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Heterodimerization of α2A- and β1-Adrenergic Receptors

2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês

10.1074/jbc.m207968200

1083-351X

Jianguo Xu, Junqi He, Amanda M. Castleberry, Srividya Balasubramanian, Anthony G. Lau, Randy A. Hall,

Olfactory and Sensory Function Studies

β- and α2–adrenergic receptors are known to exhibit substantial cross-talk and mutual regulation in tissues where they are expressed together. We have found that the β1-adrenergic receptor (β1AR) and α2A-adrenergic receptor (α2AAR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged β1AR and α2AAR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of α2AAR was found to induce substantial internalization of coexpressed β1AR, providing further evidence for a physical association between the two receptors in a cellular environment. Ligand binding assays examining displacement of [3H]dihydroalprenolol binding to the β1AR by various ligands revealed that β1AR pharmacological properties were significantly altered when the receptor was coexpressed with α2AAR. Finally, β1AR/α2AAR heterodimerization was found to be markedly enhanced by a β1AR point mutation (N15A) that blocks N-linked glycosylation of the β1AR as well as by point mutations (N10A/N14A) that blockN-linked glycosylation of the α2AAR. These data reveal an interaction between β1AR and α2AAR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors. β- and α2–adrenergic receptors are known to exhibit substantial cross-talk and mutual regulation in tissues where they are expressed together. We have found that the β1-adrenergic receptor (β1AR) and α2A-adrenergic receptor (α2AAR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged β1AR and α2AAR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of α2AAR was found to induce substantial internalization of coexpressed β1AR, providing further evidence for a physical association between the two receptors in a cellular environment. Ligand binding assays examining displacement of [3H]dihydroalprenolol binding to the β1AR by various ligands revealed that β1AR pharmacological properties were significantly altered when the receptor was coexpressed with α2AAR. Finally, β1AR/α2AAR heterodimerization was found to be markedly enhanced by a β1AR point mutation (N15A) that blocks N-linked glycosylation of the β1AR as well as by point mutations (N10A/N14A) that blockN-linked glycosylation of the α2AAR. These data reveal an interaction between β1AR and α2AAR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors. G protein-coupled receptor adrenergic receptor γ-aminobutyric acid post-synaptic density protein of 95 kDa membrane-associated guanylate kinase-like protein with an inverted domain structure hemagglutinin human embryonic kidney dihydroalprenolol phosphate-buffered saline fluorescein isothiocyanate The physiological actions of epinephrine and norepinephrine are mediated via the activation of the following three distinct classes of G protein-coupled receptors (GPCR)1: α1-, α2-, and β-adrenergic receptors. Each class of adrenergic receptor (AR) is comprised of three closely related subtypes as follows: α1A-, α1B-, and α1DAR, which couple primarily to Gq to stimulate phospholipase activity; α2A-, α2B-, and α2CAR, which couple primarily to Gi to inhibit adenylyl cyclase activity; and β1-, β2-, and β3AR, which couple primarily to Gs to stimulate adenylyl cyclase activity (1Bylund D.B. Eikenberg D.C. Hieble J.P. Langer S.Z. Lefkowitz R.J. Minneman K.P. Molinoff P.B. Ruffolo Jr., R.R. Trendelenburg U. Pharmacol. Rev. 1994; 46: 121-136PubMed Google Scholar). The adrenergic receptor subtypes are differentially distributed across various tissues, and tissue responses to epinephrine and norepinephrine are believed to be dependent upon the relative ratios of the various adrenergic receptors they express. Because β- and α2-adrenergic receptors couple to G proteins with opposing actions on adenylyl cyclase activity, the two receptors might be expected to purely antagonize each other's signaling when they are co-stimulated in the same cell. However, it has been shown that α2AR co-stimulation can in some cases paradoxically sensitize β-adrenergic signaling in brain tissue (2Northam W.J. Mobley P. Eur. J. Pharmacol. 