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- W2004755511 abstract "The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal initiation and termination. As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of G-protein-coupled receptors as probes to screen brain cytosol proteins. Peptides from the third intracellular loop (i3) of the M2-muscarinic receptor (MR) (His208–Arg387), M3-MR (Gly308–Leu497), or α2A/D-adrenergic receptor (AR) (Lys224–Phe374) were generated in bacteria as glutathione S-transferase (GST) fusion proteins, bound to glutathione-Sepharose and used as affinity matrices to detect interacting proteins in fractionated bovine brain cytosol. Bound proteins were identified by immunoblotting following SDS-polyacrylamide gel electrophoresis. Brain arrestins bound to the GST-M3fusion protein, but not to the control GST peptide or i3 peptides derived from the α2A/D-AR and M2-MR. However, each of the receptor subdomains bound purified β-arrestin and arrestin-3. The interaction of the M3-MR and M2-MR i3 peptides with arrestins was further investigated. The M3-MR i3 peptide bound in vitro translated [3H]β-arrestin and [3H]arrestin-3, but did not interact with in vitro translated or purified visual arrestin. The properties and specificity of the interaction ofin vitro translated [3H]β-arrestin, [3H]visual arrestin, and [3H]β-arrestin/visual arrestin chimeras with the M2-MR i3 peptide were similar to those observed with the intact purified M2-MR that was phosphorylated and/or activated by agonist. Subsequent binding site localization studies indicated that the interaction of β-arrestin with the M3-MR peptide required both the amino (Gly308–Leu368) and carboxyl portions (Lys425–Leu497) of the receptor subdomain. In contrast, the carboxyl region of the M3-MR i3 peptide was sufficient for its interaction with arrestin-3. The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal initiation and termination. As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of G-protein-coupled receptors as probes to screen brain cytosol proteins. Peptides from the third intracellular loop (i3) of the M2-muscarinic receptor (MR) (His208–Arg387), M3-MR (Gly308–Leu497), or α2A/D-adrenergic receptor (AR) (Lys224–Phe374) were generated in bacteria as glutathione S-transferase (GST) fusion proteins, bound to glutathione-Sepharose and used as affinity matrices to detect interacting proteins in fractionated bovine brain cytosol. Bound proteins were identified by immunoblotting following SDS-polyacrylamide gel electrophoresis. Brain arrestins bound to the GST-M3fusion protein, but not to the control GST peptide or i3 peptides derived from the α2A/D-AR and M2-MR. However, each of the receptor subdomains bound purified β-arrestin and arrestin-3. The interaction of the M3-MR and M2-MR i3 peptides with arrestins was further investigated. The M3-MR i3 peptide bound in vitro translated [3H]β-arrestin and [3H]arrestin-3, but did not interact with in vitro translated or purified visual arrestin. The properties and specificity of the interaction ofin vitro translated [3H]β-arrestin, [3H]visual arrestin, and [3H]β-arrestin/visual arrestin chimeras with the M2-MR i3 peptide were similar to those observed with the intact purified M2-MR that was phosphorylated and/or activated by agonist. Subsequent binding site localization studies indicated that the interaction of β-arrestin with the M3-MR peptide required both the amino (Gly308–Leu368) and carboxyl portions (Lys425–Leu497) of the receptor subdomain. In contrast, the carboxyl region of the M3-MR i3 peptide was sufficient for its interaction with arrestin-3. G-protein-coupled receptors possess a characteristic seven segments of hydrophobic amino acids that likely serve as membrane spans to form a core motif important for ligand recognition. The interaction of agonist with the receptor initiates an ill-defined conformational adjustment in this core motif, which is propagated to intracellular domains of the receptor resulting in the activation of G-protein and the initiation of intracellular signaling events. For most members of the superfamily of G-protein-coupled receptors, the third intracellular (i3) 1The abbreviations used are: i3, third intracellular loop; MR, muscarinic receptor; AR, adrenergic receptor; PAGE, polyacrylamide gel electrophoresis. loop and the carboxyl-terminal tail of the receptor are key sites for signal initiation and termination, and these receptor domains also exhibit the greatest variability in size among different subfamilies of these receptors. The largest i3 domains (100–240 amino acids) are found in receptors coupled to the Gi, Go, and/or Gq family of G-proteins (i.e. muscarinic, α-adrenergic), whereas shorter i3 loops are found in the photoreceptor rhodopsin or β-adrenergic receptors (20–50 amino acids). During the process of signal initiation and termination, several proteins interact with the receptor. The interaction of arrestins with G-protein-coupled receptors is a key component of signal termination (1Sterne-Marr R. Benovic J.L. Vitam. Horm. 1995; 51: 193-234Crossref PubMed Scopus (113) Google Scholar, 2Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (474) Google Scholar, 3Ranganathan R. Stevens C.F. Cell. 1995; 81: 841-848Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 4Palczewski K. Protein Sci. 1994; 3: 1355-1361Crossref PubMed Scopus (75) Google Scholar, 5Dolph P.J. Ranganathan R. Colley N.J. Hardy R.W. Socolich M. Zuker C.S. Science. 