Cloning, localization, and permanent expression of a Drosophila octopamine receptor

A cDNA for a member of the G protein-coupled receptor family was isolated from Drosophila using a probe derived from a human beta 2-adrenergic receptor cDNA. This Drosophila receptor gene is localized at 99A10-B1 on the right arm of chromosome 3 and is preferentially expressed in Drosophila heads. The insect octopamine receptor has been permanently expressed in mammalian cells, where it mediates the attenuation of adenylate cyclase activity and exhibits a pharmacological profile consistent with an octopamine type 1 receptor. Sequence and pharmacological comparisons indicate that the octopamine receptor is unique but closely related to mammalian adrenergic receptors, perhaps as an evolutionary precursor.     

GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation

Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.     

Agonist-modulated palmitoylation of beta 2-adrenergic receptor in Sf9 cells.

The palmitoylation of the human beta 2-adrenergic receptor (beta 2-AR) was studied in recombinant baculovirus-infected insect Sf9 cells. At 48 h post-infection, a high level expression of an epitope-tagged beta 2-AR (10-25 pmol/mg protein) was detected by [125I]iodocyanopindolol ([125I]CYP) binding assays. The identity of the receptor was confirmed both by photoaffinity labeling and immunoblotting. The fusion receptor displayed typical beta 2-AR pharmacological properties and conferred a beta-adrenergic sensitive adenylyl cyclase activity to the Sf9 cells. Moreover, exposure of the Sf9 cells to the beta-adrenergic agonist isoproterenol induced a rapid desensitization of the receptor-stimulated adenylyl cyclase activity. Purification of the epitope-tagged beta 2-AR by immunoprecipitation as well as by alprenolol-Sepharose affinity chromatography revealed that the receptor is covalently modified with palmitic acid in the insect cells as is observed in mammalian cells. In addition, short-term incubation of the cells with isoproterenol led to a specific increase in the incorporation of [3H]palmitate in the receptor, consistent with a rapid agonist-modulated turnover of the beta 2-AR-attached palmitic acid. These results suggest that agonist-mediated regulation of beta 2-AR post-translational palmitoylation could represent an other regulatory process for G protein-coupled receptors.     

Mutated human beta3-adrenergic receptor (Trp64Arg) lowers the response to beta3-adrenergic agonists in transfected 3T3-L1 preadipocytes.

Wild-type or mutated human beta3-adrenergic receptor (Trp64Arg) cDNAs were stably expressed in mouse 3T3-L1 cells. Saturation binding study using a beta-adrenergic ligand revealed that there was no significant difference in the receptor density and the equilibrium dissociation constant between the two cell lines. However, the ability of the mutant beta3-adrenergic receptor to accumulate cyclic AMP (cAMP) in response to isoproterenol was much reduced and Kact for cAMP accumulation was lowered as compared to the wild type receptor. The amount of alpha subunit of stimulatory GTP-binding protein (GSalpha) and adenylyl cyclase activity in response to forskolin were not different in the two cell lines. The responses of the mutant receptor to epinephrine, norepinephrine and L-755,507, a highly specific agonist for human beta3-adrenergic receptor, were also reduced, but the reduction of Kact for L-755,507 was more evident than other agonists tested. The cAMP accumulation in response to some conventional beta3 agonists was less than 10% of that to isoproterenol even in the cells expressing the wild type receptor. These results suggest that the Trp64Arg mutant beta3-adrenergic receptor has less ability to stimulate adenylyl cyclase, and that lipolytic activity through the beta3-adrenergic receptor by catecholamines in subjects carrying this mutation might be suppressed.     