1985; 113: 153-154Crossref PubMed Scopus (12) Google Scholar, 3Atkinson B.N. Minneman K.P. Mol. Pharmacol. 1992; 41: 688-694PubMed Google Scholar, 4Birnbaum A.K. Wotta D.R. Law P.Y. Wilcox G.L. Brain Res. Mol. Brain Res. 1995; 28: 72-80Crossref PubMed Scopus (18) Google Scholar). Moreover, the pharmacological properties of βARs in brain tissue are known to be regulated by α2ARs (5Woodcock E.A. Johnston C.I. Nature. 1980; 286: 159-160Crossref PubMed Scopus (16) Google Scholar, 6Nomura Y. Kawai M. Segawa T. Brain Res. 1984; 302: 101-109Crossref PubMed Scopus (20) Google Scholar), and reciprocally the pharmacological properties of α2ARs in brain tissue are known to regulated by βARs (7Maggi A. U'Prichard D.C. Enna S.J. Science. 1980; 207: 645-647Crossref PubMed Scopus (99) Google Scholar, 8Nakamura T. Tsujimura R. Nomura J. Brain Res. 1991; 542: 181-186Crossref PubMed Scopus (10) Google Scholar). These examples of cross-talk and mutual regulation between β- and α2-adrenergic receptors have been well known for more than 20 years, but the underlying molecular mechanisms remain unclear. GPCRs have traditionally been thought to exist as monomers, but recent studies (9Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (517) Google Scholar) have revealed that they can exist in the plasma membrane as both homodimers and heterodimers. At present, a key question in this field is: how widespread is the phenomenon of receptor heterodimerization? The most clear-cut case of the importance of GPCR heterodimerization comes from the GABAB receptor, a pharmacologically defined entity that is now known to be comprised of two distinct GPCRs, GABABR1 and GABABR2 (10Marshall F.H. Jones K.A. Kaupmann K. Bettler B. Trends Pharmacol. Sci. 1999; 20: 396-399Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Because GABABR1 and GABABR2 are not functional when expressed by themselves, they represent a clear example of the physiological importance of receptor heterodimerization. Although other heptahelical receptors may not absolutely require heterodimerization to be functional in the same way that the GABAB receptor does, heterodimerization of other receptors may underlie some phenomena that are major question marks in our present understanding of neurotransmitter and hormone receptors, such as unexplained forms of cross-talk between different receptor subtypes. We wondered if the previously reported cross-talk between βARs and α2ARs in brain tissue might be due in part to a physical association between these two receptor types. Many early studies (11Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, 12Hebert T.E. Loisel T.P. Adam L. Ethier N. Onge S.S. Bouvier M. Biochem. J. 1998; 330: 287-293Crossref PubMed Scopus (85) Google Scholar, 13Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3684-3689PubMed Google Scholar) of GPCR dimerization focused on the β2AR. We have found recently (14Xu J. Paquet M. Lau A.G. Wood J.D. Ross C.A. Hall R.A. J. Biol. Chem. 2001; 276: 41310-41317Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) that the β1AR also exhibits robust homodimerization in cells. Furthermore, it has been shown recently (15Lavoie C. Mercier J.F. Salahpour A. Umapathy D. Breit A. Villeneuve L.R. Zhu W.Z. Xiao R.P. Lakatta E.G. Bouvier M. Hebert T.E. J. Biol. Chem. 2002; 277: 35402-35410Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) that β1AR and β2AR can heterodimerize in a functionally important manner. β1AR is the most abundantly expressed βAR in brain (16Frielle T. Collins S. Daniel K.W. Caron M.G. Lefkowitz R.J. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7920-7924Crossref PubMed Scopus (517) Google Scholar, 17Machida C.A. Bunzow J.R. Searles R.P. Van Tol H. Tester B. Neve K.A. Teal P. Nipper V. Civelli O. J. Biol. Chem. 1990; 265: 12960-12965Abstract Full Text PDF PubMed Google Scholar), a tissue where α2ARs are found at particularly high levels (18Saunders C. Limbird L.E. Pharmacol. Ther. 1999; 84: 193-205Crossref PubMed Scopus (111) Google Scholar). The most widely expressed α2AR subtype, α2AAR, is known to be localized both pre- and post-synaptically in a number of brain regions (18Saunders C. Limbird L.E. Pharmacol. Ther. 1999; 84: 193-205Crossref PubMed Scopus (111) Google Scholar), where its pattern of expression overlaps significantly with that of the β1AR (17Machida C.A. Bunzow J.R. Searles R.P. Van Tol H. Tester B. Neve K.A. Teal P. Nipper V. Civelli O. J. Biol. Chem. 1990; 265: 12960-12965Abstract Full Text PDF PubMed Google Scholar). Based on the previously reported functional interactions between α2ARs and βARs, as well as the overlapping distribution patterns of α2AAR and β1AR, we examined the possibility that β1AR might be able to heterodimerize with α2AAR. Our findings reveal that β1AR and α2AAR robustly associate in cells and that α2AAR can regulate β1AR internalization and ligand binding. FLAG-β1AR was kindly provided by Robert J. Lefkowitz (Duke University). HA-α2AAR was kindly provided by Lee Limbird (Vanderbilt University Medical Center). HA-β1AR was kindly provided by Hitoshi Kurose (University of Tokyo). The N15A mutant β1AR was prepared via PCR amplification from the native human β1AR cDNA using a mutant sequence oligonucleotide (CTG GGC GCC TCC GAG CCC GGTGCC CTG TCG TCG GCC GCA CCG CTC). The N10A/N14A mutant α2AAR was also prepared via PCR amplification from the wild-type construct in a two-step process, first using a mutant sequence oligonucleotide (CC CTG CAG CCG GAA GCG GGC GCCGCG AGC TGG AAT GGG ACA GAG G) to make the N10A mutation, and second using a second oligonucleotide (GCG GGC GCC GCG AGC TGGGCT GGG ACA GAG GCG CCG GGG GGC) to make the N14A mutation using the N10A mutant construct as a template. The point mutations were confirmed by ABI sequencing. All tissue culture media and related reagents were purchased from Invitrogen. HEK-293 cells were maintained in complete medium (Dulbecco's modified Eagle's medium plus 10% fetal bovine serum and 1% penicillin/streptomycin) in a 37 °C, 5% CO2 incubator. For heterologous expression of receptors, 2 μg of DNA was mixed with LipofectAMINE (15 μl) and Plus reagent (20 μl) (from Invitrogen) and added to 5 ml of serum-free medium in 10-cm tissue cultures plates containing cells at ∼50–80% confluency. Following a 4-h incubation, the medium was removed, and 10 ml of fresh complete medium was added. After another 12–16 h, the medium was changed again, and the cells were harvested 24 h later. Samples (5 μg per lane) were run on 4–20% SDS-PAGE gels (Invitrogen) for 1 h at 150 V and then transferred to nitrocellulose. The blots were blocked in "blot buffer" (2% non-fat dry milk, 0.1% Tween 20, 50 mmNaCl, 10 mm Hepes, pH 7.4) for at least 30 min and then incubated with primary antibody in blot buffer for 1 h at room temperature. The primary antibodies utilized were either a 12CA5 monoclonal anti-HA antibody (Roche Molecular Biochemicals) or an M2 monoclonal anti-FLAG antibody (Sigma). The blots were then washed three times with 10 ml of blot buffer and incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) in blot buffer. Finally, the blots were washed three more times with 10 ml of blot buffer and visualized via enzyme-linked chemiluminescence using the ECL kit from AmershamBiosciences. Cells were harvested and lysed in 500 μl of ice-cold lysis buffer (10 mm Hepes, 50 mm NaCl, 0.5% Triton X-100, 5 mm EDTA, and the protease inhibitor mixture from Roche Molecular Biochemicals). The lysate was solubilized via end-over-end rotation at 4 °C for 30 min and clarified via centrifugation at 14,000 rpm for 15 min. A small fraction of the supernatant was taken at this point and incubated with SDS-PAGE sample buffer in order to examine expression of proteins in the whole cell extract. The remaining supernatant was incubated with 30 μl of beads covalently linked to anti-FLAG antibodies (Sigma) for 2 h with end-over-end rotation at 4 °C. After five washes with 1.0 ml of lysis buffer, the immunoprecipitated proteins were eluted from the beads with 1× SDS-PAGE sample buffer, resolved by SDS-PAGE, and subjected to Western blot analyses. For enzymatic deglycosylation of receptors, immunoprecipitates were separated from beads by boiling for 10 min in a denaturing buffer (0.5% SDS containing 1% β-mercaptoethanol). After cooling, Nonidet P-40 was added to the supernatants to a final concentration of 1%, and Na2HPO4/NaH2PO4 buffer (pH 7.5) was added to lysates to a final concentration of 50 mm. N-glycosidase F (1,500 units; New England Biolabs) was added to a 30-μl reaction volume, and the sample was incubated for 1 h at 37 °C. Intracellular cAMP was measured by using a non-acetylation cAMP enzyme immunoassay kit (Amersham Biosciences). Briefly, cultured cells were transfected with either FLAG-β1AR alone or FLAG-β1AR/HA-α2AAR in combination. After 24 h, cells were split into 6-well culture dishes with fresh medium. After another 48 h, cells were treated with varying concentrations of isoproterenol for 10 min and harvested with cell harvest buffer (50 mm Tris, pH 7.4, 250 μm Ro 20-1724 (Tocris, Ellisville, NJ), 5 mm MgCl2, 1 mm ATP, and 1 μm GTP). Cell lysates were sonicated, transferred to a 96-well assay plate coated with anti-rabbit IgG, and incubated with an anti-cAMP antibody at 4 °C for 2 h along with a series of cAMP standards. A cAMP-peroxidase conjugate was then added to the microtiter plate and incubated at 4 °C for 1 h. The plate was then washed four times with 400 μl of wash buffer, and the wells were incubated with 150 μl of enzyme substrate at room temperature for 1 h. When the samples were within the linear range of the standards, the reaction was stopped by adding 100 μl of 1.0m sulfuric acid. Absorbance was determined in a plate reader at 450 nm, and cAMP levels were determined using standard curves. Transfected cells were split into 35-mm dishes, grown for 48 h, and then incubated in the absence and presence of agonist for 10 min. The cells were then rinsed in PBS and fixed with 4% paraformaldehyde in PBS for 30 min and then rinsed three times in PBS and blocked with blocking buffer (2% non-fat dry milk in PBS, pH 7.4) for 30 min. The fixed cells were then incubated with primary antibody in blocking buffer for 1 h at room temperature. The dishes were subsequently washed three times with 2 ml of block buffer and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (AmershamBiosciences) in blocking buffer. Finally, the dishes were washed three times with 2 ml of blocking buffer and one time with 2 ml of PBS and then incubated with 2 ml of ECL reagent (Pierce) for exactly 15 s. The luminescence, which corresponds to the amount of receptor on the cell surface, was determined by placing the plate inside a TD 20/20 luminometer (Turner Designs). For preparation of membranes to be used in ligand binding assays, transfected cells grown on 100-mm dishes were rinsed twice with 10 ml of PBS and then scraped into 1 ml of ice-cold binding buffer (10 mm Hepes, 1 mmMgCl2, 1 mm ascorbic acid, pH 7.4). Cells were then washed three times with 1 ml of binding buffer, sonicated for 10 s, and resuspended in fresh binding buffer for use in radioligand binding assays. Membranes were incubated with increasing concentrations of [3H]DHA or [3H]RX821002 in binding buffer for saturation binding studies, or with 1 nm [3H]DHA or [3H]RX821002 in binding buffer in the absence or presence of various unlabeled ligands to generate inhibition curves. The samples were incubated for 15 min at 37 °C. Nonspecific binding was defined as [3H]DHA or [3H]RX821002 binding in the presence of either 1 mm isoproterenol or 1 mm clonidine, respectively, and represented less than 10% of total binding in all experiments. Incubations were terminated via filtration through GF/C filter paper using a Brandel cell harvester. Filters were rapidly washed three times with ice-cold wash buffer (10 mm Hepes), and radioactive ligand retained by the filters was quantified via liquid scintillation counting. The fitting of curves for one siteversus two sites was performed using Prism software (GraphPad, San Diego, CA). Goodness of fit was quantified usingF tests, comparing sum-of-squares values for the one-siteversus two-site fits. HEK-293 cells were transiently transfected with pcDNA3/FLAG-β1AR and pcDNA3/HA-α2AAR. Forty eight hours after transfection, cells were washed three times with Dulbecco's PBS and then incubated for 10 min at 37 °C in the absence or presence of 10 μm isoproterenol or 10 μm UK 14,304 (Sigma). Following this incubation, cells were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. To visualize the subcellular localization of β1AR and α2AAR, cells were blocked and permeabilized with a buffer containing 2% bovine serum albumin and 0.04% saponin in PBS ("saponin buffer") for 30 min at room temperature. The cells were then incubated with anti-β1AR polyclonal antibody (Santa Cruz Biotechnology) at 1:500 dilution and anti-HA monoclonal antibody (12CA5; Roche Molecular Biochemicals) at 1:1000 dilution for 1 h at room temperature. After three washes (1 min) with saponin buffer, the cells were incubated with a rhodamine red-conjugated anti-rabbit IgG at 1:200 dilution and FITC-conjugated anti-mouse IgG at 1:200 dilution (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. After three washes (1 min) with saponin buffer and one wash with PBS, coverslips were mounted, and rhodamine red-labeled β1AR and FITC-labeled α2AAR were visualized with a Zeiss LSM-410 laser confocal microscope. Multiple control experiments, utilizing either transfected cells in the absence of primary antibody or untransfected cells in the presence of primary antibody, revealed a very low level of background staining, indicating that the primary antibody-dependent immunostaining observed in the transfected cells was specific. To assess the potential physical association of α2A- and β-adrenergic receptors, HA-α2AAR was expressed in HEK-293 cells either alone or in combination with FLAG-β1AR or FLAG-β2AR. As shown in Fig.1, Western blotting for HA-α2AAR in cell lysates revealed multiple bands, with major species at ∼65 and 120 kDa. The higher order bands presumably represent receptor complexes resistant to separation by SDS-PAGE, as is commonly observed for many GPCRs (9Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (517) Google Scholar). When the FLAG-tagged βARs were immunoprecipitated with an anti-FLAG antibody, the co-transfected HA-α2AAR was robustly co-immunoprecipitated. All of the bands of HA-α2AAR immunoreactivity were evident in FLAG-βAR immunoprecipitates. Somewhat more co-immunoprecipitation was observed with β1AR than with β2AR, and thus further experiments in this area focused on the β1AR/α2AAR interaction. No changes in the extent of co-immunoprecipitation were observed when cells were stimulated before harvesting with various adrenergic receptor agonists (data not shown). In related control experiments, HA-α2AAR and FLAG-β1AR were transfected separately into different plates of cells, which were harvested, prepared as detergent-solubilized lysates, and then mixed together. Immunoprecipitation of FLAG-β1AR in these experiments did not yield any detectable co-immunoprecipitation of HA-α2AAR (data not shown), revealing that the two receptors need to be expressed in the same cell in order to physically associate. As a second method of assessing the physical association between α2AAR and β1AR, we expressed the two receptors in cells and studied their co-internalization. Agonist stimulation of many GPCRs induces significant internalization from the cell surface, and this process is known to be important in the desensitization and resensitization of GPCR responses (19Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). HA-α2AAR and FLAG-β1AR were expressed either separately or together in HEK-293 cells and then stimulated with one of three agonist conditions: the βAR agonist isoproterenol alone ("Iso"), the α2AR agonist UK 14,034 alone ("UK"), or Iso + UK together. When endocytosis of α2AAR was examined via a quantitative luminometer-based assay (Fig. 2 A), no significant internalization was observed in response to Iso under any condition, whereas internalization in response to UK was ∼15% whether Iso was co-applied or not. When endocytosis of β1AR was examined (Fig. 2 B), ∼25–30% receptor internalization was observed in response to Iso. The extent of internalization was not significantly different for β1AR expressed alone as compared with β1AR expressed in the presence of α2AAR. In response to UK, no significant internalization was observed for β1AR expressed alone, which is the expected result because UK does not activate β1AR. Strikingly, however, β1AR coexpressed with α2AAR exhibited ∼15% internalization in response to UK stimulation. These data indicate that stimulation of α2AAR can cause co-internalization of β1AR. The internalization of α2AAR and β1AR was also studied via immunofluorescence confocal microscopy. In cells co-transfected with HA-α2AAR and FLAG-β1AR, immunostaining for both receptors was concentrated into a smooth rim along the edge of the cells, which presumably corresponds to receptor localization in the plasma membrane of the cells (Fig.3, A–C). Stimulation with Iso resulted in the development of significant intracellular immunostaining for FLAG-β1AR (Fig. 3 D) but had no apparent effect on the pattern of immunostaining for HA-α2AAR (Fig. 3 E). In contrast, stimulation with UK resulted in mobilization of both HA-α2AAR and FLAG-β1AR inside the cell (Fig. 3, G and H), where the two receptors exhibited significant co-localization (Fig. 3 I). These data are consistent with the findings obtained using the luminometer-based assay (Fig. 2) and offer additional evidence that α2AAR and β1AR co-internalize from the cell surface following stimulation with α2-adrenergic agonists. Because coexpressed α2AAR was able to regulate β1AR internalization, we next examined if coexpression with α2AAR was able to regulate β1AR pharmacological properties. The binding of the βAR-selective antagonist [3H]DHA to lysed membranes derived from cells transfected with either β1AR alone or β1AR/α2AAR was examined. Saturation binding studies revealed that [3H]DHA bound with comparable affinity to β1AR expressed in the absence and presence of α2AAR coexpression (K D = 2.8 ± 0.5 nm, B max = 39.3 ± 7.7 pmol/mg for β1AR alone; K D = 2.3 ± 0.4 nm, B max = 33.0 ± 6.5 pmol/mg for β1AR/α2AAR). However, studies examining the displacement of [3H]DHA binding by a variety of βAR-selective ligands revealed that many of these compounds exhibited altered affinity for β1AR coexpressed with α2AAR relative to β1AR expressed alone. Inhibition curves for displacement of [3H]DHA binding to membranes expressing β1AR alone were fit extremely well by assuming one binding site (Fig.4; Table I). In contrast, curves for displacement of [3H]DHA binding to membranes expressing β1AR/α2AAR were in most cases fit significantly better by two-site analyses rather than one-site analyses. The appearance of a significant low affinity component for the displacement of [3H]DHA by metoprolol, labetalol, bisoprolol, dobutamine, and isoproterenol suggests that these ligands bind with substantially lower affinity to β1AR/α2AAR heterodimers than to β1AR alone. On the other hand, norepinephrine, an endogenous agonist for both α- and β-adrenergic receptors, exhibited slightly enhanced affinity for binding to the β1AR in the presence of α2AAR coexpression as compared with β1AR expressed alone, whereas epinephrine, which is also an endogenous agonist for both receptors, exhibited no significant change in its apparent affinity for β1AR alone versusβ1AR/α2AAR. In control experiments, membranes derived from cells expressing β1AR alone and α2AAR alone were mixed together, as in the control co-immunoprecipitation experiments described above. In these mixing experiments, no changes in the ligand binding properties of β1AR were observed for any of the ligands examined (data not shown), suggesting that β1AR and α2AAR must be expressed in the same cell for the modulation of β1AR pharmacological properties to occur. Furthermore, the effects of α2AAR coexpression on β1AR ligand binding properties were not blocked by treatment of the cells with pertussis toxin prior to harvesting (data not shown), suggesting that these effects are not due to activation of Gi/Go-dependent intracellular signaling pathways by the coexpressed α2AAR. In related experiments, the binding of various α2AR-selective ligands to membranes expressing α2AAR aloneversus β1AR/α2AAR was examined. No differences in the binding properties of the α2AR-selective agonist UK 14,034, the α2AR-selective partial agonist clonidine, or the α2AR-selective partial agonist guanfacine