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The Third Intracellular Loop of α2-Adrenergic Receptors Determines Subtype Specificity of Arrestin Interaction

2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês

10.1074/jbc.m207495200

ISSN

1083-351X

Autores

Jessica L. DeGraff, Vsevolod V. Gurevich, Jeffrey Benovic,

Tópico(s)

Nitric Oxide and Endothelin Effects

Resumo

Nonvisual arrestins (arrestin-2 and -3) serve as adaptors to link agonist-activated G protein-coupled receptors to the endocytic machinery. Although many G protein-coupled receptors bind arrestins, the molecular determinants involved in binding remain largely unknown. Because arrestins selectively promote the internalization of the α2b- and α2c-adrenergic receptors (ARs) while having no effect on the α2aAR, here we used α2ARs to identify molecular determinants involved in arrestin binding. Initially, we assessed the ability of purified arrestins to bind glutathioneS-transferase fusions containing the third intracellular loops of the α2aAR, α2bAR, or α2cAR. These studies revealed that arrestin-3 directly binds to the α2bAR and α2cAR but not the α2aAR, whereas arrestin-2 only binds to the α2bAR. Truncation mutagenesis of the α2bAR identified two arrestin-3 binding domains in the third intracellular loop, one at the N-terminal end (residues 194–214) and the other at the C-terminal end (residues 344–368). Site-directed mutagenesis further revealed a critical role for several basic residues in arrestin-3 binding to the α2bAR third intracellular loop. Mutation of these residues in the holo-α2bAR and subsequent expression in HEK 293 cells revealed that the mutations had no effect on the ability of the receptor to activate ERK1/2. However, agonist-promoted internalization of the mutant α2bAR was significantly attenuated as compared with wild type receptor. These results demonstrate that arrestin-3 binds to two discrete regions within the α2bAR third intracellular loop and that disruption of arrestin binding selectively abrogates agonist-promoted receptor internalization. Nonvisual arrestins (arrestin-2 and -3) serve as adaptors to link agonist-activated G protein-coupled receptors to the endocytic machinery. Although many G protein-coupled receptors bind arrestins, the molecular determinants involved in binding remain largely unknown. Because arrestins selectively promote the internalization of the α2b- and α2c-adrenergic receptors (ARs) while having no effect on the α2aAR, here we used α2ARs to identify molecular determinants involved in arrestin binding. Initially, we assessed the ability of purified arrestins to bind glutathioneS-transferase fusions containing the third intracellular loops of the α2aAR, α2bAR, or α2cAR. These studies revealed that arrestin-3 directly binds to the α2bAR and α2cAR but not the α2aAR, whereas arrestin-2 only binds to the α2bAR. Truncation mutagenesis of the α2bAR identified two arrestin-3 binding domains in the third intracellular loop, one at the N-terminal end (residues 194–214) and the other at the C-terminal end (residues 344–368). Site-directed mutagenesis further revealed a critical role for several basic residues in arrestin-3 binding to the α2bAR third intracellular loop. Mutation of these residues in the holo-α2bAR and subsequent expression in HEK 293 cells revealed that the mutations had no effect on the ability of the receptor to activate ERK1/2. However, agonist-promoted internalization of the mutant α2bAR was significantly attenuated as compared with wild type receptor. These results demonstrate that arrestin-3 binds to two discrete regions within the α2bAR third intracellular loop and that disruption of arrestin binding selectively abrogates agonist-promoted receptor internalization. G protein-coupled receptor G protein-coupled receptor kinase glutathione S-transferase human embryonic kidney phosphate-buffered saline adrenergic receptor luteinizing hormone/choriogonadotropin receptor N-terminal C-terminal extracellular signal-regulated kinase G protein-coupled receptors (GPCRs)1 transduce extracellular stimuli into intracellular signaling via coupling