Subtype-Specific Dimerization of 1-Adrenoceptors: Effects on Receptor Expression and Pharmacological Properties

The potential role of dimerization in controlling the expression and pharmacological properties of 1-adrenoceptor subtypes was examined using coimmunoprecipitation of epitope-tagged receptors. Human 1-adrenoceptor subtypes ( 1A, 1B, 1D) were tagged at their amino-termini with Flag or hemagglutinin epitopes and transfected into human embryonic kidney 293 cells. Homodimerization of all three subtypes was observed by coimmunoprecipitation of receptors with different tags and was not altered by norepinephrine treatment. Heterodimer formation between hemagglutinin-tagged 1B-adrenoceptors and Flag-tagged 1Aor 1D-adrenoceptors was also observed. However, no 1A/ 1D-adrenoceptor heterodimers were observed, suggesting that dimerization is subtype-specific. The extent of heterodimerization was also unaltered by norepinephrine treatment. 1-Adrenoceptor truncation mutants lacking carboxyl or amino-terminal sequences formed homoand heterodimers similarly to full-length receptors, suggesting that these domains play little or no role in dimerization. Biotinylation with a membrane-impermeable agent showed that monomers and homoand hetero-oligomers of all three subtypes are expressed on the cell surface. Radioligand binding studies showed that heterodimerization did not alter the affinity of 1-adrenoceptors for norepinephrine, prazosin, or subtype-selective antagonists, suggesting that dimerization does not result in pharmacologically distinct subtypes. However, coexpression of 1B-adrenoceptors significantly increased both binding site density and protein expression of 1Aand 1Dadrenoceptors, and increased cell surface expression of 1Dadrenoceptors, suggesting a functional role for heterodimerization. Conversely, coexpression of 1A-with 1D-adrenoceptors, which did not heterodimerize, had no effect on receptor density or protein. These studies demonstrate subtype-selective heterodimerization of 1-adrenoceptors, which does not change their pharmacological properties but seems to have functional consequences in regulating receptor expression and trafficking. Dimerization of growth factor and cytokine receptors is essential for their signaling (Heldin, 1995). However, G-protein coupled receptors (GPCRs) have traditionally been thought to function as monomers, with a single receptor binding ligand and activating G-proteins. Early studies, using muscarinic/adrenergic receptor chimeras, suggested that the monomeric GPCR model may not completely explain the mechanisms of GPCR function. These studies demonstrated that two binding-deficient muscarinic/adrenergic chimeras could form a receptor capable of specific ligand binding and signaling upon coexpression. Thus, it was suggested that a direct interaction between the two chimeras could reconstitute a ligand binding pocket (Maggio et al., 1993). Strong evidence now suggests that some GPCRs, such as GABAB and taste receptors, exist as obligate heterodimers and that dimerization may alter the functional, pharmacological or regulatory properties of many other GPCRs (Bouvier, 2001; Rios et al., 2001). These observations have revolutionized contemporary views of GPCR function, raising previously unconsidered possibilities of multiple subunits, multiple binding sites, and additional subtypes that cannot be explained by existing GPCR clones expressed in isolation. GPCR dimerization may be mediated by covalent and/or noncovalent interactions, may involve extracellular, transmembrane, or intracellular domains, and, in some cases, may be altered by agonist occupancy (Bouvier, 2001; Rios et al., 2001). Strikingly, two closely related opioid receptors ( and ) heterodimerize to form receptors with distinct ligand binding profiles and functional properties (Jordan and Devi, 1999; Jordan et al., 2000). These studies suggest that dimerSupported by National Institutes of Health grants to KPM and RAH. RAH thanks the W.M. Keck Foundation for a