Interaction of the α1B-Adrenergic Receptor with gC1q-R, a Multifunctional Protein
1999; Elsevier BV; Volume: 274; Issue: 30 Linguagem: Inglês
10.1074/jbc.274.30.21149
ISSN1083-351X
AutoresZhaojun Xu, Akira Hirasawa, Hitomi Shinoura, Gozoh Tsujimoto,
Tópico(s)Lipid Membrane Structure and Behavior
ResumogC1q-R, a multifunctional protein, was found to bind with the carboxyl-terminal cytoplasmic domain of the α1B-adrenergic receptor (173 amino acids, amino acids 344–516) in a yeast two-hybrid screen of a cDNA library prepared from the rat liver. In a series of studies with deletion mutants in this region, the ten arginine-rich amino acids (amino acids 369–378) were identified as the site of interaction. The interaction was confirmed by specific co-immunoprecipitation of gC1q-R with full-length α1B-adrenergic receptors expressed on transfected COS-7 cells, as well as by fluorescence confocal laser scanning microscopy, which showed co-localization of these proteins in intact cells. Interestingly, the α1B-adrenergic receptors were exclusively localized to the region of the plasma membrane in COS-7 cells that expressed the α1B-adrenergic receptor alone, whereas gC1q-R was localized in the cytoplasm in COS-7 cells that expressed gC1q-R alone; however, in cells that co-expressed α1B-adrenergic receptors and gC1q-R, most of the α1B-adrenergic receptors were co-localized with gC1q-R in the intracellular region, and a remarkable down-regulation of receptor expression was observed. These observations suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in regulating the cellular localization and expression of the α1B-adrenergic receptors. gC1q-R, a multifunctional protein, was found to bind with the carboxyl-terminal cytoplasmic domain of the α1B-adrenergic receptor (173 amino acids, amino acids 344–516) in a yeast two-hybrid screen of a cDNA library prepared from the rat liver. In a series of studies with deletion mutants in this region, the ten arginine-rich amino acids (amino acids 369–378) were identified as the site of interaction. The interaction was confirmed by specific co-immunoprecipitation of gC1q-R with full-length α1B-adrenergic receptors expressed on transfected COS-7 cells, as well as by fluorescence confocal laser scanning microscopy, which showed co-localization of these proteins in intact cells. Interestingly, the α1B-adrenergic receptors were exclusively localized to the region of the plasma membrane in COS-7 cells that expressed the α1B-adrenergic receptor alone, whereas gC1q-R was localized in the cytoplasm in COS-7 cells that expressed gC1q-R alone; however, in cells that co-expressed α1B-adrenergic receptors and gC1q-R, most of the α1B-adrenergic receptors were co-localized with gC1q-R in the intracellular region, and a remarkable down-regulation of receptor expression was observed. These observations suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in regulating the cellular localization and expression of the α1B-adrenergic receptors. G protein-coupled receptors interact with several classes of cytoplasmic proteins including heterotrimeric G proteins, kinases, phosphatases, and arrestins, and the binding of cytoplasmic protein with the receptor regulates receptor signaling (1Sterne M.R. Benovic J.L. Vitam. Horm. 1995; 51: 193-234Crossref PubMed Scopus (113) Google Scholar, 2Fraser C. Lee N. Pellegrino S. Kerlavage A. Prog. Nucleic Acid Res. Mol. Biol. 1994; 49: 113-156Crossref PubMed Scopus (26) Google Scholar, 3Hein L. Kobilka B.K. Neuropharmacology. 1995; 34: 357-366Crossref PubMed Scopus (105) Google Scholar, 4Kobilka B. Annu. Rev. Neurosci. 1992; 15: 87-114Crossref PubMed Scopus (313) Google Scholar). These interactions were first inferred from the functional effects of cytoplasmic proteins on receptor signaling and desensitization and were later confirmed by biochemical observation of the binding of the protein with receptor (5Goodman O.J. 