were observed (Table II), indicating that although β1AR possesses altered pharmacological properties when expressed in the presence of α2AAR, it does not seem to reciprocally be the case that α2AAR possesses altered pharmacological properties when expressed in the presence of β1AR.Table ILigand binding properties of β1AR expressed in the absence and presence of α2AARβ1AR,K iβ1AR/α2AARK HK LK Lnmnmnm%βAR-selective ligandsBisoprolol24 ± 616 ± 522,190 ± 774810Dobutamine1609 ± 971849 ± 41740,360 ± 962022Isoproterenol514 ± 46267 ± 8112,170 ± 232115Labetalol36 ± 825 ± 1011,250 ± 177312Metoprolol93 ± 588 ± 1913,380 ± 434511Propranolol5.7 ± 1.67.6 ± 1.4Endogenous ligandsEpinephrine5562 ± 5785564 ± 492Norepinephrine5010 ± 7013799 ± 404The binding of [3H]DHA to lysed membranes was studied in the presence of increasing concentrations of various adrenergic receptor ligands. The estimated K i values (in nm) are shown for each ligand. One-site and two-site fits of each data set were performed as described under "Materials and Methods." The inhibition curves for β1AR/α2AAR were significantly better fit by two-site fits rather than one-site fits for all ligands except for propranolol, norepinephrine, and epinephrine, whereas the inhibition curves for β1AR alone were not significantly better fit in any case by two-site fits relative to one-site fits. Hence, two K i values (K H for the high affinity component and K L for the low affinity component) are provided for binding to β1AR/α2AAR for most of the ligands, whereas only a single K ivalue is provided for binding to β1AR expressed alone. Note that for all of the two-site fits, the K H value for binding to β1AR/α2AAR is similar to the singleK i value for binding to α2AAR alone, suggesting that the majority of binding sites in the membranes expressing β1AR/α2AAR possess binding properties similar to the binding sites in membranes expressing β1AR alone. However, membranes expressing β1AR/α2AAR also exhibit, in most cases, a small low affinity component (K L), which was estimated between 10 and 25% of total binding sites in all cases, as shown in the right-hand column. The data for these inhibition curves were derived from 3 to 5 independent determinations for each ligand. Open table in a new tab Table IILigand binding properties of α2AAR expressed in the absence and presence of β1ARα2AAR,K iα2AAR/β1AR,K inmα2AR-selective ligandsClonidine34 ± 933 ± 8Guanfacine17 ± 526 ± 6UK 14,30491 ± 1489 ± 9 Open table in a new tab The binding of [3H]DHA to lysed membranes was studied in the presence of increasing concentrations of various adrenergic receptor ligands. The estimated K i values (in nm) are shown for each ligand. One-site and two-site fits of each data set were performed as described under "Materials and Methods." The inhibition curves for β1AR/α2AAR were significantly better fit by two-site fits rather than one-site fits for all ligands except for propranolol, norepinephrine, and epinephrine, whereas the inhibition curves for β1AR alone were not significantly better fit in any case by two-site fits relative to one-site fits. Hence, two K i values (K H for the high affinity component and K L for the low affinity component) are provided for binding to β1AR/α2AAR for most of the ligands, whereas only a single K ivalue is provided for binding to β1AR expressed alone. Note that for all of the two-site fits, the K H value for binding to β1AR/α2AAR is similar to the singleK i value for binding to α2AAR alone, suggesting that the majority of binding sites in the membranes expressing β1AR/α2AAR possess binding properties similar to the binding sites in membranes expressing β1AR alone. However, membranes expressing β1AR/α2AAR also exhibit, in most cases, a small low affinity component (K L), which was estimated between 10 and 25% of total binding sites in all cases, as shown in the right-hand column. The data for these inhibition curves were derived from 3 to 5 independent determinations for each ligand. We next examined the ability of α2AAR to modulate β1AR signaling. We utilized a transfection-based approach to study β1AR stimulation of cAMP production in the absence and presence of α2AAR coexpress