Distinguished Young Scholar in Medical Research award. ABBREVIATIONS: GPCR, G protein coupled receptor; AR, adrenoceptor; HEK, human embryonic kidney; BMY7378, 8-[2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride; HRP, horseradish peroxidase; HA, hemagglutinin, ; ECL, enhanced chemiluminescence; D M, n-dodecyl-D-maltoside; PBS, phosphate-buffered saline; PNGase F, N-glycosidase F; TBST, Tris-buffered saline containing 0.1% Tween-20; PAGE, polyacrylamide gel electrophoresis; sulfo-LC-NHS-biotin, sulfosuccinimidyl-6-(biotinamido) hexanoate; BE 2254, 2-[ -(4-hydroxyphenyl)-ethylaminomethyl]-tetralone; IBE, I-BE 2254; NE, norepinephrine. 0026-895X/03/6406-1379–1390$7.00 MOLECULAR PHARMACOLOGY Vol. 64, No. 6 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2432/1109669 Mol Pharmacol 64:1379–1390, 2003 Printed in U.S.A. 1379 at A PE T Jornals on A ril 0, 2017 m oharm .aspeurnals.org D ow nladed from ization of GPCRs may represent a novel mechanism modulating receptor pharmacology, trafficking and/or function. 1-Adrenoceptors ( 1-ARs) are Gq/11 coupled receptors that mediate responses to the neurotransmitters and hormones norepinephrine (NE) and epinephrine. 1-ARs initiate signals by activating phospholipase C and generating second messengers that release stored intracellular Ca and stimulate protein kinase C. Three human 1-AR subtypes ( 1A, 1B, and 1D) have been cloned, each encoded by different genes (Zhong and Minneman, 1999). These subtypes are highly homologous within their transmembrane domains but share little homology at their amino and carboxyl termini (Schwinn et al., 1995). Localization studies have shown that human tissues such as brain, heart, and vascular smooth muscle express mixtures of 1-ARs (Rokosh et al., 1996; Zhong and Minneman, 1999), and homogeneous cell populations, such as rat cardiomyocytes and clonal human SKN-MC neuroepithelioma cells, have also been found to coexpress all three 1-AR subtypes (Rokosh et al., 1996; Zhong and Minneman, 1999). Furthermore, there is pharmacological evidence for additional 1-AR subtypes that cannot be explained by the existing clones (Endoh, 1996; Ford et al., 1996, 1997; Rokosh et al., 1996; Zhong and Minneman, 1999). For example, 1L-AR, a subtype that has a relatively low affinity for prazosin, has been described in functional studies but has not been identified molecularly (Ford et al., 1997; Muramatsu et al., 1998). Other studies have also indicated discrepancies between native 1-ARs and recombinant subtypes (Johnson and Minneman, 1986; Han and Minneman, 1991). The possibility that native 1-AR subtypes may form dimers could partially explain such complexity. Recent studies from our laboratory suggested that epitopetagged human 1-AR subtypes can exist as both monomers and SDS-resistant homodimers and oligomers (Vicentic et al., 2002). In this study, we used biochemical and pharmacological approaches to examine the potential role of dimerization of human 1-AR subtypes in controlling their expression and pharmacological properties. Materials and Methods Materials. The human 1A-AR cDNA (Hirasawa et al., 1995) was kindly provided by Dr. Gozoh Tsujimoto (National Children’s Hospital, Tokyo, Japan), human 1B-AR cDNA (Ramarao et al., 1992) was provided by Dr. Dianne Perez (Cleveland Clinic), and human 1D-AR was cloned in our laboratory (Esbenshade et al., 1995). Other materials were obtained from the following sources: HEK293 cells (American Type Culture Collection, Manassas, VA); fura-2/acetoxymethylester and n-dodecyl-D-maltoside (Calbiochem, La Jolla, CA); ( )norepinephrine bitartrate, BMY7378, S-( )-niguldipine, Dulbecco’s modified Eagle’s medium, penicillin, streptomycin, anti-Flag M2 affinity resin, and HRP-conjugated anti-Flag M2 antibody (Sigma Chemical Co., St. Louis, MO); anti-hemagglutinin (HA) affinity matrix (Roche, Indianapolis, IN); anti-HA tag polyclonal antibody and mammalian expression vector pCMV (BD Biosciences Clontech, Palo Alto, CA); sulfo-LC-NHS-biotin, Supersignal enzyme-linked