Continuous high density expression of human beta 2-adrenergic receptors in a mouse cell line previously lacking beta-receptors

The PvuII fragment of human genomic clone LCV-517 which contains the entire coding region of a beta-adrenergic receptor gene was cloned into the SmaI site of the expression vector pMSG. The recombinant DNA was cotransfected with pRSVneo into mouse B-82 cells using the CaPO4 precipitation method. B-82 cells do not possess beta-adrenergic receptors but do contain prostaglandin E1 receptors that stimulate adenylate cyclase. Following transfection, several colonies expressing beta-adrenergic receptors were isolated. Analysis of ligand binding to expressed beta-receptors indicated that the protein encoded by the gene in clone LCV-517 was a beta 2-adrenergic subtype. Human beta 2-adrenergic receptors photoaffinity labeled with [125I]iodocyanopindolol diazirine migrated on sodium dodecyl sulfate-polyacrylamide gels consistent with a molecular mass of 68,000, demonstrating that the receptor is glycosylated to an extent of 25-30% by weight. Addition of isoproterenol to cultures of transfected cells resulted in a 3-4-fold stimulation of adenylate cyclase, an effect similar to that seen in control B-82 cells with prostaglandin E1. These data describe the production of stable murine clonal cell lines expressing human beta 2-adrenergic receptors and illustrate the utility of such lines in the biochemical and pharmacological characterization of receptor proteins.     

Expression of mRNA of beta 1- and beta 2-adrenergic receptors in Xenopus oocytes results from structurally distinct receptor mRNAs

Poly(A)+-selected RNA prepared from cells or tissues that express a homogeneous population of either beta 1- or beta 2-adrenergic receptors was isolated and then microinjected into Xenopus laevis oocytes. Following microinjection, the expression of beta-adrenergic receptors was assessed by equilibrium radioligand binding analysis using the antagonist ligand [3H]dihydroalprenolol. The pharmacology of the newly- expressed beta-adrenergic receptors in oocyte membranes was the same as that of the original tissue used as a source of RNA. Hybridization of nick-translated cDNA of hamster beta 2-adrenergic receptor to poly(A)+-selected RNA from tissues containing beta 2-adrenergic receptors was to a mRNA species of 2.2 kilobases. In contrast, hybridization of the cDNA probe to poly(A)+-selected RNA from tissues containing beta 1-adrenergic receptors was to a mRNA species of 2.0 kilobases. A single-stranded fragment of hamster beta 2-adrenergic receptor cDNA corresponding to nucleotides 730-886 was isolated and uniformly radiolabeled. This region of the gene is predicted to encode for the entire second exofacial loop (L4-5), the entire fifth transmembrane-spanning region, and the first 5 amino acid residues of the third cytoplasmic loop (L5-6) of the beta 2-adrenergic receptor. Hybridization at 48 and 56 degrees C of poly(A)+-selected RNA prepared from sources that express either beta 1 or beta 2-adrenergic receptors to the antisense orientation strand of this region of the beta 2-adrenergic receptor cDNA was followed by S1 endonuclease digestion of nonhybridized sequences. At 48 degrees C, S1-resistant hybrids from both sources of RNA protected the probe from S1 endonuclease digestion. At 56 degrees C, however, only the RNA prepared from the source of beta 2-adrenergic receptors protected the probe from S1 endonuclease digestion. These results demonstrate that the mRNAs encoding for the structurally homologous beta 1- and beta 2-adrenergic receptors are distinct in the pharmacological specificity of their translation products and in their size and structure.     

Characterization of the human transferrin receptor produced in a baculovirus expression system