to heterotrimeric guanine nucleotide-binding proteins (G proteins) (1Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar). To ensure that stimuli are translated into signals of appropriate magnitude and specificity, these signaling cascades are tightly regulated. GPCRs are subject to three principal 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, in which total cellular receptor levels are decreased (2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (857) Google Scholar, 3Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). Although multiple mechanisms contribute to these regulatory processes, GPCR phosphorylation by G protein-coupled receptor kinases (GRKs) and subsequent binding of arrestins plays an important role in the regulation of many GPCRs (2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (857) Google Scholar). Four mammalian arrestins have been identified with arrestin-1 and -4 being specific to the visual system and arrestin-2 and -3 (also termed β-arrestin-1 and -2) being ubiquitously expressed (4Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (909) Google Scholar, 5Murakami A. Yajima T. Sakuma H. McLaren M.J. Inana G. FEBS Lett. 1993; 334: 203-209Crossref PubMed Scopus (90) Google Scholar, 6Sterne-Marr R. Gurevich V.V. Goldsmith P. Bodine R.C. Sanders C. Donoso L.A. Benovic J.L. J. Biol. Chem. 1993; 268: 15640-15648Abstract Full Text PDF PubMed Google Scholar, 7Yamaki K. Takahashi Y. Sakuragi S. Matsubara K. Biochem. Biophys. Res. Commun. 1987; 142: 904-910Crossref PubMed Scopus (77) Google Scholar). Arrestin binding to activated-phosphorylated GPCRs results in the physical uncoupling of receptor from G protein, a process that functions to terminate agonist-mediated signaling. The two nonvisual arrestins also directly interact with clathrin (8Goodman 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 (1172) Google Scholar), the adaptor protein AP2 (9Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (524) Google Scholar), and phosphoinositides (10Gaidarov I. Krupnick J.G. Falck J.R. Benovic J.L. Keen J.H. EMBO J. 1999; 18: 871-881Crossref PubMed Scopus (167) Google Scholar) to promote GPCR internalization. Indeed, nonvisual arrestins have been implicated in the desensitization and internalization of a wide variety of GPCRs including members of the class 1 (rhodopsin-like) and class 2 (secretin-like) families (2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (857) Google Scholar, 11Walker J.K. Premont R.T. Barak L.S. Caron M.G. Shetzline M.A. J. Biol. Chem. 1999; 274: 31515-31523Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Nevertheless, despite numerous studies that demonstrate an essential role for arrestins in the regulation of GPCR signaling and trafficking, the precise molecular determinants within GPCRs required for arrestin binding have not been thoroughly characterized. Three α2-adrenergic receptors, α2aAR, α2bAR, and α2cAR, have been identified (12Lomasney J.W. Lorenz W. Allen L.F. King K. Regan J.W. Yang-Feng T.L. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5094-5098Crossref PubMed Scopus (274) Google Scholar, 13Kobilka B.K. Matsui H. Kobilka T.S. Yang-Feng T.L. Francke U. Caron M.G. Lefkowitz R.J. Regan J.W. Science. 1987; 238: 650-656Crossref PubMed Scopus (598) Google Scholar, 14Regan J.W. Kobilka T.S. Yang-Feng T.L. Caron M.G. Lefkowitz R.J. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6301-6305Crossref PubMed Scopus (375) Google Scholar). α2ARs are activated by the catecholamines epinephrine and norepinephrine and regulate sympathetic outflow and cardiovascular function in vivo (15Link R.E. Desai K. Hein L. Stevens M.E. Chruscinski A. Bernstein D. Barsh G.S. Kobilka B.K. Science. 1996; 273: 803-805Crossref PubMed Scopus (428) Google Scholar, 16Altman J.D. Trendelenburg A.U. MacMillan L. Bernstein D. Limbird L. Starke K. Kobilka B.K. Hein L. Mol. Pharmacol. 1999; 56: 154-161Crossref PubMed Scopus (282) Google Scholar, 17MacMillan L.B. Hein L. Smith M.S. Piascik M.T. Limbird L.E. Science. 