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 (1154) Google Scholar, 6Gurevich 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 (334) Google Scholar, 7Sohlemann P. Hekman M. Puzicha M. Buchen C. Lohse M.J. Eur. J. Biochem. 1995; 232: 464-472Crossref PubMed Scopus (41) Google Scholar, 8Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Very recently, however, several unexpected interactions between cytoplasmic proteins and receptors have been observed; for instance, the adrenergic receptor interacts with the α-subunit of the eukaryotic initiation factor 2B (9Klein U. Ramirez M.T. Kobilka B.K. von Zastrow M. J. Biol. Chem. 1997; 272: 19099-19102Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and with the Na+/H+-exchange regulatory factor (10Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (520) Google Scholar). These raise the possibility that receptors may interact with other types of cellular proteins that could play unanticipated roles in regulating the function of the receptor.We conducted a search for novel proteins that interact with the α1B-adrenergic receptor, specifically focusing on the carboxyl-terminal cytoplasmic domain, because mutations within this domain have pleiotropic effects on receptor physiology (11Valiquette M. Bonin H. Hnatowich M. Caron M.G. Lefkowitz R.J. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5089-5093Crossref PubMed Scopus (88) Google Scholar, 12Campbell P.T. Hnatowich M. O'Dowd B.F. Caron M.G. Lefkowitz R.J. Hausdorff W.P. Mol. Pharmacol. 1991; 39: 192-198PubMed Google Scholar, 13Lattion A.L. Diviani D. Cotecchia S. J. Biol. Chem. 1994; 269: 22887-22893Abstract Full Text PDF PubMed Google Scholar, 14Parker E.M. Swigart P. Nunnally M.H. Perkins J.P. Ross E.M. J. Biol. Chem. 1995; 270: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R 1The abbreviations used are: gC1q-R, receptor for globular “Heads” of c1q; PCR, polymerase chain reaction; GFP, green fluorescent protein; HA, hemagluttinin; AR, adrenergic receptor; HEAT, (2-β-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone1The abbreviations used are: gC1q-R, receptor for globular “Heads” of c1q; PCR, polymerase chain reaction; GFP, green fluorescent protein; HA, hemagluttinin; AR, adrenergic receptor; HEAT, (2-β-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone interacts with α1B-adrenergic receptors in vitro and in vivo through the specific site and that in cells that co-express α1B-adrenergic receptors and gC1q-R, the subcellular localization of α1B-adrenergic receptors is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as well as the function of α1B-adrenergic receptors.RESULTS AND DISCUSSIONThe yeast two-hybrid system (17Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4822) Google Scholar, 18Chien C.T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1222) Google Scholar) was used to identify candidate cellular proteins that interact with the carboxyl-terminal cytoplasmic tail of the hamster α1B-adrenergic receptor. The screening of approximately 2 × 107 transformants resulted in the isolation of eleven independent clones that interacted specifically with the carboxyl-terminal cytoplasmic tail of the α1B-adrenergic receptor. Sequence analysis revealed that all eleven clones encoded the same polypeptide, gC1q-R (EBI data base AJ001102) (19Ghebrehiwet B. Lim B.L. Peerschke E.I. Willis A.C. Reid K.B. J. Exp. Med. 1994; 179: 1809-1821Crossref PubMed Scopus (313) Google Scholar, 20Ghebrehiwet B. Peerschke E. Immunobiology. 