Recombinant human transferrin receptor has been produced in a baculovirus expression system. Magnetic particles coated with an anti-transferrin receptor monoclonal antibody were used to immunoselect virus-infected Sf9 insect cells expressing the human transferrin receptor on their cell surface. Recombinant virus containing the human transferrin receptor cDNA was then plaque-purified from these cells. Biosynthetic labeling studies of infected cells showed that the human transferrin receptor is one of the major proteins made 2-3 days postinfection. The recombinant receptor made in insect cells is glycosylated and is also posttranslationally modified by the addition of a fatty acid moiety. However, studies with tunicamycin and endoglycosidases H and F showed that the oligosaccharides displayed on the recombinant receptor differ from those found on the naturally occurring receptor in human cells. As a consequence, the human receptor produced in the baculovirus system has an Mr of 82,000 and is smaller in size than the authentic receptor. About 30% of human transferrin receptors made in insect cells do not form intermolecular disulfide bonds, but are recognized by the anti-transferrin receptor antibody, B3/25, and bind specifically to a human transferrin-Sepharose column. Binding studies using 125I-labeled human transferrin showed that insect cells infected with the recombinant virus expressed an average of 5.8 +/- 0.9 X 10(5) transferrin receptors (Kd = 63 +/- 9 nM) on their cell surface. Thus, the human transferrin receptor produced in insect cells is biologically active and appears suitable for structural and functional studies.     

Beta 3-adrenergic receptors regulate retinal endothelial cell migration and proliferation

Sympathetic nerves may play a role in vascular disorders of the eye. In the present study, we hypothesized that activation of beta3-adrenergic receptors on retinal endothelial cells would promote migration and proliferation of these cells, two markers of an angiogenic phenotype. We show, for the first time, expression of beta3-adrenergic receptors on cultured retinal endothelial cells. Activation of these receptors with BRL37344, a specific beta3-adrenergic receptor agonist, promoted migration that was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K), the mitogen activated protein kinase component MEK, and matrix metalloproteinases (MMPs) 2 and 9. BRL37344 stimulated proliferation, which could be blocked by inhibitors of Src, PI3K, and MEK. These cells also express the beta1-adrenergic receptor with no beta2-adrenergic receptor expression observed. Stimulation of the beta1-adrenergic receptor with xamoterol, a specific partial agonist, did not promote proliferation or migration. These results support the hypothesis that beta3-adrenergic receptors play a role in proliferation and migration of cultured human retinal endothelial cells.     

Desensitization of the human beta 1-adrenergic receptor. Involvement of the cyclic AMP-dependent but not a receptor-specific protein kinase.

Human SK-N-MC neurotumor cells express beta 1- but not beta 2-adrenergic receptors. Following exposure of the cells to isoproterenol, there was no reduction in the maximum response of adenylyl cyclase to the agonist but a 3-fold shift to less sensitivity in the concentration response. This desensitization was very rapid and dose dependent; half-maximal effects occurred at 10 nM isoproterenol. A similar shift was observed when membranes from control cells were incubated with ATP and the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). No shift, however, was observed in intact cells exposed to either dibutyryl cyclic AMP or dopamine, which stimulates adenylyl cyclase in these cells through D1 dopamine receptors. To pursue the role of protein kinases in the desensitization process, cells were made permeable, loaded with a PKA inhibitor or with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), and exposed to isoproterenol. The PKA inhibitor but not heparin blocked the agonist-mediated desensitization. In contrast, desensitized human tumor cells (HeLa and A431), which express beta 2-adrenergic receptors, exhibited both a shift in concentration response and a reduction in maximum response; the former was blocked by the PKA inhibitor and the latter by heparin. Our results indicated that whereas both human beta 1- and beta 2-adrenergic receptors are susceptible to PKA, only the beta 2 receptors are susceptible to beta ARK. These differences in desensitization may be due to differences in receptor structure as the human beta 1 receptor has fewer potential phosphorylation sites for beta ARK in the carboxyl terminus than the human beta 2 receptor.     