1993; 260: 1910-1916Crossref PubMed Scopus (268) Google Scholar). The arrestin family consists of visual arrestin, β-arrestin, arrestin-3, and a cone-specific arrestin termed C- or X-arrestin (6Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (923) Google Scholar, 7Shinohara T. Dietzschold B. Craft C.M. Wistow G. Early J.J. Donoso L.A. Horwitz J. Tao R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6975-6979Crossref PubMed Scopus (180) Google Scholar, 8Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar, 9Sterne-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, 10Murakami A. Yajima T. Sakuma H. McLaren M.J. Inana G. FEBS Lett. 1993; 334: 203-209Crossref PubMed Scopus (91) Google Scholar, 11Craft C.M. Whitmore D.H. Wiechmann A.F. J. Biol. Chem. 1994; 269: 4613-4619Abstract Full Text PDF PubMed Google Scholar). In vertebrates, visual arrestin interacts with phosphorylated rhodopsin in rod cells to terminate signal propagation by interfering with receptor coupling to transducin. β-Arrestin and arrestin-3 are widely expressed and parallel the role of visual arrestin in terms of signal termination for G-protein-coupled receptors other than rhodopsin. The affinity of arrestin binding to G-protein-coupled receptors is increased by receptor phosphorylation and/or activation by agonist. Receptors of this class are phosphorylated to varying degrees by protein kinase A and C as well as kinases specific for the activated conformation of the receptor (G-protein-coupled receptor kinases). The phosphorylation of the receptor by G-protein-coupled receptor kinases and subsequent arrestin binding are intimately associated with receptor desensitization and sequestration (12Ferguson S.S.G. Downey III, W.E. Colapietro A. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (853) Google Scholar, 13Goodman 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 (1184) Google Scholar, 14Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Resensitization of the receptor protein involves dissociation of bound arrestin and receptor dephosphorylation. The interaction of receptors with G-proteins, protein kinases, arrestins, and additional entities controlling receptor trafficking apparently involves discrete motifs in cytoplasmic domains of the receptor. The associations of these proteins with the receptor likely occur within a signal transduction complex that may also include various effector molecules and other proteins that influence signaling specificity/efficiency. To define receptor subdomains important for protein interactions and to begin to identify the components of a signal transduction complex for G-protein-coupled receptor subtypes, we generated peptides from the i3 loop of the M2-muscarinic receptor (MR), M3-MR and α2A/D-adrenergic receptor (AR) to use as probes to detect interacting proteins in bovine brain cytosol. In the present report, we determined the interaction of the i3 loop with cytosolic proteins involved in receptor regulation. Radiolabeled arrestins were in vitrotranslated as described previously (15Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Bovine β-arrestin, visual arrestin, and arrestin-3 were also expressed in BL21 cells and purified to homogeneity by successive chromatography on heparin- and Q-Sepharose (13Goodman 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 (1184) Google Scholar). Antibodies to protein kinase C isoforms were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and phosphatase 2A/C antibody was from Calbiochem. Anti-gelsolin monoclonal antibody (GS-2C4) was obtained from Sigma. Monoclonal antibody mAbF4C1, which recognizes the epitope DGVVLVD present in visual arrestin, β-arrestin, and arrestin-3, was generously provided by Dr. L. Donoso (Wills Eye Hospital, Philadelphia, PA). Glutathione-Sepharose 4B was purchased from Pharmacia Biotech Inc. Polyvinylidene difluoride membranes were obtained from Gelman Sciences (Ann Arbor, MI). Bovine brain cytosolic proteins in buffer A (10 mm Tris-HCl, pH 7.5, 0.5 mm phenylmethylsulfonyl fluoride) containing 250 mm sucrose were precipitated with 40% ammonium sulfate and pelleted by centrifugation (100,000 × g, 45 min). The precipitated proteins were resuspended in a minimal volume of 50 mm Tris-HCl, pH 8.0, followed by extensive dialysis (4 liters of buffer A; 4 liters of buffer B (20 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm EGTA, 2 mmβ-mercaptoethanol). The supernatant from the 40% ammonium sulfate precipitate was brought to 90% ammonium sulfate to precipitate additional proteins and subsequently processed as described for the 40% ammonium sulfate precipitate. The dialyzed solutions were clarified by centrifugation and applied to an anion-exchange resin (DEAE-Biogel A) equilibrated with buffer B. The column was washed with buffer B and proteins eluted sequentially with buffer B containing 100, 250, and 500 mm NaCl. Eluted proteins were desalted by dialysis, concentrated by lyophilization, and stored at −70 °C. The M3-MR i3 construct was obtained from Dr. Barry Wolfe (Department of Pharmacology, Georgetown University School of Medicine, Washington, DC) and encoded the peptide Gly308–Leu497. The M2-MR and α2A/D-AR i3 constructs were generated from the cDNA or genomic clones by amplification using the polymerase chain reaction. The M2-MR subdomain was inserted into the EcoRI restriction site of the pGEX-2T vector (Promega, Madison, WI). Using primers containing appropriate restriction sites, the rat α2A/D-AR gene (16Lanier S.M. Downing S. Duzic E. Homcy C.J. J. Biol. Chem. 1991; 266: 10470-10478Abstract Full Text PDF PubMed Google Scholar) segment encoding the peptide Lys224–Phe374 was amplified by polymerase chain reaction and subcloned into the BamHI andEcoRI restriction sites of pGEX-2T. The M3-II and M3-III were generated from M3-MR i3 construct (M3-I, Gly308–Leu497) by taking advantage of a PstI restriction site (nucleotide 1918 of the rat M3 muscarinic receptor coding region). The M3-II construct was generated by excising theBamHI/PstI fragment from the M3-I construct with subsequent subcloning of this fragment into pGEX-3X at the BamHI and EcoRI restriction sites via use of an adaptor. The M3-III construct was generated using a similar strategy with the PstI/EcoRI fragment isolated from the M3-I construct. The M3-IV construct was prepared by digesting the M3-I construct withHindIII removing the gene segment encoding amino acids Lys369–Thr424. The purified plasmid containing the amino- and carboxyl-terminal segments was then religated to yield M3-IV. The structure of each construct used in the present study was verified by restriction mapping and nucleotide sequence analysis. The fusion proteins were expressed in bacteria and purified using a glutathione affinity matrix according to the manufacturer's instructions. Immobilized fusion proteins were either used immediately or stored at 4 °C for no longer than 3 days. Each batch of fusion protein used in experiments was first analyzed by SDS-PAGE and Coomassie Blue staining. Brain cytosol (1–200 μg) fractions were incubated with ∼5 μg of GST fusion protein bound to the glutathione resin in 250 μl of buffer C (20 mmTris-HCl, pH 7.5, 70 mm NaCl) for 2.5 h at 4 °C. The resin was washed three times with 0.5 ml of buffer C, and the retained proteins were solubilized and applied to a denaturing 10% polyacrylamide gel. Polyvinylidene difluoride membrane transfers were evaluated by immunoblotting as described previously (17Sato M. Kataoka R. Dingus J. Wilcox M. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1995; 270: 15269-15276Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) using specific antibodies. The interactions of purified arrestins with the i3 peptides was determined in a similar manner. Arrestin binding to the M2-MR and M3-MR peptides was also evaluated by direct binding assays using tritiated arrestins generated by in vitro translation. Aliquots (∼2.5 μg) of GST fusion proteins bound to a glutathione resin were incubated with [3H]β-arrestin (long splice variant) (1062–1600 dpm/fmol), [3H]arrestin-3 (short splice variant) (1500 dpm/fmol), [3H]visual arrestin (1300–1871 dpm/fmol), or the [3H]β-arrestin/visual arrestin chimeras BBBA (1990 dpm/fmol) and AABB (2010 dpm/fmol) in a total volume of 20 μl of buffer C for 2.5 h at 4 °C with shaking. The resin was washed three times with 100 μl of buffer C. The washed resin was resuspended in 300 μl of buffer C, mixed with 10 ml of Ecoscint A scintillation fluid, and the retained radioactivity quantitated by scintillation spectrometry at ∼50% efficiency. Nonspecific binding was defined as the amount of ligand retained in parallel experiments using a control GST resin and represented ∼30% of total binding at a concentration of 0.75 nm arrestin. The third intracellular loop of the M2-MR, M3-MR, and the α2A/D-AR consists of 180 (His208–Arg387), 238 (Arg253–Gln490), and 157 (Arg218–Phe374) amino acids, respectively. The putative first and second intracellular loops of the three receptors are similar in size ∼11–12 amino acids, 1st loop; ∼18–19 amino acids, 2nd loop) and the carboxyl-terminal tails of the M2-MR, M3-MR, and α2A/D-AR are 23, 44, and 20 amino acids in length, respectively. As an initial attempt to define proteins that may interact with the intracellular domains of these G-protein-coupled receptors, we focused on the i3 loop as it is the largest intracellular domain in this receptor group. The juxtamembrane segments of the i3 domain are of critical importance for receptor coupling to G-protein, whereas other segments participate in receptor phosphorylation, receptor trafficking, and other aspects of signal propagation. The M2-MR (His208–Arg387), M3-MR (Gly308–Leu497), and α2A/D-AR (Lys224–Phe374) i3 peptides were expressed in bacteria as a GST fusion protein and used to generate an affinity matrix by saturating a glutathione-Sepharose resin with the fusion protein (Fig. 1). The M2-MR peptide corresponded to the entire i3 loop of the receptor. The M3-MR i3 peptide began 45 amino acids downstream of the amino terminus of the i3 loop and terminated seven amino acids into the VI membrane span. The α2A/D-AR peptide began six amino acids downstream of the amino terminus of the i3 loop and terminated at the beginning of the VI membrane span. To determine the interaction of these receptor-derived peptides with cytosolic proteins, we first fractionated bovine brain cytosol to enrich for potential interacting proteins.Figure 1Generation of receptor subdomain probes.Peptides corresponding to segments of the third intracellular loop of the human M2-MR (180 amino acids), rat M3-MR (190 amino acids), and rat α2A/D-AR (151 amino acids) (A) were generated as GST fusion proteins in bacteria and purified as described under “Experimental Procedures”. InA, the black segments correspond to the putative fifth and sixth membrane spans of the receptor. B, the purified fusion proteins were electrophoresed on denaturing polyacrylamide gels (10%) and visualized by Coomassie Blue stain of the proteins. The calculated molecular weights of GST and the M2-MR, M3-MR, and α2A/D-AR GST fusion proteins were 28,146, 47,864, 49,796, and 43,343, respectively, including adapter amino acids. The arrows indicate the migration of GST and each of the GST fusion proteins. Thenumbers to the left of the gel indicate the migration of standards of known molecular weight × 10−3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the first series of experiments, we determined the interaction of receptor subdomains with brain arrestins. Bovine brain cytosol was fractionated by ammonium sulfate precipitation and ion exchange chromatography and the fractions enriched for arrestins were determined by immunoblotting (Fig. 2 A, left panel). The cytosol fraction enriched for arrestin was incubated with the M2-MR, M3-MR or the α2A/D-AR i3 subdomains as well as the control GST affinity matrix and arrestins retained by the matrices were determined by immunoblotting. Brain arrestins were adsorbed by the M3-MR affinity matrix but did not interact with matrices constructed of the GST control peptide or the i3 peptides derived from the M2-MR or the α2A/D-AR (Fig.2 A, right panel). The amount of arrestin retained by the M3-MR matrix was directly related to the amount of cytosol present during incubation (Fig. 2 B). Brain arrestins were not retained by GST fusion proteins containing subdomains of the tyrosine phosphatase Syp (Src homology 2 domains, 215 amino acids), the Na+/H+ exchanger (carboxyl terminus, 178 amino acids), a subdomain of the M2-MR i3 loop (56 amino acids), or the transregulatory protein c-Jun (amino terminus, 79 amino acids) (Fig. 2 C). These data indicated that the interaction of brain arrestins with the M3-MR affinity matrix was specific for the M3-MR peptide. The specificity of arrestin binding to the M3-MR peptide was further investigated by determining the interaction of the peptide with other cytosolic proteins. The distribution of protein kinase C isoforms, phosphatase 2A, and the actin-binding protein gelsolin in the fractionated bovine brain cytosol was determined by immunoblotting (Fig. 3, left panel). Two protein kinase C isoforms fractionated in the 250 mm NaCl elution of the 90% ammonium sulfate precipitate. The phosphatase 2A immunoreactive species was identified in the 250 mm NaCl elution of the 40% ammonium sulfate precipitate. Gelsolin was enriched in the 100 mm NaCl elution of the 40% ammonium sulfate precipitate. The appropriate fraction was then incubated with the M3-MR or the α2A/D-AR i3 affinity matrix and processed as described for arrestins. Although the M3-MR affinity matrix adsorbed brain arrestins (Fig. 2), neither the M3-MR or the α2A/D-AR i3 peptides interacted with the protein kinase C isoforms, phosphatase 2A, or gelsolin (Fig. 3, right panel). The interaction of arrestins with the i3 peptides was investigated in more detail to define issues of arrestin selectivity and sites of arrestin association. In the first series of experiments, we evaluated arrestin binding to the M3-MR using radiolabeled arrestins. Increasing concentrations of radiolabeled β-arrestin, arrestin-3, and visual arrestin were incubated with the M3-MR or the control GST resin. Both [3H]β-arrestin and [3H]arrestin-3 exhibited specific binding to the M3-MR peptide relative to the binding of the arrestins to the control GST peptide (Fig. 4 A). In contrast, the binding of [3H]visual arrestin to the M3-MR matrix was only slightly increased above that observed with the GST peptide itself, consistent with the lower affinity of visual arrestin at G-protein-coupled receptors other than rhodopsin. Interestingly, the affinities exhibited by [3H]β-arrestin and [3H]arrestin-3 (0.5–1.0 nm) are in the range of the K d for arrestin binding to purified β2-AR and M2-MR (15Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). The selectivity of the binding of [3H]arrestins to the M3-MR peptide was also observed with purified β-arrestin and visual arrestin (Fig.4 B). In the second series of experiments, the interaction of arrestins with the i3 peptides was further evaluated using purified β-arrestin and arrestin-3. Purified β-arrestin and arrestin-3 were adsorbed to the M3-MR matrix in a concentration-dependent manner (Fig.5 A). Neither arrestin was retained by the control GST resin (Fig. 5 A). As indicated above, brain arrestins were not retained by the M2-MR or α2A/D-AR affinity matrices. However, both the M2-MR and the α2A/D-AR i3 peptides bound purified β-arrestin and arrestin-3 (Fig.5 B). 2Due to differences in the degree of resin substitution for various fusion proteins, variable membrane transfer efficiencies, and relative signal intensities, it is difficult to accurately determine the affinity of arrestin binding to the resins. However, data obtained thus far indicate no dramatic differences in the affinity of the purified arrestins for the three i3 peptides. Figure 5Interaction of purified β-arrestin and arrestin-3 with third intracellular loop peptides. InA, the M3 matrix (∼5 μg of protein) was incubated with increasing concentrations of purified β-arrestin or arrestin-3 and the samples processed as described in the legend to Fig.2 for brain arrestin preparations. Similar results were obtained in three experiments. GST, glutathione S-transferase matrix. In B, GST-receptor subdomain fusion proteins (∼5 μg of protein) were incubated with 200 μg of brain cytosol enriched for arrestins or 50 ng of either purified β-arrestin or arrestin-3. Similar results were obtained in three experiments using different preparations of fusion protein. The aberrant migration of β-arrestin in the M2-MR lane relative to control input is due to comigration of the fusion protein and β-arrestin. The last three lanes on the right of the immunoblot contain aliquots of the material incubated with the resins (cytosol, 5 μg; β-arrestin, 25 ng; arrestin-3, 25 ng). The results are representative of three individual experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The interaction of arrestins with the intact purified M2-MR was previously characterized using in vitro translated arrestins and β-arrestin/visual arrestin chimeras (15Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). We thus compared the interaction of arrestins with the M2-MR i3 peptide and the intact receptor relative to the influence of ionic strength and the selectivity of binding for the different arrestins. As observed for the intact purified receptor that was phosphorylated and/or activated by agonist (15Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar), [3H]β-arrestin binding to the i3 peptide was first increased and then decreased with increasing ionic strength of the incubation buffer (Fig.6 A). Previous studies using a series of [3H]β-arrestin/visual arrestin chimeras indicated that the selectivity of β-arrestin and visual arrestin binding to the intact purified M2-MR and the β2-adrenergic receptor involved specific domains of the two arrestins (15Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). To determine if this selectivity of arrestin binding was maintained with the M2-MR i3 peptide, we evaluated the binding of two [3H]β-arrestin/visual arrestin chimeras. The BBBA chimera consists of amino acids 1–340 of β-arrestin and amino acids 346–404 of visual arrestin. The AABB chimera consists of amino acids 1–213 of visual arrestin and amino acids 208–418 of β-arrestin. The binding of BBBA to the intact M2-MR was similar or greater than that observed with β-arrestin, whereas the binding of AABB was intermediate between β-arrestin and visual arrestin (15Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). The relative binding of [3H]β-arrestin, [3H]visual arrestin, and the [3H]β-arrestin/visual arrestin chimeras to the M2-MR i3 peptide, as well as the M3-MR peptide, was similar to that observed with the intact purified M2-MR (Fig. 6 B). The next series of experiments were designed to localize subdomains of the M3-MR i3 loop required for interaction with arrestins. The M3-MR i3 peptide (M3-I) was divided into three segments: an amino-terminal region (M3-II, Gly308–Gln389), the carboxyl-terminal region (M3-III, Val390–Leu497), and a middle portion (Lys369–Thr424) (Fig.7 A). The M3 peptide possesses a concentrated negative charge in the amino-terminal third and a concentrated positive charge in the carboxyl-terminal third of the peptide. The contribution of the middle portion was determined using construct M3-IV in which this segment was deleted and the peptide Gly308–Leu368 was fused to the peptide Lys425–Leu497 (Fig. 7 A). Neither construct II or III interacted with brain arrestins or purified β-arrestin under these incubation conditions (Fig.7 B). 3Based on the relative migration of β-arrestin and arrestin-3 in 10% denaturing polyacrylamide gels and the comigration of the partially purified brain arrestin with β-arrestin (Fig. 5), the arrestin identified in the 100 mm NaCl elution of the 90% ammonium sulfate precipitate is predominantly β-arrestin (G. Wu and S. M. Lanier, unpublished data). However, the construct containing both the amino and carboxyl regions of the M3-I peptide (M3-IV) retained the ability to interact with brain arrestins and purified β-arrestin (Fig. 7 B). These data suggest that there are at least two sites on the M3-I peptide for β-arrestin binding and that one of these sites by itself is insufficient. In contrast to the results with β-arrestin, the carboxyl region of the M3-MR peptide was sufficient for interaction with arrestin-3 (Fig. 7 B). Arrestin-3 bound to the M3-I, -III, and -IV, but not to construct II, suggesting that the binding motif for arrestin-3 is different from that of β-arrestin. As is apparently the case with most complex signal processing systems, G-protein-coupled receptors likely operate within a signal transduction complex that is either preexisting or is generated by the biological stimuli. The components of such a signaling complex are unclear but might include proteins that influence events at the receptor-G-protein or G-protein-effector interface or contribute to the formation of the signal transduction complex itself (Refs. 17Sato M. Kataoka R. Dingus J. Wilcox M. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1995; 270: 15269-15276Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar and 18Sato M. Ribas C. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1996; 271: 30052-30060Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, and references therein). As part of an effort to identify components of this signal transduction complex, we initiated two experimental approaches. The first approach was based on a biological activity and was designed to detect factors that might influence the transfer of signal from receptor to G-protein (17Sato M. Kataoka R. Dingus J. Wilcox M. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1995; 270: 15269-15276Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 18Sato M. Ribas C. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1996; 271: 30052-30060Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The second approach, described in the present report, involved the identification of proteins capable of associating with receptors of this class. In the latter approach, we used intracellular domains of G-protein-coupled receptors as “bait” to search for interacting proteins in brain cytosol. Receptor subdomains were generated from the i3 loop of the M2-MR, M3-MR, and α2-AR. Both the muscarinic receptor subtypes and the α2-AR are capable of coupling to multiple G-proteins and effectors in a cell type-specific manner. Activation of the M2-MR results in inhibition of adenylyl cyclase, whereas the M3-MR couples to the inositol phosphate/protein kinase C signaling pathway. The α2A/D-AR is generally associated with inhibition of adenylyl cyclase, but activation of this receptor also influences signal transduction pathways involving phospholipases, p21 ras, the mitogen-activated protein kinase signaling pathway, and various ion channels (see Ref. 17Sato M. Kataoka R. Dingus J. Wilcox M. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1995; 270: 15269-15276Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, and references therein). We first evaluated the interaction of the i3 loops with the arrestin family of proteins, which are implicated in receptor uncoupling and internalization. The arrestins exhibited a specific interaction with the i3 receptor subdomains. The receptor subdomain probes did not interact with selected protein kinase C isoforms, cytosolic phosphatase 2A, or the actin-binding protein gelsolin. However, both β-arrestin and arrestin-3 bound to the i3 loops of the muscarinic receptor subtypes and the α2A/D-AR. The major observations concerning this interaction are as follows. First, the interaction of β-arrestin and arrestin-3 with the M3-MR involved different regions of the i3 loop. Second, the i3 domains from the muscarinic receptors and the α2A/D-AR differed in their ability to interact with endogenous bovine brain arrestins in a crude cytosol fraction versus purified recombinant β-arrestin and arrestin-3. Third, the interaction of arrestins with the i3 loop peptides occurred in the absence of peptide phosphorylation. Both β-arrestin and arrestin-3 are widely expressed, but exhibit a heterogeneous intra-tissue distribution, and both arrestins are alternatively spliced to generate a short and long form of the protein. Although arrestins clearly interact with the receptor protein (15Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar,19Gurevich V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar, 20Krupnick 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, 21Gurevich V.V. Richardson R.M. Kim C.M. Hosey M.M. Benovic J.L. J. Biol. Chem. 1993; 268: 16879-16882Abstract Full Text PDF PubMed Google Scholar, 22Sohlemann P. Hekman M. Puzicha M. Buchen C. Lohse M.J. Biochemistry. 1995; 232: 464-472Google Scholar), the sites of interaction and the selectivity among the different arrestins and receptor families outside the visual system are undefined. However, the present study indicates that there are receptor motifs that are indeed capable of distinguishing the two types of arrestin. Within the M3-MR i3 loop, amino acids Gly308–Leu368 and Lys425–Leu497 are required for binding of β-arrestin and the partially purified brain arrestin, whereas amino acids Gly308–Leu368 are not required for binding of arrestin-3. The differential interaction of the two arrestins with the M3-MR subdomain may relate to the charge distribution within the receptor peptide segments and the structural properties of the two arrestins. Although the i3 peptides from the M2-MR, M3-MR and α2A/D-AR all bound purified arrestins, only the M3-MR was capable of interacting with brain cytosol arrestins. The inability of brain arrestins to interact with the M2-MR and α2-AR may simply reflect differences in the relative affinities of the different receptor subdomains for the arrestins. However, there were no apparent differences in the efficiency of the interaction of the three receptor subdomains with the purified arrestins under these experimental conditions, suggesting that other factors may be regulating this interaction. It is possible that the brain arrestins are slightly different from the recombinant arrestins, and that this difference contributes to selective interactions with receptor families. Alternatively, perhaps there are additional proteins in the fractionated brain cytosol that impede arrestin binding to the M2-MR and α2A/D-AR, but not the M3-MR i3 peptides. Such proteins may interact with motifs in the i3 peptide or perhaps with arrestin itself. Interaction of visual arrestin with rhodopsin or β-arrestin and arrestin-3 with the β2-AR or the M2-MR involves multiple contact sites that impart apparent positive cooperativity to the reaction (15Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 19Gurevich V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar, 20Krupnick 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, 21Gurevich V.V. Richardson R.M. Kim C.M. Hosey M.M. Benovic J.L. J. Biol. Chem. 1993; 268: 16879-16882Abstract Full Text PDF PubMed Google Scholar). Arrestin binding to the receptor is proposed to first involve an ionic interaction that senses the phosphorylation and activation state of the receptor. If the receptor is phosphorylated or agonist-occupied, the apparent affinity of arrestin binding to the receptor is increased. The phosphorylated receptor subdomain likely forms a component of the arrestin binding site, although phosphorylation-dependent conformational shifts in the intracellular regions of the receptor may also reveal sites that participate in arrestin binding. Whereas the binding of arrestin to rhodopsin is highly dependent upon receptor phosphorylation and activation, a truncated splice variant of visual arrestin binds to nonphosphorylated rhodopsin. The truncated and full-length visual arrestins differ in their subcellular distribution, with the former constitutively localized to disc membranes, while the latter associates with the disc membranes in a light-dependent manner (23Smith W.C. Milam A.H. Dugger D. Arendt A. Hargrave P.A. Palczewski K. J. Biol. Chem. 1994; 269: 15407-15410Abstract Full Text PDF PubMed Google Scholar). β-Arrestin and arrestin-3 also bind to nonphosphorylated M2-MR or β2-AR and high affinity binding is less dependent upon the activation state of the receptor than is the interaction between visual arrestin and rhodopsin (15Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 23Smith W.C. Milam A.H. Dugger D. Arendt A. Hargrave P.A. Palczewski K. J. Biol. Chem. 1994; 269: 15407-15410Abstract Full Text PDF PubMed Google Scholar). The possible interaction of arrestins with nonphosphorylated receptors is also suggested by the ability of phosphorylation-defective mutants to undergo receptor desensitization and sequestration. An important role for arrestin interaction with a nonphosphorylated receptor is also apparent for visual signaling in invertebrates (24Plangger A. Malicki D. Whitney M. Paulsen R. J. Biol. Chem. 1994; 269: 26969-26975Abstract Full Text PDF PubMed Google Scholar). In the dipteranCalliphora, arrestin interaction with the receptor protein clearly precedes receptor phosphorylation, and indeed the arrestin-receptor complex is the preferred kinase substrate (24Plangger A. Malicki D. Whitney M. Paulsen R. J. Biol. Chem. 1994; 269: 26969-26975Abstract Full Text PDF PubMed Google Scholar). As the interaction of arrestins with the i3 peptides in the present study occurred without peptide phosphorylation, the i3 loop or perhaps other domains of G-protein-coupled receptors appear capable of serving as a docking site for arrestin independent of receptor phosphorylation. Indeed, the properties of arrestin binding to the M2-MR i3 peptide appeared similar to those exhibited by the purified receptor protein that was phosphorylated and/or activated by agonist. The binding of β-arrestin and arrestin-3 to the i3 peptides may also relate to the agonist-induced conformational changes responsible for initiating the signaling cascade. In the absence of agonist, the regions of the receptor involved in G-protein activation are stabilized in a conformation that acts as a “brake” on signal initiation (25Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar). Such a conformational “brake” may be released by discrete mutations in the i3 loop of some receptors, such that the receptor becomes constitutively active (26Allen L.F. Lefkowitz R.J. Caron M.G. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11354-11358Crossref PubMed Scopus (295) Google Scholar). An analogous situation may occur when the i3 loop is separated from the conformational restrictions imposed by membrane spans of the receptor and assumes an “activated and/or accessible conformation” that is recognized by arrestin. The demonstration that the experimental approach using receptor subdomains as probes for receptor-associated proteins resulted in the detection of protein-protein interactions of clear biological relevance underscores the potential utility of the system to identify additional interacting proteins that may contribute to the formation of a signal transduction complex. Such interacting proteins may play an important role in directing the receptor-initiated signal to a specific effector pathway. In contrast to signaling events in the visual system where the components are localized and the interactions between the individual molecules are relatively specific, other G-protein-coupled receptors operate in diverse cell types and couple to multiple G-proteins and effectors. Perhaps, such receptors have evolved larger i3 loops to maintain the fidelity of the signaling system by providing sites for interaction with additional accessory proteins that influence receptor trafficking and/or signaling specificity and efficiency. We thank Dr. Barry Wolfe, Dr. Tatsuya Haga (Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, University of Tokyo, Japan), Dr. Larry Fliegel (Department of Biochemistry and Pediatrics, University of Alberta, Alberta, Canada), Dr. Gen-Shen Feng (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine), and Dr. Steven Rosenzweig (Department of Pharmacology, Medical University of South Carolina, Charleston, SC) for providing the M3-MR, M2-MR (56-amino acid segment for the third intracellular loop), Na+/H+ exchanger, Syp, and c-Jun fusion protein constructs, respectively. We also thank Dr. Larry Donoso for mAbF4C1 and Dr. Vsevolod Gurevich (Sun Health Research Institute, Sun City, AZ) for purified arrestins. The expression vector containing the peptide derived from the third intracellular loop of the α2A/D-adrenergic receptor was generated by Dr. Sally Kadkhodayan in the laboratory of Dr. Lanier." @default.
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