1996; 273: 801-803Crossref PubMed Scopus (449) Google Scholar). All three α2AR subtypes primarily couple to the Gi family of G proteins and modulate a variety of signaling pathways including activation of phospholipase A2 (18Jones S.B. Halenda S.P. Bylund D.B. Mol. Pharmacol. 1991; 39: 239-245PubMed Google Scholar), phospholipase D (19MacNulty E.E. McClue S.J. Carr I.C. Jess T. Wakelam M.J. Milligan G. J. Biol. Chem. 1992; 267: 2149-2156Abstract Full Text PDF PubMed Google Scholar), and extracellular regulated kinases ERK1/2 (20Alblas J. van Corven E.J. Hordijk P.L. Milligan G. Moolenaar W.H. J. Biol. Chem. 1993; 268: 22235-22238Abstract Full Text PDF PubMed Google Scholar, 21Flordellis C.S. Berguerand M. Gouache P. Barbu V. Gavras H. Handy D.E. Bereziat G. Masliah J. J. Biol. Chem. 1995; 270: 3491-3494Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 22Seuwen K. Magnaldo I. Kobilka B.K. Caron M.G. Regan J.W. Lefkowitz R.J. Pouyssegur J. Cell Regul. 1990; 1: 445-451Crossref PubMed Scopus (35) Google Scholar) and inhibition of adenylyl cyclase (23Limbird L.E. FASEB J. 1988; 2: 2686-2695Crossref PubMed Scopus (277) Google Scholar). α2AR signaling is also subject to dynamic regulation. The α2aAR and α2bAR are subject to agonist-dependent phosphorylation by GRKs (24Kurose H. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10093-10099Abstract Full Text PDF PubMed Google Scholar). Moreover, previous studies have revealed a role for arrestins in agonist-promoted internalization of α2ARs with arrestin-3 promoting internalization of the α2bAR and α2cAR and arrestin-2 selectively promoting internalization of the α2bAR. Interestingly, α2aAR internalization was not promoted by either arrestin, suggesting arrestin/receptor binding specificity (25DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Previous studies have demonstrated an important role for the third intracellular loops of the α2ARs in mediating protein-protein interaction. These loops are quite large (>150 amino acids) and include sites for GRK phosphorylation (26Liggett S.B. Ostrowski J. Chesnut L.C. Kurose H. Raymond J.R. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 4740-4746Abstract Full Text PDF PubMed Google Scholar), Gi activation (27Wade S.M. Lim W.K. Lan K.L. Chung D.A. Nanamori M. Neubig R.R. Mol, Pharmacol. 1999; 56: 1005-1013Crossref PubMed Scopus (63) Google Scholar), and binding of 14-3-3 (28Prezeau L. Richman J.G. Edwards S.W. Limbird L.E. J. Biol. Chem. 1999; 274: 13462-13469Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), sphinophilin (29Richman J.G. Brady A.E. Wang Q. Hensel J.L. Colbran R.J. Limbird L.E. J. Biol. Chem. 2001; 40: 19Google Scholar), and arrestin (30Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Although arrestin binding to GPCRs is dependent on both the phosphorylation and activation state of the receptor (31Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar), the receptor domains that mediate the agonist-dependent nature of arrestin binding have not been thoroughly characterized. Because third intracellular loops mediate agonist-dependent binding and activation of heterotrimeric G proteins (32Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar, 33Wade S.M. Scribner M.K. Dalman H.M. Taylor J.M. Neubig R.R. Mol. Pharmacol. 1996; 50: 351-358PubMed Google Scholar), it seems likely that specific regions of the third intracellular loop might also confer the agonist dependence and selectivity of arrestin binding. Indeed, the third intracellular loop has been implicated in arrestin interaction for a number of GPCRs including rhodopsin (34Krupnick J.G. Gurevich V.V. Schepers T. Hamm H.E. Benovic J.L. J. Biol. Chem. 1994; 269: 3226-3232Abstract Full Text PDF PubMed Google Scholar), α2a-adrenergic (30Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), M2 and M3muscarinic (30Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), δ-opioid (35Appleyard S.M. Celver J. Pineda V. Kovoor A. Wayman G.A. Chavkin C. J. Biol. Chem. 1999; 274: 23802-23807Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), 5-hydroxytryptamine2A(36Gelber E.I. Kroeze W.K. Willins D.L. Gray J.A. Sinar C.A. Hyde E.G. Gurevich V. Benovic J. Roth B.L. J. Neurochem. 