Cloning, sequence analysis, and permanent expression of a human alpha 2-adrenergic receptor in Chinese hamster ovary cells. Evidence for independent pathways of receptor coupling to adenylate cyclase attenuation and activation

The gene encoding a human alpha 2-adrenergic receptor was isolated from a human genomic DNA library using a 367-base pair fragment of Drosophila genomic DNA that exhibited 54% identity with the human beta 2-adrenergic receptor and 57% identity with the human alpha 2-adrenergic receptor. The nucleotide sequence of a fragment containing the human alpha 2-receptor gene and 2.076 kilobases of untranslated 5' sequence was determined, and potential upstream regulatory regions were identified. This gene encodes a protein of 450 amino acids and was identified as an alpha 2-adrenergic receptor by homology with published sequences and by pharmacological characterization of the protein expressed in cultured cells. Permanent expression of the alpha 2-receptor was achieved by transfecting Chinese hamster ovary (CHO) cells which lack adrenergic receptors with a 1.5-kilobase NcoI-HindIII fragment of the genomic clone containing the coding region of the gene. The alpha 2-receptor expressed in CHO cells displayed pharmacology characteristic of an alpha 2 A-receptor subtype with a high affinity for yohimbine (Ki = 1 nM) and a low affinity for prazosin (Ki = 10,000 nM). Agonists displayed a rank order of potency in radioligand binding assays of para-aminoclonidine greater than or equal to UK-14304 greater than (-)-epinephrine greater than (-)-norepinephrine greater than (-)-isoproterenol, consistent with the identification of this protein as an alpha 2-receptor. The role of the alpha 2-receptor in modulating intracellular cyclic AMP concentrations was investigated in three transfected cell lines expressing 50, 200, and 1200 fmol of receptor/mg membrane protein. At low concentrations (1-100 nM), (-)-epinephrine attenuated forskolin-stimulated cyclic AMP accumulation by up to 60% in a receptor density-dependent manner. At epinephrine concentrations above 100 nM, cyclic AMP levels were increased up to 140% of the forskolin-stimulated level. Pertussis toxin pretreatment of cells eliminated alpha 2-receptor-mediated attenuation of forskolin-stimulated cyclic AMP levels and enhanced the receptor density-dependent potentiation of forskolin-stimulated cyclic AMP concentrations from 3 to 8-fold. Potentiation of forskolin-stimulated cyclic AMP levels was also elicited by the alpha 2-adrenergic agonists, UK-14304 and para-aminoclonidine, and blocked by the alpha 2-adrenergic antagonist yohimbine, but not by the alpha 1-adrenergic antagonist prazosin or the beta-adrenergic antagonist propranolol.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distinct roles for Src tyrosine kinase in beta2-adrenergic receptor signaling to MAPK and in receptor internalization

G protein-coupled receptors form the largest family of membrane receptors and transmit diverse ligand signals to modulate various cellular responses. After activation by their ligands, some of these G protein-coupled receptors are desensitized, internalized (endocytosed), and down-regulated (degraded). In HEK 293 cells, the G(s)-coupled beta2-adrenergic receptor was postulated to initiate a second wave of signaling, such as the activation of the mitogen-activated protein kinase (MAPK) pathway after the receptor is internalized. The tyrosine kinase c-Src plays a critical role in these events. Here we used mouse embryonic fibroblast (MEF) cells deficient in Src family tyrosine kinases to examine the role of Src in beta2-adrenergic receptor signaling to the MAPK pathway and in receptor internalization. We found that in Src-deficient cells the beta2-adrenergic receptor could activate the MAPK pathway. However, the internalization of beta2-adrenergic receptors was blocked in Src-deficient MEF cells. Furthermore, we observed that in MEF cells deficient in beta-arrestin 2 the internalization of the beta2-adrenergic receptor was impaired, whereas the activation of the MAPK pathway by the beta2-adrenergic receptor was normal. Our data demonstrate that although Src and beta-arrestin 2 play essential roles in beta2-adrenergic receptor internalization, they are not required for the activation of the MAPK pathway by the beta2-adrenergic receptor. In other words, our finding suggests that receptor internalization is not required for beta2-adrenergic receptor signaling to the MAPK pathway in MEF cells.     

Indirect immunofluorescence localization of beta-adrenergic receptors and G-proteins in human A431 cells.

