Differential Roles of Arrestin-2 Interaction with Clathrin and Adaptor Protein 2 in G Protein-coupled Receptor Trafficking
2002; Elsevier BV; Volume: 277; Issue: 34 Linguagem: Inglês
10.1074/jbc.m204528200
ISSN1083-351X
Autores Tópico(s)Advanced Proteomics Techniques and Applications
ResumoThe non-visual arrestins, arrestin-2 and arrestin-3, play a critical role in regulating the signaling and trafficking of many G protein-coupled receptors (GPCRs). Molecular insight into the role of arrestins in GPCR trafficking has suggested that arrestin interaction with clathrin, β2-adaptin (the β-subunit of the adaptor protein AP2), and phosphoinositides contributes to this process. In the present study, we have attempted to better define the molecular basis and functional role of arrestin-2 interaction with clathrin and β2-adaptin. Site-directed mutagenesis revealed that the C-terminal region of arrestin-2 mediated β2-adaptin and clathrin interaction with Phe-391 and Arg-395 having an essential role in β2-adaptin binding and LIELD (residues 376–380) having an essential role in clathrin binding. Interestingly, arrestin-2-R169E, an activated form of arrestin that binds to GPCRs in a phosphorylation-independent manner, has significantly enhanced binding to β2-adaptin and clathrin. This suggests that receptor-induced conformational changes in the C-terminal tail of arrestin-2 will likely play a major role in mediating arrestin interaction with clathrin-coated pits. In an effort to clarify the role of these interactions in GPCR trafficking we generated arrestin mutants that were completely and selectively defective in either clathrin (arrestin-2-ΔLIELD) or β2-adaptin (arrestin-2-F391A) interaction. Analysis of these mutants in COS-1 cells revealed that arrestin/clathrin interaction was essential for agonist-promoted internalization of the β2-adrenergic receptor, while arrestin/β2-adaptin interaction appeared less critical. Arrestin-2 mutants defective in both clathrin and β2-adaptin binding functioned as effective dominant negatives in HEK293 cells and significantly attenuated β2-adrenergic receptor internalization. These mutants should prove useful in better defining the role of arrestins in mediating receptor trafficking. The non-visual arrestins, arrestin-2 and arrestin-3, play a critical role in regulating the signaling and trafficking of many G protein-coupled receptors (GPCRs). Molecular insight into the role of arrestins in GPCR trafficking has suggested that arrestin interaction with clathrin, β2-adaptin (the β-subunit of the adaptor protein AP2), and phosphoinositides contributes to this process. In the present study, we have attempted to better define the molecular basis and functional role of arrestin-2 interaction with clathrin and β2-adaptin. Site-directed mutagenesis revealed that the C-terminal region of arrestin-2 mediated β2-adaptin and clathrin interaction with Phe-391 and Arg-395 having an essential role in β2-adaptin binding and LIELD (residues 376–380) having an essential role in clathrin binding. Interestingly, arrestin-2-R169E, an activated form of arrestin that binds to GPCRs in a phosphorylation-independent manner, has significantly enhanced binding to β2-adaptin and clathrin. This suggests that receptor-induced conformational changes in the C-terminal tail of arrestin-2 will likely play a major role in mediating arrestin interaction with clathrin-coated pits. In an effort to clarify the role of these interactions in GPCR trafficking we generated arrestin mutants that were completely and selectively defective in either clathrin (arrestin-2-ΔLIELD) or β2-adaptin (arrestin-2-F391A) interaction. Analysis of these mutants in COS-1 cells revealed that arrestin/clathrin interaction was essential for agonist-promoted internalization of the β2-adrenergic receptor, while arrestin/β2-adaptin interaction appeared less critical. Arrestin-2 mutants defective in both clathrin and β2-adaptin binding functioned as effective dominant negatives in HEK293 cells and significantly attenuated β2-adrenergic receptor internalization. These mutants should prove useful in better defining the role of arrestins in mediating receptor trafficking. G protein-coupled receptor adaptor protein β2-adrenergic receptor β-subunit of the adaptor protein AP2 bovine serum albumin glutathione S-transferase human embryonic kidney Tris-buffered saline Many transmembrane signaling systems consist of specific G protein-coupled receptors (GPCRs)1 that transduce the binding of a diverse array of extracellular stimuli into intracellular signaling events (1Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar). GPCRs modulate the activity of numerous effector molecules including adenylyl cyclases, phosphoinositide 3-kinase, non-receptor tyrosine kinases, small G proteins, phosphodiesterases, phospholipases, and ion channels. To ensure that extracellular stimuli are translated into intracellular signals of appropriate magnitude and specificity, these signaling cascades are tightly regulated. GPCRs are subject to three principle modes of regulation: desensitization, in which a receptor becomes refractory to continued stimuli; endocytosis, whereby receptors are removed from the cell surface; and down-regulation, where total cellular receptor levels are decreased (2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (858) Google Scholar, 3Ferguson S.S.G. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). GPCR desensitization is primarily mediated by second messenger-dependent kinases, such as protein kinase A and protein kinase C, and by G protein-coupled receptor kinases. G protein-coupled receptor kinases specifically phosphorylate activated GPCRs, initiating recruitment of arrestins. Arrestins are divided into two major classes, visual and non-visual, on the basis of localization. The non-visual arrestins, arrestin-2 and arrestin-3 (also termed β-arrestin-1 and -2), are broadly distributed and have been implicated in regulating multiple processes including GPCR desensitization (4Wilden U. Hall S.W. Kuhn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (577) Google Scholar, 5Lohse M.J. Benovic J.L. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (919) Google Scholar), trafficking (6Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (853) Google Scholar, 7Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1179) Google Scholar), and signaling via non-receptor tyrosine kinases (8Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Kawakatsu H Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar, 9Barlic J. Andrews J.D. Kelvin A.A. Bosinger S.E. Dobransky T. Feldman R.D. Ferguson S.S.G. Kelvin D.J. Nat. Immunol. 2000; 1: 227-233Crossref PubMed Scopus (198) Google Scholar) and mitogen-activated protein kinases (10DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (690) Google Scholar, 11DeFea K.A. Vaughn Z.D. O'Bryan E.M. Nishijima D. Dery O. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11086-11091Crossref PubMed Scopus (353) Google Scholar, 12McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar). Multiple interactions contribute to arrestin-mediated trafficking of GPCRs including a C-terminal insert region that interacts with the N-terminal domain of the clathrin heavy chain (13Krupnick JG. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar,14Goodman Jr., O.B. Krupnick J.G. Gurevich V.V. Benovic J.L. Keen J.H. J. Biol. Chem. 1997; 272: 15017-15022Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), a C-terminal region that interacts with the β-subunit of the heterotetrameric adaptor protein 2 (AP2) complex (β2-adaptin) (15Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (529) Google Scholar, 16Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar), and a basic region that binds phosphoinositides (17Gaidarov I. Krupnick J.G. Falck J.R. Benovic J.L. Keen J.H. EMBO J. 1999; 18: 871-881Crossref PubMed Scopus (170) Google Scholar). While each of these interactions appears to contribute to the ability of non-visual arrestins to mediate GPCR trafficking, the mechanistic basis of this process remains unclear. Here we have attempted to better define the interactions of arrestin-2 with clathrin and β2-adaptin. We were able to generate arrestin mutants that were selectively and completely defective in either clathrin or β2-adaptin interaction. These mutants were then used to elucidate the role of these interactions in β2-adrenergic receptor (β2AR) trafficking. Our studies suggested that arrestin interaction with clathrin, but not β2-adaptin, is critical for β2AR internalization. Moreover, we found that arrestin-2 mutants defective in both clathrin and β-adaptin binding functioned as effective dominant negative mutants. These mutants should prove useful in better defining the role of arrestins in receptor trafficking. Reagents were generously provided by Drs. James Keen (glutathione S-transferase (GST)-clathrin-TD), Harvey McMahon (wild type and mutant GST-β2-adaptin and GST-α2-adaptin), Juan Bonifacino (GST-β3-adaptin), and Larry Donoso (mouse monoclonal antibody F4C1). A GST-β2-adaptin appendage (residues 700–937) fusion protein, several mutant GST-β2-adaptin fusions, and a GST-clathrin terminal domain (residues 1–579) fusion were expressed and purified on glutathione-agarose as described previously (13Krupnick JG. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 18Owen D.J. Vallis Y. Pearse B.M.F. McMahon H.T. Evans P.R. EMBO J. 2000; 19: 4216-4227Crossref PubMed Google Scholar). Arrestin-2 and additional β2-adaptin mutations were generated by PCR and confirmed by DNA sequencing. Wild type and mutant arrestins were expressed in COS-1 cells by transient transfection as described previously (19Orsini M.J. Benovic J.L. J. Biol. Chem. 1998; 273: 34616-34622Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Arrestin extracts were prepared by lysing the cells by freeze/thaw and Polytron disruption in 20 mm Hepes, pH 7.4, 0.1 m NaCl, 0.02% Triton X-100, 10 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 0.02 mg/ml leupeptin, 0.2 mg/ml benzamidine. The extracts were centrifuged (55,000 × g, 20 min), and the supernatants were aliquoted, frozen, and stored at −80 °C until needed (extracts contained ∼2 μg of arrestin/ml). Arrestin-containing lysates (∼20 ng of arrestin) were incubated with 5 μl of glutathione-agarose beads (containing ∼50 pmol of bound GST or GST fusion protein) in binding buffer (20 mm Hepes, pH 7.2, 120 mm potassium acetate, 0.1 mmdithiothreitol, 0.1% Triton X-100) for 1 h at 4 °C (in a total volume of 100 μl). The beads were then pelleted (1000 rpm, 5 min at 4 °C) and washed two to three times with 0.5 ml of ice-cold binding buffer, and bound arrestin was eluted by boiling the beads in SDS sample buffer for 10 min. The samples were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose, and arrestin was detected by immunoblotting using mouse monoclonal (F4C1) or rabbit polyclonal (178) anti-arrestin antibodies, horseradish peroxidase-labeled goat anti-mouse or anti-rabbit secondary antibodies, and chemiluminescence. To prepare brain extracts, fresh bovine brain was stripped of connective tissue and minced in ∼1 ml of homogenization buffer (40 mm Tris, pH 8, 150 mm NaCl, 2 mmEDTA, 0.2 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 0.02 mg/ml aprotinin, 0.02 mg/ml leupeptin, 0.2 mg/ml benzamidine, 0.02 mg/ml pepstatin) per mg of tissue using a Brinkman Polytron (20,000 rpm, 30 s). The homogenate was centrifuged at 30,000 × g for 30 min, and the resulting pellet was resuspended in homogenization buffer containing 1% Triton X-100, homogenized again (20,000 rpm, 30 s), and centrifuged at 30,000 × g for 30 min. The supernatant was then centrifuged at 55,000 × g for 30 min, and the final supernatant (∼20 mg of protein/ml) was aliquoted and stored at −80 °C until use. For binding of bovine brain proteins to GST-β2-adaptin, 20 μl of brain extract (∼400 μg of total protein) were incubated with 50 pmol of various GST-β2-adaptin mutant proteins bound to glutathione-agarose beads. The beads were washed, and bound proteins were eluted, electrophoresed, and transferred to a nitrocellulose membrane. Binding of AP180 and Eps15 to GST-β2-adaptin was visualized by probing the membrane with either anti-AP180 or anti-Eps15 monoclonal antibodies (Transduction Laboratories). COS-1 and HEK293 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin sulfate in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Transient transfection was done using FuGENE 6 (Roche Molecular Biochemicals) according to the manufacturer's recommendations. COS-1 cells transiently transfected with pcDNA3-FLAG-β2AR and various pcDNA3-arrestin-2 constructs were stimulated with 10 μm (−)-isoproterenol for 2 min at 37 °C and then lysed in 1 ml of co-immunoprecipitation buffer (coIP buffer) containing 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm EDTA, 10% glycerol, 1% Triton X-100, and protease inhibitors. Insoluble materials were removed by centrifugation at 55,000 × g for 30 min, and the supernatants were incubated with 5 μg of purified polyclonal anti-β2AR antibody (Santa Cruz Biotechnology) for 1 h followed by incubation with 20 μl of 50% protein A-agarose for 16 h at 4 °C to immunoprecipitate the β2AR. Immunocomplexes were then extensively washed with coIP buffer, and samples were subjected to SDS-PAGE and Western blot analysis for β2AR (using M1 anti-FLAG antibody, Sigma) or arrestin-2 (using mouse monoclonal anti-arrestin antibody F4C1). Internalization of FLAG-β2AR was assessed by enzyme-linked immunosorbent assay as described previously (20Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar) with some minor modifications. Briefly, COS-1 cells transfected with pcDNA3-FLAG-β2AR and various arrestin constructs were split into poly-l-lysine-coated 24-well tissue culture plates after 24 h. The next day (48 h post-transfection), cells were treated with 10 μm (−)-isoproterenol and 0.1 mm ascorbate at 37 °C for 0, 15, or 30 min, fixed with 3.7% formaldehyde in Tris-buffered saline (TBS) for 5 min at ambient temperature, and washed three times with TBS. Cells were then blocked with TBS containing 1% bovine serum albumin (TBS/BSA) for 45 min, incubated with a primary antibody (M2 anti-FLAG conjugated with alkaline phosphatase (Sigma), 1:2000 dilution in TBS/BSA) for 1 h, and washed three times with TBS. Colorimetric visualization of antibody binding was performed using an alkaline phosphatase substrate kit (Bio-Rad), and samples were read at 405 nm in a microplate reader using Microplate Manager software (Bio-Rad). The reading from cells that did not express FLAG-β2AR was used as a blank. The percentage of surface receptor loss was determined by calculating the change of antibody-accessible FLAG-β2AR. Previous studies have suggested an important role for non-visual arrestin interaction with clathrin and β2-adaptin in GPCR trafficking (7Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1179) Google Scholar, 13Krupnick JG. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 15Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (529) Google Scholar, 16Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Arrestin interaction with clathrin is thought to occur primarily via a LφXφE motif (where φ is a bulky aliphatic residue) found within a C-terminal insert unique to non-visual arrestins (residues 376–380 in arrestin-2) (13Krupnick JG. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Mutation of the aliphatic or acidic residues within this motif results in significant reduction in arrestin binding to clathrin (13Krupnick JG. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Non-visual arrestin interaction with β2-adaptin was first characterized by yeast two-hybrid and co-immunoprecipitation analysis (15Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (529) Google Scholar). Additional studies revealed that Arg-394 and Arg-396 in arrestin-3 are critical for β2-adaptin binding (16Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar), while we recently found that Phe-391 and Arg-395 (analogous to Arg-396 in arrestin-3) are essential for arrestin-2 binding to β2-adaptin (21Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar). In an attempt to more completely define the binding interface of arrestin and β2-adaptin we constructed several additional C-terminal mutant arrestin-2 proteins including K397E, M399A, K400E, D401R, D402R, and K403E (Fig.1a). The mutant arrestins were then expressed in COS-1 cells and tested for binding to a GST fusion protein containing the β2-adaptin appendage domain (residues 700–937) (Fig. 1b, upper panel). The appendage domain has been implicated in interactions with multiple proteins including clathrin, AP180, Eps15, epsin, and non-visual arrestins (18Owen D.J. Vallis Y. Pearse B.M.F. McMahon H.T. Evans P.R. EMBO J. 2000; 19: 4216-4227Crossref PubMed Google Scholar, 21Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar, 22Laporte S.A. Miller W.E. Kim K.M. Caron M.G. J. Biol. Chem. 2002; 277: 9247-9254Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Subsequent densitometric analysis of the blots revealed that mutation of Lys-397, Lys-400, or Lys-403 resulted in a 65–90% loss in β2-adaptin binding (Fig. 1c, left panel). The various mutants were also tested for their binding to a GST fusion protein containing residues 1–579 of the clathrin terminal domain (Fig. 1b, middle panel). Quantification of the blots suggested that mutation of Lys-397, Lys-400, or Lys-403 resulted in an ∼40% loss in clathrin binding (Fig. 1c, right panel). Overall these results suggest that Lys-397, Lys-400, and Lys-403 may play a role in β2-adaptin and, to a lesser extent, clathrin binding. It is somewhat surprising that several C-terminal residues (Lys-397, Lys-400, Lys-403, and Arg-395; Ref. 21Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar) contribute to interaction with both β2-adaptin and clathrin. While one possible explanation is that charge inversion of these residues results in partial misfolding of the C-terminal region of arrestin, this seems unlikely since individual mutations elicited a more profound inhibitory effect on β2-adaptin binding compared with clathrin. Since binding surfaces on components of clathrin-mediated endocytic pathways are often shared by multiple interacting proteins (18Owen D.J. Vallis Y. Pearse B.M.F. McMahon H.T. Evans P.R. EMBO J. 2000; 19: 4216-4227Crossref PubMed Google Scholar, 31ter Haar E. Harrison S.C. Kirchhausen T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1096-1100Crossref PubMed Scopus (238) Google Scholar), we favor the explanation that these particular residues directly contribute to β2-adaptin and clathrin binding. The partial overlap of the binding surfaces for β2-adaptin and clathrin on arrestin-2 may contribute to spatial and temporal regulation of GPCR targeting to clathrin-coated pits. We next attempted to further address the specificity of arrestin interaction with APs. Four AP complexes have been identified, each of which consists of two large (γ-, α-, δ-, ε-adaptin and β1–4-adaptin), one medium (μ1–4-adaptin), and one small (φ1–4-adaptin) subunit (23Robinson M.S. Bonifacino J.S. Curr. Opin. Cell Biol. 2001; 13: 444-453Crossref PubMed Scopus (444) Google Scholar). AP2 is localized in cell surface clathrin-coated pits and mediates endocytosis from the plasma membrane, while AP1, AP3, and AP4 are associated with intracellular membranes and mediate sorting at the trans-Golgi network and/or endosomes (23Robinson M.S. Bonifacino J.S. Curr. Opin. Cell Biol. 2001; 13: 444-453Crossref PubMed Scopus (444) Google Scholar). Since arrestins interact with the appendage domain of β2-adaptin and arrestin interaction with other adaptins could potentially contribute to receptor trafficking, we compared the ability of arrestin-2 to bind to GST fusion proteins containing the appendage domains of α-adaptin, β2-adaptin, and β3-adaptin. These studies revealed that arrestin-2 was highly specific for binding to β2-adaptin and suggested that the primary function of this interaction will involve GPCR endocytosis (Fig.2a). In an effort to elucidate the molecular basis for β2-adaptin binding to arrestin-2, we systematically mutated 10 surface residues in β2-adaptin, several of which have been implicated in β2-adaptin binding to clathrin, AP180, Eps15, and epsin (18Owen D.J. Vallis Y. Pearse B.M.F. McMahon H.T. Evans P.R. EMBO J. 2000; 19: 4216-4227Crossref PubMed Google Scholar). The various β2-adaptin mutants were expressed as GST fusions and then tested for binding to purified arrestin-2. These studies suggested that arrestin-2 interacts with multiple residues on the β2-adaptin surface including Arg-834, Trp-841, Glu-849, Tyr-888, and Glu-902 (Fig. 2b). These results confirm and extend a recent study that suggested a role for Glu-849, Tyr-888, and Glu-902 in arrestin-3 binding to β2-adaptin (22Laporte S.A. Miller W.E. Kim K.M. Caron M.G. J. Biol. Chem. 2002; 277: 9247-9254Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) and also confirm many of the features of our proposed model of β2-adaptin interaction with arrestin-2 (21Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar). To verify that the various adaptins were functional, we also analyzed the binding of AP180 and Eps15 to the GST-β2-adaptin mutants. These studies largely recapitulated previous findings (18Owen D.J. Vallis Y. Pearse B.M.F. McMahon H.T. Evans P.R. EMBO J. 2000; 19: 4216-4227Crossref PubMed Google Scholar) and revealed that Lys-917 and Tyr-888 contributed to AP180 binding, while Trp-841 and Tyr-888 were involved in Eps15 binding (Fig.2c). Overall our results suggest that Phe-391 and Arg-395 in arrestin-2 play an essential role in interacting with β2-adaptin since mutation of either of these residues completely disrupts binding (21Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar). Indeed, our modeling predicted that Phe-391 interacts with Trp-841 and Tyr-888 in β2-adaptin, while Arg-395 interacts with Glu-849 and Glu-902. However, our results suggest that Leu-396, Lys-397, Lys-400, and Lys-403 also contribute to β2-adaptin binding (Ref. 21Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar and Fig. 1). While the binding analysis of the β2-adaptin mutants fully supports this model (Ref. 22Laporte S.A. Miller W.E. Kim K.M. Caron M.G. J. Biol. Chem. 2002; 277: 9247-9254Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar and Fig. 2b), structural analysis will be required to completely define the binding interface of these proteins. Structural insight has revealed that arrestin-2 contains two major domains made up of β-sheets that are joined by a polar core of buried salt bridges (21Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar, 24Han M. Gurevich V.V. Vishnivetskiy S.A. Sigler P.B. Schubert C. Structure. 2001; 9: 869-880Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). The region of arrestin-2 that interacts with β2-adaptin includes one residue in the last β-sheet (Phe-391) as well as several residues that are C-terminal to this region (Arg-395, Leu-396, Lys-397, Lys-400, and Lys-403). The C-terminal region in arrestin has been suggested to undergo significant conformational change upon receptor binding (25Schleicher A. Kuhn H. Hofmann K.P. Biochemistry. 1989; 28: 1770-1775Crossref PubMed Scopus (167) Google Scholar,26Ohguro H. Palczewski K. Walsh K.A. Johnson R.S. Protein Sci. 1994; 3: 2428-2434Crossref PubMed Scopus (84) Google Scholar) and potentially loses intramolecular contacts between β-sheets in the N-terminal and C-terminal regions of the protein (27Vishnivetskiy S.A. Schubert C. Climaco G.C. Gurevich Y.V. Velez M.-G. Gurevich V.V. J. Biol. Chem. 1999; 275: 41049-41057Abstract Full Text Full Text PDF Scopus (154) Google Scholar). In addition, the conformational change induced by receptor binding is also thought to reflect disruption of the polar core of arrestin potentially mediated by interactions between basic residues within the polar core and phosphoserines from the receptor (28Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 29Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). In particular, Arg-169 in arrestin-2 appears to play a key role in stabilizing the polar core since mutation of this residue results in an activated form of arrestin-2 that binds to GPCRs independent of their phosphorylation state (30Kovoor A. Celver J. Abdryashitov R.I. Chavkin C. Gurevich V.V. J. Biol. Chem. 1999; 274: 6831-6834Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Based on the importance of the polar core in stabilizing the basal state of arrestin, we hypothesized that mutation of Arg-169 would not only result in a less constrained C-terminal domain but more avid binding to β2-adaptin. Indeed, purified arrestin-2-R169E bound to GST-β2-adaptin much better than wild type arrestin-2 (Fig. 3a). Dose-response analysis suggested that arrestin-2-R169E bound to β2-adaptin with much higher affinity than wild type arrestin-2 (Fig. 3b, left panel). Interestingly, arrestin-2-R169E also bound with higher affinity to GST-clathrin compared with wild type arrestin-2 (Fig. 3, a and b). Since the R169E mutant most likely better represents the conformation of arrestin-2 that would bind to β2-adaptin and clathrin in cells, we also assessed whether the various arrestin mutants that were defective in β2-adaptin and/or clathrin binding remained so in the background of the R169E mutation. Thus, we compared the ability of F391A, R393E, R395E, L396A, K397E, K400E, and K403E mutants of arrestin-2-R169E to bind to GST-β2-adaptin (Fig. 3c, upper panel) and GST-clathrin (Fig. 3c, lower panel). These studies revealed that the Phe-391 mutation retained its selective and complete disruption in β2-adaptin binding, while the Arg-395 mutation remained completely disrupted in β2-adaptin binding and partially disrupted in clathrin binding. Interestingly, the reduced binding of the L396A, K397E, K400E, and K403E mutants to β2-adaptin and clathrin previously observed (Ref. 21Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar and Fig. 1) appeared to be largely attenuated by the R169E mutation. Overall these results suggest that the conformational change induced by GPCR binding will likely play a major role in mediating β2-adaptin and clathrin binding and that the F391A mutant should prove useful in selectively dissecting the functional role of arrestin/β2-adaptin interaction. We next focused on further characterizing the binding of arrestin-2 to clathrin. Previous work has implicated a C-terminal LφXφE motif in clathrin binding and demonstrated that mutation of the three aliphatic residues within this motif results in an ∼85% reduction in clathrin binding (13Krupnick JG. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Indeed, such a motif appears to play an important role in the ability of numerous proteins to bind clathrin (31ter Haar E. Harrison S.C. Kirchhausen T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1096-1100Crossref PubMed Scopus (238) Google Scholar). In an effort to generate an arrestin-2 mutant that was completely defective in clathrin binding we deleted the five-amino acid clathrin binding motif (LIELD) in arrestin-2. This mutant, arrestin-2-ΔLIELD, was expressed and initially tested for its ability to bind GST-clathrin and GST-β2-adaptin. As expected, arrestin-2-ΔLIELD was completely defective in clathrin binding (Fig. 4, middle panel), while it was largely unchanged in β2-adaptin binding (Fig. 4, top panel). These studies confirmed that the LφXφE motif plays an essential role in arrestin-2 binding to clathrin. A number of additional mutations were tested in the background of arrestin-2-ΔLIELD including F391A, R395E, and F391A/R395E. As expected, each of these mutants was completely defective in clathrin and β2-adaptin binding (Fig. 4). Since arrestin-2 binding to clathrin appeared to be conformationally sensitive (Fig. 3), we also tested whether incorporation of the R169E mutation in arrestin-2-ΔLIELD affected clathrin binding. Arrestin-2-R169E-ΔLIELD remained completely defective in clathrin binding and unaltered in β2-adaptin binding (not shown). This suggests that the conformational change in arrestin-2 induced by the Arg-169 mutation most likely mediates clathrin binding via the LφXφE motif. Nevertheless, to further address whether the C-terminal domain is involved in mediating clathrin binding we analyzed binding of C-terminally truncated arrestin-2 and arrestin-2-ΔLIELD to clathrin. Arrestin-2-(1–393) was completely defective in β2-adaptin binding, while it appeared to have modestly increased binding to clathrin (Fig. 4). As expected, arrestin-2-ΔLIELD-(1–393) did not bind to clathrin or β2-adaptin. Although we cannot rule out the possibility of secondary clathrin binding sites on arrestin-2, our data suggest that the LIELD motif plays an essential role in clathrin binding and that arrestin-2-ΔLIELD is an effective mutant for assessing the functional role of clathrin binding. For the various arrestin mutants to be useful in dissecting the role of clathrin and β2-adaptin binding in cells, we also needed to analyze whether these mutants were altered in receptor binding. The various arrestin constructs were co-expressed with a FLAG-tagged β2AR in COS-1, the cells were incubated with agonist for 2 min and lysed, and the receptor was immunoprecipitated using a β2AR-specific polyclonal antibody. The samples were then run on an SDS-polyacrylamide gel and analyzed by immunoblotting using an anti-arrestin monoclonal antibody to detect arrestin-2 in the immunoprecipitate (Fig. 5, upper panel) or in the initial lysate (Fig. 5, lower panel). Subsequent densitometric analysis of blots from three independent experiments suggested that all of the arrestin mutants interacted with the β2AR to a similar extent (data not shown). However, there was an ∼40–50% reduction in co-immunoprecipitation of the β2AR with the ΔLIELD and R395E mutants compared with wild type arrestin-2 suggesting that these mutants might have a modestly reduced ability to bind the β2AR. In contrast, the various double and triple mutants (ΔLIELD/F391A, ΔLIELD/R395E, and ΔLIELD/F391A/R395E) had ∼30–35% higher binding to the β2AR compared with wild type arrestin-2 suggesting that the ΔLIELD and R395E mutations do not attenuate receptor binding. Taken together these results suggest that the C-terminal mutations in arrestin-2 do not significantly affect interaction with receptor and that the ΔLIELD, F391A, and ΔLIELD/F391A arrestin-2 mutants should prove effective in further dissecting the role of clathrin and β2-adaptin interaction in arrestin function. Previous studies using an arrestin-3 construct containing mutations of the three aliphatic residues within the LφXφE motif suggested that binding of non-visual arrestins to clathrin contributes to GPCR internalization (13Krupnick JG. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). A similar line of investigation identified a role for arrestin-3 interaction with β2-adaptin in GPCR internalization and further concluded that the β2-adaptin binding, and not clathrin, is necessary for the initial targeting of receptor to clathrin-coated pits (16Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). One concern with these previous studies is that the arrestin mutants used to dissect the role of clathrin and β2-adaptin binding in GPCR internalization were potentially flawed. For example, the LIFA mutant (containing the three aliphatic residues in the LφXφE motif in arrestin-3 mutated to alanines) is only ∼85% reduced in clathrin binding (13Krupnick JG. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), while the R396E mutant (which corresponds to R395E in arrestin-2) used by Laporte et al. (16Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar) is completely defective in β2-adaptin binding but is likely also reduced in clathrin binding (21Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar). Thus, these are not ideal mutants to draw conclusions about the respective involvement of clathrin and β2-adaptin binding in arrestin function. Since our studies demonstrate that the F391A (Ref. 21Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar and Fig.3c) and ΔLIELD (Fig. 4) mutants are selectively defective in β2-adaptin and clathrin binding, respectively, we used these mutants to further dissect the role of these interactions in GPCR internalization. When co-expressed in COS-1 cells, wild type arrestin-2 promoted an ∼2-fold increase in agonist-induced internalization of the β2AR (Fig. 6). In striking contrast, the ΔLIELD mutant was completely defective in promoting β2AR internalization demonstrating the important role of arrestin/clathrin interaction in this process. By comparison, arrestin-2-F391A promoted an ∼2-fold increase in β2AR internalization suggesting that arrestin-2 interaction with β2-adaptin is not essential in this process. Interestingly, arrestin-2-R395E, which was completely defective in β2-adaptin binding and attenuated in clathrin binding, was also ineffective at promoting β2AR internalization (Fig. 6). This finding largely recapitulates the results of Laporte et al. who used the analogous mutation in arrestin-3 (R396E) to conclude that the binding of a receptor-arrestin complex to AP2 is necessary for the initial targeting of receptor to clathrin-coated pits (16Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Analysis of additional mutants defective in either β2-adaptin binding (residues 1–393) or β2-adaptin and clathrin binding (ΔLIELD/F391A, ΔLIELD/R395E, and 1–393/ΔLIELD) confirmed that arrestin-2 binding to clathrin plays the primary role in agonist-promoted internalization of the β2AR (Fig. 6). We next analyzed the ability of the various arrestin-2 mutants to regulate β2AR internalization in HEK293 cells. HEK293 cells have higher endogenous levels of arrestin and have proven useful for testing whether arrestin mutants can function as dominant negatives to inhibit GPCR internalization (6Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (853) Google Scholar, 32Menard L. Ferguson S.S. Zhang J. Lin F.T. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. 