated receptor endocytosis and mitogen-activated protein kinase activation. Taken together the results indicate that palmitoylation enhances the recruitment of beta-arrestin to the activated V2 vasopressin receptor thus facilitating processes requiring the scaffolding action of beta-arrestin.