1998; 199: 225-238Crossref PubMed Scopus (74) Google Scholar); two clones encoded the full-length gC1q-R, whereas the remaining nine clones encoded gC1q-R from the 26th residue to beyond the stop codon. The gC1q-R open reading frame encodes a prepro-protein of 279 amino acid residues (Fig.1 A). The mature protein is preceded by a 13-residue-long leader peptide, which probably contains the signal peptide. The precise function of the 57 residues immediately preceding the mature protein has not been determined, but is predicted to play a role in cellular translocation. The mature protein is presumed to be generated by site-specific cleavage and removal during post-translational processing. We, therefore, constructed the prepro form of gC1q-R (gC1qR), the amino terminus fragment cleaved from prepro gC1q-R (gC1qR1), and the mature form of gC1q-R (gC1qR71) by PCR and compared the binding of each construct with the carboxyl-terminal cytoplasmic tail of the α1B-adrenergic receptor in the yeast two-hybrid assay. As shown in Fig. 1 B, both the prepro form and mature form of gC1q-R, but not the amino-terminal fragment, were found to interact with the α1B-adrenergic receptor; however, the prepro form of gC1q-R (gC1qR) interacted with the α1B-adrenergic receptor to a much lesser degree than the mature form of gC1q-R (gC1qR71).We next examined the interaction between the α1B-adrenergic receptor and gC1q-R in vivo. Both gC1qR and gC1qR71 were epitope-tagged with HA at the carboxyl terminus (gC1qR/HA and gC1qR71/HA), and the α1B-adrenergic receptor was transiently co-expressed with either gC1qR/HA or gC1qR71/HA in COS-7 cells. The cell lysate was then immunoprecipitated with the anti-α1B-adrenergic receptor polyclonal antibody and subjected to Western blot analysis with anti-HA antibody (Fig. 2). Anti-α1B-adrenergic receptor antibody coprecipitated gC1qR and gC1qR71 only in the cells that co-expressed the α1B-adrenergic receptor, demonstrating in vivothat the α1B-adrenergic receptor interacts with gC1q-R (Fig. 2). Furthermore, in the cells transfected with the full-length gC1q-R cDNA and in the cells transfected with gC1qR71, a 32-kDa protein was detected, indicating that the HA-tagged prepro-gC1q-R was efficiently processed to the mature form in the COS-7 cells (Fig. 2). Taken together with the results from the yeast two-hybrid assay (Fig.1) and immunoprecipitation analysis (Fig. 2), the mature form of gC1q-R (gC1qR71) rather than prepro-gC1q-R (gC1qR) is considered to bind more efficiently with the α1B-adrenergic receptor. Therefore, in the following experiments, we examined the interaction of gC1qR71 with the α1B-adrenergic receptor.Figure 2Interaction of the α1B-adrenergic receptor with gC1qR.COS-7 cells were transfected with the expression vector gC1qR (lane 1 and 2) or gC1qR71 (lanes 3–6) with (lanes 1-4) or without α1B-adrenergic receptor (lanes 5 and6). Forty-eight hours after transfection, the cells were harvested and lysed. Extracts were immunoprecipitated with (lanes 1, 3, and 5) or without (lanes 2, 4, and 6) anti-α1B-adrenergic receptor polyclonal antibody at 4 °C overnight. The samples were then subjected to SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with anti-HA monoclonal antibody. The position of the molecular mass standard in kDa is shown on theleft. AR, adrenergic receptor.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Furthermore, to search the site of interaction within the carboxyl-terminal cytoplasmic tail of the α1B-adrenergic receptor (α1B-AR), which interacts with gC1qR71, we constructed a series of deletion mutants of the carboxyl-terminal cytoplasmic tail of the α1B-adrenergic receptor. As shown in Fig. 3 A, the carboxyl-terminal cytoplasmic tail of the hamster α1B-AR344–516 (amino acids 344–516) that we had used as a bait contains the NPXXY motif at amino acids 344–348, which is highly conserved among G protein-coupled receptors, the putative acidic/dihydrophobic sequence motif (amino acids 349–364), which was recently shown to mediate cell surface delivery of a vasopressin receptor (21Schulein R. Hermosilla R. Oksche A. Dehe M. Wiesner B. Krause G. Rosenthal W. Mol. Pharmacol. 1998; 54: 525-535Crossref PubMed Scopus (133) Google Scholar), and the arginine-rich region (amino acids 371–378). Thus, we constructed thirteen truncated forms of the α1B-adrenergic receptor carboxyl-terminal tail as shown in Fig. 3 A and compared the binding of each construct with gC1qR71 in the yeast two-hybrid assay. As shown in Fig. 3 B, gC1qR71 could bind with the α1B-adrenergic receptor carboxyl-terminal tail that contains the arginine-rich region.Figure 3Two-hybrid assay of gC1qR with carboxyl-terminal truncated α1B-adrenergic receptor. A, constructs of fourteen carboxyl-terminal truncated α1B-adrenergic receptors (numbers denote amino acid residues of the α1B-adrenergic receptor primary sequence). B, each α1B-adrenergic receptor indicated in A and gC1qR71 were expressed in the yeast SFY526 strain as fusion proteins with an amino-terminal GAL4 activation domain and GAL4 DNA binding domain, respectively. After incubation, the level of β-galactosidase activity of three colonies was analyzed employing a colony-lift filter assay using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside as the substrate. AR, adrenergic receptor.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, we examined the subcellular localization of the α1B-adrenergic receptor (FLAG-tagged α1B-adrenergic receptor) using a fluorescent antibody, as well as the endogenous fluorescence (gC1qR71/GFP) by fluorescent confocal microscopy. As seen in Fig. 4(A, C, E, H, andK) immunocytochemical analysis of cells transiently transfected with only FLAG-tagged α1B-adrenergic receptor showed that the fluorescence distribution was typical of that of a plasma membrane-labeling pattern (Fig. 4 A, red by Cy3). The fluorescence distribution of gC1qR71/GFP in cells transiently transfected with only gC1qR71/GFP was characteristic of that of a cytoplasmic distribution (enhanced perinuclear fluorescence) (Fig. 4,middle, green by GFP). In COS-7 cells transfected with both gC1qR71/GFP and FLAG-tagged α1B-adrenergic receptor, a marked change in the subcellular localization of α1B-adrenergic receptor was observed; the α1B-adrenergic receptor was co-localized with gC1qR71/GFP in the cytoplasm (Fig. 4 G). Furthermore, a remarkable decrease in the fluorescent signal of FLAG-tagged α1B-adrenergic receptor was observed in these cells, compared with that in the cells expressing only FLAG-tagged α1B-adrenergic receptor (Fig. 4, E andG). Further, as shown in Fig. 4, K-M, in COS-7 cells transfected with both gC1qR71/GFP and FLAG/α1B-T378-adrenergic receptor, a similar change in cellular localization of the FLAG-tagged α1B-adrenergic receptor as seen in COS-7 cells transfected with both gC1qR71/GFP and the FLAG-tagged α1B-adrenergic receptor (Fig.4 G) was observed; however, the FLAG/α1B-T368-adrenergic receptor, which lacked the amino acid region 379–516, was found not to be co-localized with gC1qR71/GFP (Fig. 4, H-J), confirming the results of the yeast two-hybrid assays that gC1qR71 could interact with the α1B-adrenergic receptor carboxyl-terminal tail that contains the arginine-rich region.Figure 4Co-localization of gC1qR71 with α1B-adrenergic receptors. COS-7 cells were transfected with the carboxyl-terminal FLAG-tagged full-length α1B-adrenergic receptor (α1B-adrenergic receptor/FLAG) alone (A andB), gC1qR71/GFP alone (C and D), α1B-adrenergic receptor/FLAG and gC1qR71/GFP (E-G), FLAG/α1B-T368-adrenergic receptor and gC1qR71/GFP (H-J), or FLAG/α1B-T378-adrenergic receptor and gC1qR71/GFP (K-M), and immunostaining and fluorescence microscopy were carried out as described under “Experimental Procedures.” Cells transfected with the α1B-adrenergic receptor/FLAG were incubated with mouse anti-FLAG monoclonal antibody, stained with Cy-3-labeled anti-mouse antibody, and observed using the redfluorescence channel (A, C, E, H, and K). GFP fluorescence was observed using the green fluorescence channel to detect gC1qR71/GFP (B, D, F, I, andL). In cells transfected with α1B-adrenergic receptor/FLAG alone, the α1B-adrenergic receptor was distributed over the cell surface (A and E), whereas in cells transfected with gC1qR71 alone, gC1qR71 was localized throughout the cytoplasm (D and F). In cells co-transfected with α1B-adrenergic receptor/FLAG and gC1qR71/GFP or FLAG/α1B-T378-adrenergic receptor and gC1qR71/GFP, α1B-adrenergic receptors were co-localized with gC1qR71 throughout the cytoplasm, as shown in yellow in the two-color merged image (G and M); however, in cells co-transfected with FLAG/α1B-T368-adrenergic receptor and gC1qR71/GFP, α1B-adrenergic receptors were found not to be co-localized with gC1qR71 (J). Thearrows indicate the cells co-expressing the α1B-adrenergic receptor/FLAG and gC1qR71-GFP. Scale bar, 10 μm. AR, adrenergic receptor.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Using the carboxyl-terminal FLAG/GFP-tagged α1B-adrenergic receptor (α1B-adrenergic receptor/GFP), we further examined the effect that gC1qR71 has on the subcellular localization of the α1B-adrenergic receptor upon co-transfection. GFP fusion in this manner does not perturb normal ligand binding nor the subcellular localization of α1B-adrenergic receptor (22Awaji T. Hirasawa A. Kataoka M. Shinoura H. Nakayama Y. Sugawara T. Izumi S. Tsujimoto G. Mol. Endocrinol. 1998; 12: 1099-1111PubMed Google Scholar). Flow cytometry analysis of GFP fluorescence enables us to detect the α1B-adrenergic receptors. Cells transfected with the α1B-adrenergic receptor/GFP and gC1qR71/HA were examined by flow cytometry. In cells transfected with the α1B-adrenergic receptor/GFP alone and in cells co-transfected with the α1B-adrenergic receptor/GFP and gC1qR71/HA, approximately 10–20% of the COS-7 cells were positively detected as having GFP-associated fluorescence, indicating successful transfection with α1B-adrenergic receptor/GFP (Fig. 5 A). However, as shown in Fig. 5 A, the GFP fluorescence of cells, which co-expressed the α1B-adrenergic receptor/GFP and gC1qR71, was significantly lower than the GFP fluorescence of cells, which expressed only α1B-adrenergic receptor/GFP. The mean value of the fluorescence intensity of GFP in cells co-expressing α1B-adrenergic receptor/GFP and gC1qR71 was 87% lower than that in the cells expressing only the α1B-adrenergic receptor-GFP (Fig. 5 B). Additionally, the mean value of the fluorescence intensity of α1B-adrenergic receptor-GFP in cells that co-expressed β-galactosidase as a negative control did not differ significantly from that of cells expressing α1B-adrenergic receptor/GFP alone (Fig.5 B).To further assess the effect that gC1qR71 has on the level of expression of the α1B-adrenergic receptor upon co-transfection, a radioligand binding assay was performed on COS-7 cells transiently expressing the α1B-adrenergic receptor with or without gC1qR71. Membrane preparations of these cells were used to determine the saturation binding isotherms for125I-labeled HEAT (Table I). Although the K d value was not significantly altered by co-expression of α1B-adrenergic receptor with gC1qR71, the B max value of the α1B-adrenergic receptor dramatically decreased when co-transfected with gC1qR71 (Table I). These results are in agreement with the results from the flow cytometry analysis in that the level of expression of α1B-adrenergic receptors on the cell surface is lower in the cells co-expressing gC1qR71. Co-expression of β-galactosidase did not significantly affect the125I-labeled HEAT binding site (Table I).Table IEffect of gC1q-R on α1B-adrenergic receptor bindingSaturation isothermsα1B-ARα1B-AR/β-galactosidaseα1B-AR/gC1qR71B max (pmol/mg protein)5.3 ± 1.24.3 ± 1.50.7 ± 2.6K d (pm)61.3 ± 1350 ± 23.230 ± 21.8COS-7 cells were transfected with α1B-adrenergic receptor alone or co-transfected with α1B-adrenergic receptor and either gC1qR71 or β-galactosidase. Forty-eight hours after transfection, the cells were harvested, and membranes prepared from the COS-7 cells were exposed to increasing concentrations of the radiolabeled antagonist 125I-labeled HEAT (range, 0–500 pm). Each value in the table represents the mean ± S.D. of three independent experiments. Open table in a new tab In this study, we identified a novel cellular protein that interacts with the α1B-adrenergic receptor, gC1q-R. Expression studies indicated that gC1q-R regulates the expression level and cellular localization of the α1B-adrenergic receptor through its carboxyl terminus. gC1q-R was previously identified as a protein that binds to the globular heads of C1q. Recent accumulating evidence suggests that gC1q-R is a multiligand-binding, multifunctional protein with affinity for diverse ligands including thrombin, vitronectin, and high molecular weight kininogen (23Lim B.L. Reid K.B.M. Ghebrehiwet B. Peerschke E.I.B. Leigh L.A.E. Preissner K.T. J. Biol. Chem. 1996; 271: 26739-26744Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 24Herwald H. Dedio J. Kellner R. Loos M. Muller-Esterl W. J. Biol. Chem. 1996; 271: 13040-13047Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Moreover, the gC1q-R molecule was found to be identical with the splicing factor SF-2 and with a protein that interacts with the human immunodeficiency virus, type I Tat transactivator designated the Tat-associated protein or TAP (25Krainer A.R. Mayeda A. Kozak D. Binns G. Cell. 1991; 66: 383-394Abstract Full Text PDF PubMed Scopus (411) Google Scholar, 26Yu L. Zhang Z. Loewenstein P.M. Desai K. Tang Q. Mao D. Symington J.S. Green M. J. Virol. 1995; 69: 3007-3016Crossref PubMed Google Scholar); however, the biological function of gC1q-R has not been clearly defined. Our present results suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in the regulation of the cellular localization and expression of the α1B-adrenergic receptor.The carboxyl-terminal cytoplasmic region of the adrenergic receptor has a pleiotropic function, because mutations within this region affect receptor physiology (11Valiquette M. Bonin H. Hnatowich M. Caron M.G. Lefkowitz R.J. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5089-5093Crossref PubMed Scopus (88) Google Scholar, 12Campbell P.T. Hnatowich M. O'Dowd B.F. Caron M.G. Lefkowitz R.J. Hausdorff W.P. Mol. Pharmacol. 1991; 39: 192-198PubMed Google Scholar, 13Lattion A.L. Diviani D. Cotecchia S. J. Biol. Chem. 1994; 269: 22887-22893Abstract Full Text PDF PubMed Google Scholar, 14Parker E.M. Swigart P. Nunnally M.H. Perkins J.P. Ross E.M. J. Biol. Chem. 1995; 270: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and because several domains within this region have been shown to interact with several classes of cytoplasmic proteins. One domain that is conserved in both α2- and β2-adrenergic receptors, the carboxyl-terminal DFRXXFXXXL motif, interacts with the α-subunit of the eukaryotic initiation factor 2B (9Klein U. Ramirez M.T. Kobilka B.K. von Zastrow M. J. Biol. Chem. 1997; 272: 19099-19102Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). This protein interaction enhances the β2-adrenergic receptor-mediated activation of adenylyl cyclase (9Klein U. Ramirez M.T. Kobilka B.K. von Zastrow M. J. Biol. Chem. 1997; 272: 19099-19102Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The glutamate/dileucine sequence motif conserved in many G protein-coupled receptors is involved in the cell surface transport of receptors (21Schulein R. Hermosilla R. Oksche A. Dehe M. Wiesner B. Krause G. Rosenthal W. Mol. Pharmacol. 1998; 54: 525-535Crossref PubMed Scopus (133) Google Scholar). Also, the carboxyl terminus of the β2-adrenergic receptor interacts with the Na+/H+ exchange regulatory factor family of PDZ proteins (10Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (520) Google Scholar). Our study demonstrated that gC1q-R binds with the carboxyl tail of the α1B-adrenergic receptor at the arginine-rich region, which differs from the known domains described above, and that gC1q-R regulates the cellular localization and expression of the α1B-adrenergic receptor through their interaction. Further studies to clarify the functional significance of this protein interaction in the regulation of α1B-adrenergic receptor signaling would be of value. G protein-coupled receptors interact with several classes of cytoplasmic proteins including heterotrimeric G proteins, kinases, phosphatases, and arrestins, and the binding of cytoplasmic protein with the receptor regulates receptor signaling (1Sterne M.R. Benovic J.L. Vitam. Horm. 1995; 51: 193-234Crossref PubMed Scopus (113) Google Scholar, 2Fraser C. Lee N. Pellegrino S. Kerlavage A. Prog. Nucleic Acid Res. Mol. Biol. 1994; 49: 113-156Crossref PubMed Scopus (26) Google Scholar, 3Hein L. Kobilka B.K. Neuropharmacology. 1995; 34: 357-366Crossref PubMed Scopus (105) Google Scholar, 4Kobilka B. Annu. Rev. Neurosci. 1992; 15: 87-114Crossref PubMed Scopus (313) Google Scholar). These interactions were first inferred from the functional effects of cytoplasmic proteins on receptor signaling and desensitization and were later confirmed by biochemical observation of the binding of the protein with receptor (5Goodman O.J. 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 (1154) Google Scholar, 6Gurevich 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 (334) Google Scholar, 7Sohlemann P. Hekman M. Puzicha M. Buchen C. Lohse M.J. Eur. J. Biochem. 1995; 232: 464-472Crossref PubMed Scopus (41) Google Scholar, 8Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Very recently, however, several unexpected interactions between cytoplasmic proteins and receptors have been observed; for instance, the adrenergic receptor interacts with the α-subunit of the eukaryotic initiation factor 2B (9Klein U. Ramirez M.T. Kobilka B.K. von Zastrow M. J. Biol. Chem. 1997; 272: 19099-19102Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and with the Na+/H+-exchange regulatory factor (10Hall R.A. Premont R.T. Chow C.W. Blitzer J.T. Pitcher J.A. Claing A. Stoffel R.H. Barak L.S. Shenolikar S. Weinman E.J. Grinstein S. Lefkowitz R.J. Nature. 1998; 392: 626-630Crossref PubMed Scopus (520) Google Scholar). These raise the possibility that receptors may interact with other types of cellular proteins that could play unanticipated roles in regulating the function of the receptor. We conducted a search for novel proteins that interact with the α1B-adrenergic receptor, specifically focusing on the carboxyl-terminal cytoplasmic domain, because mutations within this domain have pleiotropic effects on receptor physiology (11Valiquette M. Bonin H. Hnatowich M. Caron M.G. Lefkowitz R.J. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5089-5093Crossref PubMed Scopus (88) Google Scholar, 12Campbell P.T. Hnatowich M. O'Dowd B.F. Caron M.G. Lefkowitz R.J. Hausdorff W.P. Mol. Pharmacol. 1991; 39: 192-198PubMed Google Scholar, 13Lattion A.L. Diviani D. Cotecchia S. J. Biol. Chem. 1994; 269: 22887-22893Abstract Full Text PDF PubMed Google Scholar, 14Parker E.M. Swigart P. Nunnally M.H. Perkins J.P. Ross E.M. J. Biol. Chem. 1995; 270: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R 1The abbreviations used are: gC1q-R, receptor for globular “Heads” of c1q; PCR, polymerase chain reaction; GFP, green fluorescent protein; HA, hemagluttinin; AR, adrenergic receptor; HEAT, (2-β-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone1The abbreviations used are: gC1q-R, receptor for globular “Heads” of c1q; PCR, polymerase chain reaction; GFP, green fluorescent protein; HA, hemagluttinin; AR, adrenergic receptor; HEAT, (2-β-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone interacts with α1B-adrenergic receptors in vitro and in vivo through the specific site and that in cells that co-express α1B-adrenergic receptors and gC1q-R, the subcellular localization of α1B-adrenergic receptors is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as well as the function of α1B-adrenergic receptors. RESULTS AND DISCUSSIONThe yeast two-hybrid system (17Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4822) Google Scholar, 18Chien C.T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1222) Google Scholar) was used to identify candidate cellular proteins that interact with the carboxyl-terminal cytoplasmic tail of the hamster α1B-adrenergic receptor. The screening of approximately 2 × 107 transformants resulted in the isolation of eleven independent clones that interacted specific
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