Polyclonal antibodies directed against (i) rodent lung beta 2-adrenergic receptor, (ii) a synthetic fragment of an extracellular domain of the receptor, and (iii) human placenta G-protein beta-subunits, were used to localize these antigens in situ in intact and permeabilized human epidermoid carcinoma A431 cells. Antibodies directed against beta 2-adrenergic receptors showed a punctate immunofluorescence staining throughout the cell surface of fixed intact cells. Punctate staining was also observed in clones of Chinese hamster ovary cells transfected with an expression vector harbouring the gene for the hamster beta 2-adrenergic receptor. The immunofluorescence observed with anti-receptor antibodies paralleled the level of receptor expression. In contrast, the beta-subunits common to G-proteins were not stained in fixed intact cells, presumably reflecting their intracellular localization. In detergent-permeabilized fixed cells, strong punctate staining of G beta-subunits was observed throughout the cytoplasm. This is the first indirect immunofluorescence localization of beta-adrenergic receptors and G-proteins. Punctate immunofluorescence staining suggests that both antigens are distributed in clusters.     

Characterization and Regulation of β1-Adrenergic Receptors in a Human Neuroepithelioma Cell Line

Intact human neuroepithelioma SK-N-MC cells bound the beta-adrenergic antagonist (-)-[3H]-CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to beta-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol much greater than norepinephrine greater than epinephrine, which is indicative of a beta 1-subtype receptor. Membranes prepared from the cells bound (-)-3-[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the beta 1-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the beta 2-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only beta 1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the beta 1- and beta 2-adrenergic receptors revealed the presence of a very strong beta 1-adrenergic receptor mRNA signal, while under the same conditions no beta 2-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of beta 1-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.     

scFv single chain antibody variable fragment as inverse agonist of the beta2-adrenergic receptor

Antibodies directed against the second extracellular loop of G protein-coupled receptors were shown to possess functional activities. Using a functional monoclonal antibody against the human beta2-adrenergic receptor, a scFv fragment with high affinity for the target epitope was constructed and produced. The fragment recognized the beta2-adrenergic receptors on A431 cells, blocked cAMP accumulation induced by the beta2-agonist salbutamol, and decreased basal cAMP accumulation in the same cells. Their in vitro activity was tested on neonatal rat cardiomyocytes. The antibody fragments blocked the chronotropic activity induced by the beta2-agonist clenbuterol. They also decreased the in vivo heart beating frequency of mice pretreated with bisoprolol (a beta1-adrenergic receptor antagonist) for 4 min after injection. The immunological approach presented here may serve as a strategy for the synthesis of a new class of allosteric modulators for G protein-coupled receptors.     

重组人卵泡刺激素在昆虫细胞中的表达

为了研究在昆虫细胞中表达重组人卵泡刺激素,我们以人胎盘组织提取的染色体DNA为模板,利用重叠PCR方法扩增出hFSHβ亚基的cDNA的编码区。将此cDNA克隆入核型多角体病毒(AcNPV)非融合蛋白基因表达载体pVLl393,我们得到了表达载体pVLl393-hFSHβ,然后与BaculoGold^TM线性杆状病毒DNA共转染昆虫细胞SF9,经多次扩增后获得高滴度的重组病毒AcNPV-hFSHβ。将此重组病毒感染昆虫细胞,我们得到了在胞浆中表达的hFSHβ亚基,Western blot显示分子量大约为21kDa。以重组病毒AcNPV-hFSHβ与AcNPV-hCGoL一同感染昆虫细胞得到了具有分泌性的重组hFSH异二聚体,在非还原的条件下Western blot显示分子量大约为33kDa。     

Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer

Quantitative bioluminescence resonance energy transfer (BRET) analysis was applied to the study of beta(1)- and beta(2)-adrenergic receptor homo- and heterodimerization. To assess the relative affinity between each of the protomers, BRET saturation experiments were carried out in HEK-293T cells. beta(1)- and beta(2)-adrenergic receptors were found to have similar propensity to engage in homo- and heterotropic interactions suggesting that, at equivalent expression levels of the two receptor subtypes, an equal proportion of homo- and heterodimers would form. Analysis of the data also revealed that, at equimolar expression levels of energy donor and acceptor, more than 80% of the receptor molecules exist as dimers and that this high incidence of receptor dimerization is insensitive to receptor density for expression levels varying between 1.4 and 26.9 pmol of receptor/mg of membrane protein. Taken together, these results indicate that most of the receptors expressed in cells exist as constitutive dimers and that, at least in undifferentiated fibroblasts, the proportion of homo- and heterodimers between the closely related beta(1)- and beta(2)-adrenergic receptors is determined by their relative levels of expression.     

Insulin activation of mitogen-activated protein kinases Erk1,2 is amplified via beta-adrenergic receptor expression and requires the integrity of the Tyr350 of the receptor

Insulin activates a complex set of intracellular responses, including the activation of mitogen-activated protein kinases Erk1,2. The counterregulatory actions of insulin on catecholamine action are well known and include phosphorylation of the beta(2)-adrenergic receptor on Tyr(350), Tyr(354), and Tyr(364) in the C-terminal cytoplasmic domain, as well as enhanced sequestration of the beta(2)-adrenergic receptor. Both beta-adrenergic agonists and insulin provoke sequestration of beta(2)-adrenergic receptors in a synergistic manner. In the current work, cross-talk between insulin action and beta(2)-adrenergic receptors revealed that insulin activation of Erk1,2 was amplified via beta(2)-adrenergic receptors. In Chinese hamster ovary cells, expression of beta(2)-adrenergic receptors enhanced 5-10-fold the activation of Erk1,2 by insulin and prolonged the activation, the greatest enhancement occurring at 5 min post-insulin. The potentiation of insulin signaling on Erk1,2 was proportional to the level of expression of beta(2)-adrenergic receptor. The potentiation of insulin signaling requires the integrity of Tyr(350) of the beta(2)-adrenergic receptor, a residue phosphorylated in response to insulin. beta(2)-adrenergic receptors with a Y350F mutation failed to potentiate insulin activation of Erk1,2. Expression of the C-terminal domain of the beta(2)-adrenergic receptor (Pro(323)-Leu(418)) in cells expressing the intact beta(2)-adrenergic receptor acts as a dominant negative, blocking the potentiation of insulin activation of Erk1,2 via the beta(2)-adrenergic receptor. Blockade of beta(2)-adrenergic receptor sequestration does not alter the ability of the beta(2)-adrenergic receptor to potentiate insulin action on Erk1,2. We propose a new paradigm in which a G-protein-linked receptor, such as the beta(2)-adrenergic receptor, itself acts as a receptor-based scaffold via its binding site for Src homology 2 domains, facilitating signaling of the mitogen-activated protein kinase pathway by insulin.     

Heterodimerization of alpha 2A- and beta 1-adrenergic receptors

beta- and alpha(2)-adrenergic receptors are known to exhibit substantial cross-talk and mutual regulation in tissues where they are expressed together. We have found that the beta(1)-adrenergic receptor (beta(1)AR) and alpha(2A)-adrenergic receptor (alpha(2A)AR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged beta(1)AR and alpha(2A)AR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of alpha(2A)AR was found to induce substantial internalization of coexpressed beta(1)AR, providing further evidence for a physical association between the two receptors in a cellular environment. Ligand binding assays examining displacement of [(3)H]dihydroalprenolol binding to the beta(1)AR by various ligands revealed that beta(1)AR pharmacological properties were significantly altered when the receptor was coexpressed with alpha(2A)AR. Finally, beta(1)AR/alpha(2A)AR heterodimerization was found to be markedly enhanced by a beta(1)AR point mutation (N15A) that blocks N-linked glycosylation of the beta(1)AR as well as by point mutations (N10A/N14A) that block N-linked glycosylation of the alpha(2A)AR. These data reveal an interaction between beta(1)AR and alpha(2A)AR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors.