Interaction of the α1B-Adrenergic Receptor with gC1q-R, a Multifunctional Protein*

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 (1)(2)(3)(4). 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 (5)(6)(7)(8). Very recently, however, several unexpected interactions between cytoplasmic proteins and receptors have been observed; for instance, the adrenergic receptor interacts with the ␣-subunit of the eukaryotic initiation factor 2B (9) and with the Na ϩ /H ϩexchange regulatory factor (10). 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 ␣ 1B -adrenergic receptor, specifically focusing on the carboxyl-terminal cytoplasmic domain, because mutations within this domain have pleiotropic effects on receptor physiology (11)(12)(13)(14). Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R 1 interacts with ␣ 1B -adrenergic receptors in vitro and in vivo through the specific site and that in cells that co-express ␣ 1B -adrenergic receptors and gC1q-R, the subcellular localization of ␣ 1B -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 ␣ 1B -adrenergic receptors.

EXPERIMENTAL PROCEDURES
Yeast Two-hybrid Cloning-All components were purchased from CLONTECH, and all assays were carried out as suggested by the manufacturer. The Matchmaker TM two-hybrid system was used to screen a rat liver cDNA library (complexity ϳ3 ϫ 10 6 total recombinants) constructed in pGAD10 (CLONTECH) with the carboxyl-terminal cytoplasmic domain of the hamster ␣ 1B -adrenergic receptor. Bait plasmids were constructed in pGBT9 (CLONTECH) using the carboxylterminal cytoplasmic domain of the hamster ␣ 1B -adrenergic receptor, starting from the "NPXXY-motif" in the seventh transmembrane domain (e.g. amino acids 344 -516 of the ␣ 1B -adrenergic receptor, Gen-Bank TM /EBI data base accession number J04084) followed by amplification with polymerase chain reaction (PCR). Screening of ϳ2 ϫ 10 7 transformants yielded eleven independent clones that interacted with the ␣ 1B -adrenergic receptor tail but not with the murine p53 control (CLONTECH protocol). The sequences of the eleven clones were identical with that of rat gC1q-R (EBI data base AJ001102).
Co-immunoprecipitation and Western Blotting-To construct the carboxyl-terminal FLAG-tagged ␣ 1B -adrenergic receptor, the stop codon of the sequence that encodes the ␣ 1B -adrenergic receptor was altered to a KpnI site by site-directed mutagenesis PCR; a FLAG fragment was cloned at the carboxyl-terminal KpnI site of the ␣ 1B -adrenergic receptor. This was subsequently subcloned into the mammalian expression vector pME18s. Carboxyl-terminal HA epitope-tagged versions of the full-length (gC1qR/HA) and amino-terminal truncated (gC1qR71/HA) forms of gC1q-R were constructed by PCR and each was subcloned into pME18s. Each of these constructs was co-expressed with a hamster ␣ 1B -adrenergic receptor in COS-7 cells by transient transfection using the electroporation method. Forty-eight hours after transfection, cells were harvested and lysed in 10 mM Tris-HCl (pH 8.0), 1% Nonidet P-40, 0.15 M NaCl, 1 mM EDTA, and protease inhibitors phenylmethanesulfonyl fluoride and pepstatin A. The receptors were immunoprecipitated by adding rabbit anti-␣ 1B -adrenergic receptor polyclonal antibody (15) and protein A-Sepharose (Amersham Pharmacia Biotech). The samples were then subjected to SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with anti-HA monoclonal antibody (Roche Molecular Biochemicals). Epitopetagged gC1q-R was detected using horseradish peroxidase-conjugated sheep anti-mouse secondary antibody (Amersham Pharmacia Biotech) * This work was supported in part by research grants from the Scientific Fund of the Ministry of Education, Science, and Culture of Japan, the Japan Health Science Foundation, and the Ministry of Human Health and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Confocal Laser Scanning Microscope Analysis-Carboxyl-terminal green fluorescent protein (GFP)-tagged gC1qR71/GFP was constructed and subcloned into the pEGFP-N3 vector (CLONTECH). Either a carboxyl-terminal FLAG-tagged full-length ␣ 1B -adrenergic receptor (␣ 1Badrenergic receptor/FLAG), an amino-terminal FLAG-tagged truncated ␣ 1B -adrenergic receptor, which lacked the amino acids 369 -516 (FLAG/ ␣ 1B -T368-adrenergic receptor), or an amino-terminal FLAG-tagged truncated ␣ 1B -adrenergic receptor, which lacked the amino acids 379 -516 (FLAG/␣ 1B -T378-adrenergic receptor), was constructed and then subcloned into the mammalian expression vector pME18s. COS-7 cells expressing ␣ 1B -adrenergic receptor/FLAG, FLAG/␣ 1B -T368-adrenergic receptor, or FLAG/␣ 1B -T378-adrenergic receptor and gC1qR71/GFP were seeded at 1 ϫ 10 5 /well in an 8-well Lab-tek chamber slide (Nunc, Naperville, IL) in 0.5 ml of medium. Fixation was performed by placing the slide in 80% acetone for 5 min. The cells were then incubated with 0.05% Triton X-100 in phosphate-buffered saline. The primary antibody, 10 g/ml of anti-FLAG monoclonal antibody (Eastman Kodak) in phosphate-buffered saline containing 10% bovine serum albumin and 0.05% Triton X-100, was applied to the cells, which were subsequently placed in a humidified chamber for 1 h at room temperature. Cy3conjugated donkey anti-mouse IgG (Chemicon International, Temecura, CA) was diluted 1/200 in phosphate-buffered saline containing 10% bovine serum albumin and 0.05% Triton X-100 and applied to the cells for 1 h at room temperature. The cells were then washed twice with phosphate-buffered saline, and coverslips were applied using Gel/ Mount (Biomeda, Foster City, CA). After immunocytochemical staining, the cells were examined using the LSM-GB200 Laser Scanning Microscope (Olympus, Tokyo, Japan) with the argon-ion laser set at 514 nm for excitation of Cy3 and 488 nm for excitation of GFP.
Flow Cytometry Analysis-To construct the carboxyl-terminal FLAG/ GFP-tagged ␣ 1B -adrenergic receptor, the stop codon of the sequence that encodes the ␣ 1B -adrenergic receptor was altered to a KpnI site by site-directed mutagenesis PCR, and a FLAG/GFP fragment was cloned at the carboxyl-terminal KpnI site of the ␣ 1B -adrenergic receptor. This was subsequently subcloned into the mammalian expression vector pME18s. To construct ␤-galactosidase/pME18s, the full-length fragment of ␤-galactosidase from the plasmid pCMV⅐ SPORT ␤-gal (Life Technologies, Inc.) was inserted into the vector pME18s. ␣ 1B -Adrenergic receptor-FLAG/GFP and either the gC1qR71 or ␤-galactosidase expression construct were co-transfected into COS-7 cells by the electroporation method using Cell-Porator (Life Technologies, Inc.) according to the manufacturer's instructions, and the cells were assayed. Forty-eight hours after transfection, the cells were analyzed with FAC-Scan flow cytometry for excitation of GFP (Becton Dickinson, Mountain View, CA) as described previously (16). Routinely, data from fluorescence of 10 4 cells were subjected to two-dimensional dot-plot analysis with FL-1 and FL-3 (Fig. 5A); GFP-positive cells were further gated, and the average value of FL-1 fluorescence intensities of these cells was calculated by the Cell Quest software (Becton Dickinson, Mountain View, CA). 125 I-labeled HEAT Binding Assay-Membrane preparation of the cells was performed as described previously (15). Briefly, cells were collected and disrupted by a sonicator (Model SONIFIER 250, setting 5 for 8 s) in ice-cold buffer A (250 mM sucrose, 5 mM Tris-HCl, 1 mM MgCl 2 , pH 7.4) and then centrifuged at 3000 ϫ g at 4°C for 10 min to remove the nuclei. The supernatant fraction was centrifuged at 125,000 ϫ g for 30 min at 4°C. The resulting pellet was resuspended in binding buffer B (50 mM Tris-HCl, 12.5 mM MgCl 2 , 10 mM EGTA, pH 7.4) and was frozen at Ϫ80°C until assay.
The 125 I-labeled HEAT binding assay was performed as described previously (15). Briefly, membrane aliquots (ϳ1 g of protein) were incubated with various concentrations of 125 I-labeled HEAT in a final volume of 150 l of binding buffer for 60 min at 25°C. The incubation was terminated by adding ice-cold buffer B, and this was immediately filtered through Whatman GF/C glass fiber filters with a Brandel cell harvester (Model-30) (Gaithersburg, MD). Each filter was collected, and the radioactivity was measured. Binding assays were always performed in duplicate, and specific 125 I-labeled HEAT binding was experimentally determined by calculating the difference between counts in the absence and presence of 10 M phentolamine. B max and K d values were obtained by fitting rectangular hyperbolic functions to the experimental data using computer-assisted iterative nonlinear regression analysis. The protein concentration was measured using the BCA Protein Assay Kit (Pierce). Values are expressed as the mean Ϯ S.D.

RESULTS AND DISCUSSION
The yeast two-hybrid system (17,18) was used to identify candidate cellular proteins that interact with the carboxylterminal cytoplasmic tail of the hamster ␣ 1B -adrenergic receptor. The screening of approximately 2 ϫ 10 7 transformants resulted in the isolation of eleven independent clones that interacted specifically with the carboxyl-terminal cytoplasmic tail of the ␣ 1B -adrenergic receptor. Sequence analysis revealed that all eleven clones encoded the same polypeptide, gC1q-R (EBI data base AJ001102) (19,20); 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 in the yeast two-hybrid system. A, constructs of gC1q-R: the fulllength form (gC1qR), prepro form (gC1qR1), and mature form (gC1qR71) (numbers denote amino acid residues in the gC1q-R sequence). B, the cDNA that encodes the ␣ 1B -adrenergic receptor carboxyl-terminal tail was inserted into the yeast expression plasmid pGBT9. Each gC1qR fragment indicated in A was inserted into the yeast expression plasmid pGAD424 and co-expressed with the ␣ 1B -adrenergic receptor carboxyl-terminal tail in the SFY526 strain. After incubation, the level of ␤-galactosidase activity was estimated by the liquid culture method with o-nitrophenyl ␤-D-galactopyranoside as the substrate. Each value represents the mean Ϯ S.D. of three independent experiments. AR, adrenergic receptor.  6) with (lanes 1-4) or without ␣ 1B -adrenergic receptor (lanes 5 and 6). 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-␣ 1B -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 the left. AR, adrenergic receptor.
gC1q-R open reading frame encodes a prepro-protein of 279 amino acid residues (Fig. 1A). 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 carboxylterminal cytoplasmic tail of the ␣ 1B -adrenergic receptor in the yeast two-hybrid assay. As shown in Fig. 1B, both the prepro form and mature form of gC1q-R, but not the amino-terminal fragment, were found to interact with the ␣ 1B -adrenergic receptor; however, the prepro form of gC1q-R (gC1qR) interacted with the ␣ 1B -adrenergic receptor to a much lesser degree than the mature form of gC1q-R (gC1qR71).
We next examined the interaction between the ␣ 1B -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 ␣ 1B -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-␣ 1B -adrenergic receptor polyclonal antibody and subjected to Western blot analysis with anti-HA antibody (Fig. 2). Anti-␣ 1B -adrenergic receptor antibody coprecipitated gC1qR and gC1qR71 only in the cells that co-expressed the ␣ 1B -adrenergic receptor, demonstrating in vivo that the ␣ 1B -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 FIG. 3. Two-hybrid assay of gC1qR with carboxyl-terminal truncated ␣ 1B -adrenergic receptor. A, constructs of fourteen carboxylterminal truncated ␣ 1B -adrenergic receptors (numbers denote amino acid residues of the ␣ 1B -adrenergic receptor primary sequence). B, each ␣ 1B -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 ␤-galactosidase activity of three colonies was analyzed employing a colony-lift filter assay using 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside as the substrate. AR, adrenergic receptor. 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 ␣ 1B -adrenergic receptor. Therefore, in the following experiments, we examined the interaction of gC1qR71 with the ␣ 1B -adrenergic receptor.
Furthermore, to search the site of interaction within the carboxyl-terminal cytoplasmic tail of the ␣ 1B -adrenergic receptor (␣ 1B -AR), which interacts with gC1qR71, we constructed a series of deletion mutants of the carboxyl-terminal cytoplasmic tail of the ␣ 1B -adrenergic receptor. As shown in Fig. 3A, the carboxyl-terminal cytoplasmic tail of the hamster ␣ 1B -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 (21), and the arginine-rich region (amino acids 371-378). Thus, we constructed thirteen truncated forms of the ␣ 1B -adrenergic receptor carboxyl-terminal tail as shown in Fig. 3A and compared the binding of each construct with gC1qR71 in the yeast two-hybrid assay. As shown in Fig. 3B, gC1qR71 could bind with the ␣ 1B -adrenergic receptor carboxylterminal tail that contains the arginine-rich region.
Next, we examined the subcellular localization of the ␣ 1Badrenergic receptor (FLAG-tagged ␣ 1B -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, and K) immunocytochemical analysis of cells transiently transfected with only FLAG-tagged ␣ 1Badrenergic receptor showed that the fluorescence distribution was typical of that of a plasma membrane-labeling pattern , and immunostaining and fluorescence microscopy were carried out as described under "Experimental Procedures." Cells transfected with the ␣ 1B -adrenergic receptor/FLAG were incubated with mouse anti-FLAG monoclonal antibody, stained with Cy-3-labeled anti-mouse antibody, and observed using the red fluorescence channel (A, C, E, H, and K). GFP fluorescence was observed using the green fluorescence channel to detect gC1qR71/GFP (B, D, F, I, and L). In cells transfected with ␣ 1B -adrenergic receptor/FLAG alone, the ␣ 1B -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 ␣ 1B -adrenergic receptor/FLAG and gC1qR71/GFP or FLAG/␣ 1B -T378-adrenergic receptor and gC1qR71/GFP, ␣ 1B -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/␣ 1B -T368-adrenergic receptor and gC1qR71/GFP, ␣ 1B -adrenergic receptors were found not to be co-localized with gC1qR71 (J). The arrows indicate the cells co-expressing the ␣ 1B -adrenergic receptor/FLAG and gC1qR71-GFP. Scale bar, 10 m. AR, adrenergic receptor. (Fig. 4A, 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 ␣ 1B -adrenergic receptor, a marked change in the subcellular localization of ␣ 1B -adrenergic receptor was observed; the ␣ 1B -adrenergic receptor was co-localized with gC1qR71/GFP in the cytoplasm (Fig. 4G). Furthermore, a remarkable decrease in the fluorescent signal of FLAG-tagged ␣ 1B -adrenergic receptor was observed in these cells, compared with that in the cells expressing only FLAG-tagged ␣ 1B -adrenergic receptor (Fig. 4, E and G). Further, as shown in Fig. 4, K-M, in COS-7 cells transfected with both gC1qR71/GFP and FLAG/␣ 1B -T378-adrenergic receptor, a similar change in cellular localization of the FLAG-tagged ␣ 1B -adrenergic receptor as seen in COS-7 cells transfected with both gC1qR71/GFP and the FLAG-tagged ␣ 1B -adrenergic receptor (Fig. 4G) was observed; however, the FLAG/␣ 1B -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 ␣ 1B -adrenergic receptor carboxyl-terminal tail that contains the arginine-rich region.
Using the carboxyl-terminal FLAG/GFP-tagged ␣ 1B -adrenergic receptor (␣ 1B -adrenergic receptor/GFP), we further examined the effect that gC1qR71 has on the subcellular localization of the ␣ 1B -adrenergic receptor upon co-transfection. GFP fusion in this manner does not perturb normal ligand binding nor the subcellular localization of ␣ 1B -adrenergic receptor (22). Flow cytometry analysis of GFP fluorescence enables us to detect the ␣ 1B -adrenergic receptors. Cells transfected with the ␣ 1B -adrenergic receptor/GFP and gC1qR71/HA were examined by flow cytometry. In cells transfected with the ␣ 1B -adrenergic receptor/GFP alone and in cells co-transfected with the ␣ 1B -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 ␣ 1B -adrenergic receptor/GFP (Fig.  5A). However, as shown in Fig. 5A, the GFP fluorescence of cells, which co-expressed the ␣ 1B -adrenergic receptor/GFP and gC1qR71, was significantly lower than the GFP fluorescence of cells, which expressed only ␣ 1B -adrenergic receptor/GFP. The mean value of the fluorescence intensity of GFP in cells coexpressing ␣ 1B -adrenergic receptor/GFP and gC1qR71 was 87% lower than that in the cells expressing only the ␣ 1Badrenergic receptor-GFP (Fig. 5B). Additionally, the mean value of the fluorescence intensity of ␣ 1B -adrenergic receptor-GFP in cells that co-expressed ␤-galactosidase as a negative control did not differ significantly from that of cells expressing ␣ 1B -adrenergic receptor/GFP alone (Fig. 5B).