1997; 51: 800-808Crossref PubMed Scopus (214) Google Scholar, 33Krupnick J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 32507-32512Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). β2AR internalization in HEK293 cells was ∼25% following a 30-min treatment with agonist and was increased to ∼35% by wild type arrestin-2 (Fig. 7). In contrast, arrestin-2 mutants that were completely defective in β2-adaptin binding (F391A, R395E, and residues 1–393) did not promote any additional internalization. This suggests that arrestin binding to β2-adaptin contributes to β2AR internalization in HEK293 cells, although loss of such interaction did not produce an effective dominant negative mutant. Selective loss of clathrin binding (ΔLIELD) produced a modestly effective dominant negative, although in these studies it appeared as effective as a C-terminal arrestin-2 construct (residues 319–418) that is widely used to study the role of arrestins in receptor trafficking (33Krupnick J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 32507-32512Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Interestingly, mutants that were defective in both clathrin and β2-adaptin binding functioned as potent dominant negative mutants with ΔLIELD/F391A and ΔLIELD/F391A/R395E being as effective at inhibiting β2AR internalization as dominant negative dynamin I. The ΔLIELD/F391A/R169E mutant was also an effective dominant negative and should be particularly useful in future studies since it overcomes the requirement for GPCR phosphorylation for arrestin binding. We conclude that arrestin-2 binding to clathrin is essential for promoting GPCR endocytosis, while binding to β2-adaptin appears less important. These findings appear to contradict Laporteet al. (16Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar) who concluded that binding of a receptor-arrestin complex to AP2 was critical for targeting the receptor to coated pits. How do we explain these differences in the two studies? We favor the explanation that the arrestin mutants used by Laporte et al. were either incomplete in disrupting clathrin interaction (13Krupnick JG. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar) or were not completely selective in disrupting β2-adaptin binding (21Milano S.K. Pace H.C. Kim Y.-M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar). This would result in an apparent diminished role of clathrin binding and exaggerated role of β2-adaptin binding. However, we cannot exclude the possibility that mechanistic differences between arrestin-2 (used in the present study) and arrestin-3 (used by Laporte et al.) may have contributed to the different findings. In addition, non-visual arrestins also interact with several additional proteins that have been implicated in GPCR endocytosis including ADP-ribosylation factor-6, ADP-ribosylation factor nucleotide binding site opener, and N-ethylmaleimide-sensitive factor (34Claing A. Chen W. Miller W.E. Vitale N. Moss J. Premont R.T. Lefkowitz R.J. J. Biol. Chem. 2001; 276: 42509-42513Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 35McDonald P.H. Cote N.L. Lin F.T. Premont R.T. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 10677-10680Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Since the arrestin residues that mediate binding to these proteins have not been identified, we also cannot exclude the possibility that our arrestin mutants may be altered in their interaction with one or more of these proteins, although this seems unlikely. Overall, our studies are compatible with non-visual arrestin binding to activated phosphorylated GPCRs mediating a conformational change in arrestin that promotes binding to clathrin and β2-adaptin. Although our studies do not address whether receptor-bound arrestins directly recruit clathrin or AP2 to inducede novo coated pit formation, we favor a mechanism whereby the receptor-arrestin complex moves laterally within the membrane and binds to clathrin and AP2 in a pre-existing coated pit. This mechanism is consistent with our earlier studies that demonstrated that arrestins do not promote clathrin assembly (7Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1179) Google Scholar) as well as more recent studies that demonstrate that a receptor-arrestin-3 complex does not promote new coated pit formation (36Santini F. Gaidarov I. Keen J.H. J. Cell Biol. 2002; 156: 665-676Crossref PubMed Scopus (93) Google Scholar). Our results suggest that arrestin-2 binding to clathrin is essential for mediating GPCR trafficking, while the interaction with β2-adaptin appears less critical. Nevertheless, arrestin interaction with β2-adaptin may participate in the cooperative formation of an arrestin-AP2-clathrin complex and thereby contribute to the dynamics of receptor/arrestin association with clathrin-coated pits. We are grateful to Dr. James Keen for the GST-clathrin construct, Dr. Larry Donoso for the arrestin monoclonal antibody, Dr. Harvey McMahon for the GST-β2-adaptin and α-adaptin constructs, and Dr. Juan Bonifacino for the GST-β3-adaptin construct. We also thank Kirby Steger, Rosey Stracquatanio, Joseph Kubik, and William McCormick for technical assistance and Drs. Adriano Marchese and James Keen for helpful discussions.
Referência(s)