immunosorbent assay Pico chemiluminescent substrate, and Immunopure immobilized streptavidin (Pierce, Rockford, IL); prazosin (Pfizer, Groton, CT); ECL reagent, [I]arylazidoprazosin and carrier-free Na I (Amersham, Chicago, IL). G418 (geneticin), all electrophoresis reagents, and precast 4 to 20% Tris-Glycine polyacrylamide gels were obtained from Invitrogen (Carlsbad, CA), and Superfect Transfection Reagent was from QIAGEN (Valencia, CA) . Epitope-Tagged 1-AR Constructs. Human 1-AR cDNAs were generated by polymerase chain reaction and subcloned into the mammalian expression vector pDT containing in-frame N-terminal hexahistidine and Flag epitope tags as described previously (Robeva et al., 1996; Vicentic et al., 2002). These subtypes were also subcloned into the mammalian expression vector pCMV containing an in-frame N-terminal HA tag (YPYDVPDYA). After sequencing, unique restriction enzyme sites were used to replace the Flag-hexahistidine tag in pDT with the HA-epitope tag to allow generation of stable cell lines under the selection of 400 g/ml G418. Cell Culture and Transfection. HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 10 mg/ml streptomycin, and 100 U/ml penicillin at 37°C in a humidified atmosphere with 5% CO2 (Esbenshade et al., 1993). Cells were transfected with the tagged human 1-AR subtypes by calcium phosphate precipitation (30 g/15-cm plate). Transfected cells were propagated for several weeks in the presence of 400 g/ml G418, and subclones were screened by radioligand binding for receptor expression. HA-tagged receptors were transiently transfected into a stable cell line expressing individual Flag-tagged receptors for receptor coexpression studies using 20 to 30 g of cDNA, and cells were harvested 48 to 72 h after transfection. Radioligand Binding and Ca Measurements. For radioligand binding, confluent 15-cm plates were washed with phosphatebuffered saline (PBS; 20 mM NaPO4, 154 mM NaCl, pH 7.6) and harvested by scraping. Cells were collected by centrifugation and homogenized with a Polytron homogenizer (Kinematica, Basel, Switzerland). Cell membranes were collected by centrifugation at 30,000g for 20 min and resuspended by homogenization in 1 buffer (25 mM HEPES, 150 mM NaCl, pH 7.4, 5 mM EDTA) with a protease inhibitor cocktail (1 mM benzamidine, 3 M pepstatin, 3 M phenylmethylsulfonyl fluoride, 3 M aprotinin, and 3 M leupeptin). Radioligand binding sites were measured by saturation analysis of specific binding of the 1-AR antagonist radioligand I-BE 2254 (IBE; 20–800 pM). Nonspecific binding was defined as binding in the presence of 10 M phentolamine. The pharmacological specificity of radioligand binding sites was determined by displacement of IBE (50–70 pM) by selected agonists and antagonists, and data were analyzed by nonlinear regression (Theroux et al., 1996). Intracellular Ca mobilization was measured using fura-2 as described previously (Theroux et al., 1996). Photoaffinity Labeling. Photoaffinity labeling was performed on membranes from HEK293 cells expressing Flagor HA-tagged 1-adrenoceptors. Membranes were prepared as described above for radioligand binding (1 g protein/ l), and treated in the dark for 1 h at room temperature with 6 nM [I]arylazidoprazosin (Vicentic et al., 2002). Nonspecific labeling was determined in the presence of 1 M unlabeled prazosin. While still in the dark, open tubes were exposed to 6000 J/cm ultraviolet light for 3 min using a Stratalinker. Membranes were then washed once with 1 ml of 1 buffer A with protease inhibitors, centrifuged at 16,000g for 5 min and the pellet homogenized in 0.5 ml of 2 buffer A (50 mM HEPES, 300 mM NaCl, pH 7.4) containing protease inhibitors. Radiolabeled membranes were solubilized with 2% n-dodecyl-D-maltoside (D M) in 1 buffer supplemented with protease inhibitor cocktail for 2 h at 4°C with gentle agitation. After solubilization, the soluble fractions (16,000g) and remaining insoluble material were counted on an automatic gamma counter (1470; PerkinElmer Wallac, Gaithers-