To further assess the effect that gC1qR71 has on the level of expression of the ␣ 1B -adrenergic receptor upon co-transfection, a radioligand binding assay was performed on COS-7 cells transiently expressing the ␣ 1B -adrenergic receptor with or without gC1qR71. Membrane preparations of these cells were used to determine the saturation binding isotherms for 125 Ilabeled HEAT (Table I). Although the K d value was not significantly altered by co-expression of ␣ 1B -adrenergic receptor with gC1qR71, the B max value of the ␣ 1B -adrenergic receptor dramatically decreased when co-transfected with gC1qR71 (Table   FIG. 5. Effect of gC1q-R on ␣ 1B -adrenergic receptor expression (flow cytometry analysis). COS-7 cells were transfected with ␣ 1B -adrenergic receptor/GFP alone or co-transfected with ␣ 1B -adrenergic receptor/GFP and either gC1qR71/HA or ␤-galactosidase. Forty-eight hours after transfection, cells were harvested and analyzed by a fluorescence-activated cell sorter flow cytometer. A, results are shown as a two-dimensional dot plot of GFP fluorescence. GFPϩ, cells expressing GFP-fused ␣ 1B -adrenergic receptor; GFPϪ, cells that do not express GFP-fused ␣ 1B -adrenergic receptor. The x axis represents the relative fluorescence of GFP. B, the mean value of fluorescence intensity in each experimental condition. The values represent the mean Ϯ S.D. of at least three independent experiments. AR, adrenergic receptor.

TABLE I
Effect of gC1q-R on ␣ 1B -adrenergic receptor binding COS-7 cells were transfected with ␣ 1B -adrenergic receptor alone or co-transfected with ␣ 1B -adrenergic receptor and either gC1qR71 or ␤-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 125 I-labeled HEAT (range, 0 -500 pM). Each value in the I). These results are in agreement with the results from the flow cytometry analysis in that the level of expression of ␣ 1Badrenergic receptors on the cell surface is lower in the cells co-expressing gC1qR71. Co-expression of ␤-galactosidase did not significantly affect the 125 I-labeled HEAT binding site (Table I).
In this study, we identified a novel cellular protein that interacts with the ␣ 1B -adrenergic receptor, gC1q-R. Expression studies indicated that gC1q-R regulates the expression level and cellular localization of the ␣ 1B -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 (23,24). 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 (25,26); 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 ␣ 1B -adrenergic receptor.
The carboxyl-terminal cytoplasmic region of the adrenergic receptor has a pleiotropic function, because mutations within this region affect receptor physiology (11)(12)(13)(14) 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 ␣ 2 -and ␤ 2 -adrenergic receptors, the carboxyl-terminal DFRXXFXXXL motif, interacts with the ␣-subunit of the eukaryotic initiation factor 2B (9). This protein interaction enhances the ␤ 2 -adrenergic receptor-mediated activation of adenylyl cyclase (9). The glutamate/dileucine sequence motif conserved in many G protein-coupled receptors is involved in the cell surface transport of receptors (21). Also, the carboxyl terminus of the ␤ 2 -adrenergic receptor interacts with the Na ϩ /H ϩ exchange regulatory factor family of PDZ proteins (10). Our study demonstrated that gC1q-R binds with the carboxyl tail of the ␣ 1B -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 ␣ 1B -adrenergic receptor through their interaction.
Further studies to clarify the functional significance of this protein interaction in the regulation of ␣ 1B -adrenergic receptor signaling would be of value.