1999; 72: 2206-2214Crossref PubMed Scopus (70) Google Scholar), CXCR4 (37Cheng Z.J. Zhao J. Sun Y., Hu, W., Wu, Y.L. Cen B., Wu, G.X. Pei G. J. Biol. Chem. 2000; 275: 2479-2485Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), and the luteinizing hormone/choriogonadotropin (LH/CG) receptor (38Mukherjee S. Palczewski K. Gurevich V.V. Hunzicker-Dunn M. J. Biol. Chem. 1999; 274: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The subtype-specific differences in arrestin sensitivity for the three α2ARs provides a useful model to identify specific regions involved in arrestin binding. To address this issue, we studied arrestin binding to a series of glutathione S-transferase (GST) fusion proteins containing various regions of the third intracellular loops of the three α2AR subtypes. Our results revealed arrestin binding specificity that recapitulates the arrestin selectively observed previously in α2AR trafficking (25DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Truncation and site-directed mutagenesis revealed that arrestin-3 binds to two discrete regions within the α2bAR third intracellular loop. Moreover, disruption of arrestin binding selectively abrogated agonist-promoted internalization of the α2bAR. These studies help to address questions of specificity in GPCR/arrestin interaction that ultimately will lead to a better understanding of the role of arrestins in regulating receptor-mediated signaling. FLAG-tagged α2AAR, α2BAR, and α2CAR were cloned into pcDNA3 as described previously (25DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The third intracellular loops of the α2aAR (residues 218–374), α2bAR (residues 194–368), and α2cAR (residues 232–379) were amplified by PCR using the full-length receptors as template. The PCR products were cut with EcoRI/XhoI (α2bAR and α2cAR) orEcoRI/SalI (α2aAR), gel-purified, inserted into the plasmid pGEX4T-2 in-frame with GST, and sequenced. The following α2bAR third loop truncation mutants were made and cloned into pGEX4T-2 using the same strategy: N-terminal (NT) (residues 194–292), NT1 (194–214), NT2 (206–292), C-terminal (CT) (293–368), CT1 (293–312), CT2 (313–358), and CT3 (344–368). α2bAR point mutations were constructed by oligonucleotide-directed PCR mutagenesis in either the NT1 construct (K200A, R201A, K200A/R201A, S202A, N203A, R204A, R205A, R204A/R205A, P207A, and R208A), the CT3 construct (L344A/L345A, R347A, Q355A, W356A/W357A, R358A, R359A, R360A, Q362A, T364A, R365A, E366A, K367A, and R268A), or the third loop construct (K200A/R201A (KR), R204A/R205A (RR), R358A/R359A/R360A (3R), and RR/3R). PCR products were cut withEcoRI/XhoI, ligated intoEcoRI/XhoI-digested pGEX4T-2, and sequenced. The K200A/R201A, R204A/R205A, and R358A/R359A/R360A mutations were also generated in full-length α2bAR by two-step PCR using the expand high fidelity PCR system according to the manufacturer's recommendations. Briefly, using pcDNA3-α2bAR as template, an N-terminal fragment of the mutant α2bAR was created using a cytomegalovirus (5′-tgt acg gtg gga ggt-3′) sense primer and a mutagenic antisense primer, and a C-terminal fragment of the mutant α2bAR was generated using a mutagenic sense primer and an Sp6 (5′-gat aag ata tca cag tgg att tac-3′) antisense primer. Mutagenic primers used were: KR sense (5′-ctg atc gcc gca gcc agc aac cgc-3′) and antisense (5′-gcg gtt gct ggc tgc gta gat cag-3′); RR sense (5′-cgc agc aac gcc gca ggt ccc agg-3′) and antisense (5′-cct ggg acc tgc ggc gtt gct gcg-3′); 3R sense (5′-ggg cag tgg tgg gct gca gcg gcg cag ctg acc cgg-3′) and antisense (5′-ggt cag ctg cgc cgc tgc agc cca cca ctg ccc-3′). PCR products were purified, and N-terminal and C-terminal products (100 ng of each) were then used as template using the cytomegalovirus and Sp6 primers. PCR products were cut withEcoRI/XhoI, purified, and ligated into pcDNA3. An RR/3R mutation in full-length α2bAR was generated as described above using RR as template for the first round of PCR and then proceeding as specified above for the 3R mutation. BL21 (De3) lysS cells transformed with a GST-α2AR fusion construct were grown overnight at 37 °C, diluted 1:100 into LB containing ampicillin, grown for 3 h at 37 °C, and then induced with 0.1 mmisopropyl-thiogalactyl-pyranosidase for 2 h at 30 °C. Cells were pelleted (3000 × g for 30 min) and washed with phosphate-buffered saline (PBS) containing 1 mmdithiothreitol and protease inhibitors (1 mmphenylmethylsulfonyl fluoride, 0.2 mg/ml benzamidine, 20 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin). Cells were pelleted, resuspended in 1–3 ml of PBS plus protease inhibitors (per 100 ml of culture), lysed by incubation for 10 min on ice with 1 mg/ml lysozyme, and then aliquoted (0.4 ml), frozen, and stored at −80 °C until needed. Aliquots were thawed on ice, Triton X-100 (2% final) and sarcosyl (0.5% final) were added, and the cells were frozen, thawed, and centrifuged for 1 h (30,000 rpm in TLA-45 rotor). The supernatant (∼200 μl) was then incubated with 200 μl of 50% glutathione-agarose bead slurry for 1 h at 4 °C, and the beads were washed twice with PBS containing protease inhibitors and 1% Triton X-100 and resuspended in 200 μl of PBS with protease inhibitors. To quantify protein amounts, 20 μl of resuspended beads were incubated with SDS sample buffer and centrifuged, and the supernatant was electrophoresed on a 10% SDS-polyacrylamide gel. The gel was stained with Coomassie Blue, and protein levels were quantified using bovine serum albumin as standard. GST-α2AR fusion proteins (typically ∼500 ng/incubation) bound to glutathione-agarose were washed with arrestin binding buffer (20 mm Tris-HCl, pH 7.4, 100 mmNaCl, 0.04% Triton X-100, 1 mm dithiothreitol, and protease inhibitors) and then incubated with 300 ng of purified bovine arrestin-1, -2, or -3 in arrestin binding buffer for 1 h at 4 °C. Incubation mixtures were centrifuged (1000 ×g) for 1 min and washed three times in binding buffer, and the proteins were released with SDS sample buffer. Samples were centrifuged, and the supernatants were electrophoresed on a 10% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose and detected by immunoblotting using a general arrestin antibody (F4C1, 1:5000 dilution), an arrestin-3-specific antibody (182, 1:5000), or an arrestin-2-specific antibody (178, 1:5000). GST bound to glutathione-agarose was used in all experiments as a control. HEK 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C in a humidified atmosphere containing 5% CO2. HEK 293 cells grown to 50–75% confluence in 60-mm dishes were transfected with 3 μg of FLAG-tagged wild type or mutant (KR, RR, 3R, RR/3R) α2bAR using 10 μl of FuGENE-6 reagent according to the manufacturer's protocol. Briefly, cells were incubated with the FuGENE-DNA mixture for 5 h and then split into poly-l-lysine-coated 12-well dishes (for ERK1/2 assays) or 24-well dishes (for enzyme-linked immunosorbent assay). Enzyme-linked immunosorbent assays were performed 24 h after transfection as described previously (25DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), whereas ERK1/2 assays were done 48 h after transfection. HEK 293 cells in 60-mm dishes were transfected as described above and then split into three wells of a poly-l-lysine-coated 12-well dish. The following day, cells were serum-starved overnight in Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum. The next day, cells were stimulated with 10 μm UK14304 at 37 °C for 0–30 min and then rinsed with PBS, lysed by addition of SDS sample buffer, and scraped off the plates. Samples were boiled and then electrophoresed on 10% SDS-polyacrylamide gels. The gels were transferred to nitrocellulose and blocked for 30 min in Tris-buffered saline containing 0.1% Tween 20 and 5% non-fat dry milk. Phospho-ERK1/2 was detected as described previously (25DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Previous studies have demonstrated subtype-specific differences in arrestin-promoted internalization of α2ARs with arrestin-3 enhancing internalization of α2bAR and α2cAR and arrestin-2 only acting on the α2bAR (25DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). To explore the mechanistic basis of arrestin binding selectivity for α2ARs, we initially focused on the third intracellular loop of the α2ARs. The first and second intracellular loops of the α2ARs share sequence homology; however, the third intracellular loops have very divergent sequences, and this region likely directly contributes to arrestin binding specificity (27Wade S.M. Lim W.K. Lan K.L. Chung D.A. Nanamori M. Neubig R.R. Mol, Pharmacol. 1999; 56: 1005-1013Crossref PubMed Scopus (63) Google Scholar, 30Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The third intracellular loops of the α2aAR (residues 218–374), α2bAR (residues 194–368), and α2CAR (residues 232–379) were expressed as GST fusion proteins, purified on glutathione-agarose, and then used in direct binding assays with purified arrestin-1, -2, or -3 (Fig. 1 A). Arrestin-1 did not bind to any of the α2AR fusion proteins (data not shown), arrestin-2 bound only to the GST-α2bAR third loop, and arrestin-3 bound to both the GST-α2bAR and the GST-α2cAR third loops but not to the α2aAR (Fig. 1 B). A dose-response analysis was next performed to determine whether there were binding differences between arrestin-2 and -3 and the α2bAR. Arrestin-3 was found to bind much more effectively to the α2bAR as compared with arrestin-2 with ∼20-fold more binding at the highest concentrations of arrestin (Fig.1 C). Overall, these results largely recapitulate the selectivity of arrestins in promoting internalization of the α2ARs (25DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and suggest that this selectivity is mediated by differences in arrestin binding to the third intracellular loops of these receptors. In an effort to identify specific arrestin binding domains, we further investigated the interaction of arrestin-3 with the α2bAR third loop. Initially, the third loop was bisected into NT (residues 194–292) and CT (residues 293–368) pieces and tested for arrestin binding (Fig. 2 A). These studies revealed that both the NT and CT regions of the α2bAR third loop bind arrestin-3, albeit not as well as the intact third loop (Fig. 2 B). Truncation mutagenesis of the NT construct revealed that arrestin-3 binding was primarily localized to the first ∼20 residues as NT1 (residues 194–214) bound arrestin-3 as well as NT, whereas NT2 (residues 206–292) did not bind (Fig. 2 B). Similar analysis of CT revealed that arrestin-3 binding was primarily localized to the last ∼25 amino acids since CT3 (residues 344–368) bound arrestin-3, whereas CT1 (residues 293–312), which contains a long stretch of acidic residues implicated previously in receptor desensitization (39Jewell-Motz E.A. Liggett S.B. Biochemistry. 1995; 34: 11946-11953Crossref PubMed Scopus (42) Google Scholar), and CT2 (residues 313–358) did not (Fig. 2 B). These results suggest that the proximal and distal ends of the third intracellular loop of the α2bAR contain the major arrestin-3 binding domains. To define specific residues within NT1 and CT3 that contribute to arrestin binding, a series of alanine point mutants were generated and tested for their ability to bind arrestin-3. Point mutations introduced into the NT1 and CT3 are indicated by an asterisk in Fig.3, A and B, respectively, whereas double mutations are underlined. These studies revealed that mutation of basic residues within the NT1 and CT3 constructs resulted in a dramatic reduction in arrestin-3 binding. Specifically, mutation of Arg-201, Arg-204, and Arg-205 in NT1 effectively disrupted arrestin-3 binding, whereas mutation of Arg-207 partially disrupted binding (Fig. 3 A). In contrast, mutation of Lys-200, Ser-202, Asn-203, Pro-205, and Arg-206 in NT1 had no significant effect on arrestin-3 binding. The placement of essential residues involved in arrestin binding was interesting given recent studies demonstrating that mutation of Lys-382 in the third loop of the parathyroid hormone receptor reduced arrestin-promoted internalization (40Vilardaga J.P. Krasel C. Chauvin S. Bambino T. Lohse M.J. Nissenson R.A. J. Biol. Chem. 2002; 277: 8121-8129Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), whereas residues in the LH/CG receptor important for arrestin-promoted internalization are localized within the analogous region of the third intracellular loop (38Mukherjee S. Palczewski K. Gurevich V.V. Hunzicker-Dunn M. J. Biol. Chem. 1999; 274: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Mutational analysis of CT3 also revealed the involvement of basic residues in arrestin binding with mutation of several arginines (358, 359, 360, 365, and 368) as well as Lys-367, resulting in a significant reduction in arrestin-3 binding (Fig. 3 B). In contrast, mutation of Leu-344 and -345 and Gln-355 had a partial effect on binding, whereas Gln-362, Thr-364, and Glu-366 mutations had no effect on arrestin-3 binding. The important role of the proximal and distal ends of the third intracellular loop in arrestin binding is reminiscent of the domains implicated in G protein binding and activation (27Wade S.M. Lim W.K. Lan K.L. Chung D.A. Nanamori M. Neubig R.R. Mol, Pharmacol. 1999; 56: 1005-1013Crossref PubMed Scopus (63) Google Scholar). In fact, previous studies have identified a BBXXB motif (where B is a basic residue and X is any residue) in the α2AR involved in Gi activation with Lys-367 and Arg-368 contributing to this motif (27Wade S.M. Lim W.K. Lan K.L. Chung D.A. Nanamori M. Neubig R.R. Mol, Pharmacol. 1999; 56: 1005-1013Crossref PubMed Scopus (63) Google Scholar, 32Okamoto T. Nishimoto I. J. Biol. Chem. 1992; 267: 8342-8346Abstract Full Text PDF PubMed Google Scholar). This suggests that significant overlap between the arrestin-3 and Gi binding sites on the α2bAR will likely contribute to the mechanism by which arrestin mediates desensitization (i.e. G protein uncoupling). To verify that specific arrestin binding mutations made within the NT1 and CT3 constructs were important for arrestin binding in the context of the whole third loop, a series of GST-α2bAR third loop mutants were generated. Four different GST-α2bAR fusions incorporating either the K200A/R201A (KR), R204A/R205A (RR), R358A/R359A/R360A (3R), or RR/3R mutations were used in binding assays with purified arrestin-3 and compared with the wild type third loop. The ability of the KR and 3R mutants to bind arrestin-3 was modestly reduced as compared with the wild type α2bAR (Fig.4 A). However, RR and RR/3R mutant binding to arrestin-3 was strongly attenuated with an 80–90% reduction in binding. To detect potential binding differences between the RR and RR/3R mutations, a dose-response analysis was performed. The RR mutation alone had a very similar binding pattern to RR/3R with the R204A/R205A mutation almost completely disrupting arrestin-3 binding to the α2bAR third loop (Fig.4 B). Taken together, these results suggest that the α2bAR third intracellular loop contains two arrestin-3 binding domains with the N-terminal region playing the predominant role. We next incorporated the various third loop mutations (KR, RR, 3R, and RR/3R) into the holo-α2bAR. Because arrestins are involved in agonist-promoted internalization of the α2bAR (25DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), we anticipated that disrupting arrestin binding to the α2bAR third intracellular loop would attenuate receptor internalization. HEK 293 cells expressing FLAG-tagged wild type or mutant α2bARs were incubated with agonist for 30 min and then analyzed for cell surface receptors by enzyme-linked immunosorbent assay (Fig.5). Internalization of the wild type α2bAR was ∼30% after agonist treatment, consistent with previous studies of α2bAR internalization in HEK 293 cells (41Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar). Internalization of the KR and 3R mutant receptors was very similar to that of the wild type α2bAR, consistent with the in vitro data showing that these mutations did not severely disrupt arrestin binding. In contrast, internalization of the RR mutant was reduced ∼50%, whereas the RR/3R mutant was decreased ∼65% as compared with the wild type receptor. These data suggest that disrupting arrestin binding to the third intracellular loop of the α2bAR has an inhibitory effect on agonist-promoted receptor internalization. These results also help to confirm the important role of arrestins in mediating internalization of the α2bAR. To ensure that the various mutations did not directly affect signaling of the α2bAR, we next analyzed the ability of the wild type and mutant α2bARs to activate ERK1/2. Our previous studies demonstrated that all three α2AR subtypes activate ERK1/2 in an agonist-dependent manner via a pathway that is Gi- and Ras-dependent but arrestin- and internalization-independent (25DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). HEK 293 cells expressing wild type or mutant (RR, 3R, RR/3R) α2bARs were incubated with agonist for 0, 5, or 30 min and then analyzed for ERK activation by immunoblotting for phospho-ERK1/2. All receptors activated ERK1/2 from 5- to 7-fold after a 5-min treatment with agonist, suggesting that the mutations that inhibit arrestin binding and receptor internalization have no significant effect on α2bAR activation of signaling (Fig.6, A and B). Our results on the NT1 region of the α2bAR suggest the importance of a BXXBB binding motif that is essential for arrestin-3 binding. Mutation of Arg-201, Arg-204, or Arg-205 completely disrupted arrestin-3 binding, whereas mutation of surrounding residues had minimal effect on arrestin binding (Fig. 3). Interestingly, the analogous region of the α2cAR, but not the α2aAR, can also directly bind arrestin-3 (data not shown). Although the three α2ARs share significant homology within the N-terminal 20 residues of the third intracellular loops, a key basic residue present in both the α2bAR (Arg-201) and the α2cAR (Arg-234) is replaced with Gln-221 in the α2AAR (Fig.7). The absence of this particular basic residue within the α2AAR may disrupt arrestin binding. It is also interesting to note that mutation of Arg-239 (the last B in the BXXBB motif) within the NT1 region of the α2cAR completely disrupts arrestin-3 binding (data not shown), further establishing the importance of basic residues within this region for arrestin binding. Several recent studies have also suggested a role for receptor third intracellular loops in arrestin binding (30Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 34Krupnick J.G. Gurevich V.V. Schepers T. Hamm H.E. Benovic J.L. J. Biol. Chem. 1994; 269: 3226-3232Abstract Full Text PDF PubMed Google Scholar, 35Appleyard S.M. Celver J. Pineda V. Kovoor A. Wayman G.A. Chavkin C. J. Biol. Chem. 1999; 274: 23802-23807Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 36Gelber E.I. Kroeze W.K. Willins D.L. Gray J.A. Sinar C.A. Hyde E.G. Gurevich V. Benovic J. Roth B.L. J. Neurochem. 1999; 72: 2206-2214Crossref PubMed Scopus (70) Google Scholar, 37Cheng Z.J. Zhao J. Sun Y., Hu, W., Wu, Y.L. Cen B., Wu, G.X. Pei G. J. Biol. Chem. 2000; 275: 2479-2485Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 38Mukherjee S. Palczewski K. Gurevich V.V. Hunzicker-Dunn M. J. Biol. Chem. 1999; 274: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). For example, the third loops of the δ-opioid receptor (35Appleyard S.M. Celver J. Pineda V. Kovoor A. Wayman G.A. Chavkin C. J. Biol. Chem. 1999; 274: 23802-23807Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and the LH/CG receptor (38Mukherjee S. Palczewski K. Gurevich V.V. Hunzicker-Dunn M. J. Biol. Chem. 1999; 274: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) can directly bind arrestins, and third loop peptides from the LH/CG receptor inhibit receptor desensitization by sequestering arrestin-2 (38Mukherjee S. Palczewski K. Gurevich V.V. Hunzicker-Dunn M. J. Biol. Chem. 1999; 274: 12984-12989Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The domains involved in these interactions share similar homology and generally contain several basic residues as well as serines and/or threonines (Fig. 7). Although the δ-opioid receptor studies suggested a role for two serines in arrestin binding (35Appleyard S.M. Celver J. Pineda V. Kovoor A. Wayman G.A. Chavkin C. J. Biol. Chem. 1999; 274: 23802-23807Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), all of these domains contain the BXXBB motif and help to establish the importance of such a motif in arrestin binding.

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