Heterodimerization of alpha 2A- and beta 1-Adrenergic Receptors*

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

From the Department of Pharmacology, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia 30322

Received for publication, August 5, 2002, and in revised form, December 23, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 1AR) and alpha 2A-adrenergic receptor (alpha 2AAR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged beta 1AR and alpha 2AAR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of alpha 2AAR was found to induce substantial internalization of coexpressed beta 1AR, 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 beta 1AR by various ligands revealed that beta 1AR pharmacological properties were significantly altered when the receptor was coexpressed with alpha 2AAR. Finally, beta 1AR/alpha 2AAR heterodimerization was found to be markedly enhanced by a beta 1AR point mutation (N15A) that blocks N-linked glycosylation of the beta 1AR as well as by point mutations (N10A/N14A) that block N-linked glycosylation of the alpha 2AAR. These data reveal an interaction between beta 1AR and alpha 2AAR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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: alpha 1-, alpha 2-, and beta -adrenergic receptors. Each class of adrenergic receptor (AR) is comprised of three closely related subtypes as follows: alpha 1A-, alpha 1B-, and alpha 1DAR, which couple primarily to Gq to stimulate phospholipase activity; alpha 2A-, alpha 2B-, and alpha 2CAR, which couple primarily to Gi to inhibit adenylyl cyclase activity; and beta 1-, beta 2-, and beta 3AR, which couple primarily to Gs to stimulate adenylyl cyclase activity (1). 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 beta - and alpha 2-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 alpha 2AR co-stimulation can in some cases paradoxically sensitize beta -adrenergic signaling in brain tissue (2-4). Moreover, the pharmacological properties of beta ARs in brain tissue are known to be regulated by alpha 2ARs (5, 6), and reciprocally the pharmacological properties of alpha 2ARs in brain tissue are known to regulated by beta ARs (7, 8). These examples of cross-talk and mutual regulation between beta - and alpha 2-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 (9) 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 (10). 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 beta ARs and alpha 2ARs in brain tissue might be due in part to a physical association between these two receptor types. Many early studies (11-13) of GPCR dimerization focused on the beta 2AR. We have found recently (14) that the beta 1AR also exhibits robust homodimerization in cells. Furthermore, it has been shown recently (15) that beta 1AR and beta 2AR can heterodimerize in a functionally important manner. beta 1AR is the most abundantly expressed beta AR in brain (16, 17), a tissue where alpha 2ARs are found at particularly high levels (18). The most widely expressed alpha 2AR subtype, alpha 2AAR, is known to be localized both pre- and post-synaptically in a number of brain regions (18), where its pattern of expression overlaps significantly with that of the beta 1AR (17). Based on the previously reported functional interactions between alpha 2ARs and beta ARs, as well as the overlapping distribution patterns of alpha 2AAR and beta 1AR, we examined the possibility that beta 1AR might be able to heterodimerize with alpha 2AAR. Our findings reveal that beta 1AR and alpha 2AAR robustly associate in cells and that alpha 2AAR can regulate beta 1AR internalization and ligand binding.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmids-- FLAG-beta 1AR was kindly provided by Robert J. Lefkowitz (Duke University). HA-alpha 2AAR was kindly provided by Lee Limbird (Vanderbilt University Medical Center). HA-beta 1AR was kindly provided by Hitoshi Kurose (University of Tokyo). The N15A mutant beta 1AR was prepared via PCR amplification from the native human beta 1AR cDNA using a mutant sequence oligonucleotide (CTG GGC GCC TCC GAG CCC GGT GCC CTG TCG TCG GCC GCA CCG CTC). The N10A/N14A mutant alpha 2AAR 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 GCC GCG AGC TGG AAT GGG ACA GAG G) to make the N10A mutation, and second using a second oligonucleotide (GCG GGC GCC GCG AGC TGG GCT 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.

Cell Culture and Transfection-- 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.

Western Blotting-- 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 mM NaCl, 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 Amersham Biosciences.

Immunoprecipitation-- 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.

Enzymatic Deglycosylation-- For enzymatic deglycosylation of receptors, immunoprecipitates were separated from beads by boiling for 10 min in a denaturing buffer (0.5% SDS containing 1% beta -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.

Cyclic AMP Assay-- Intracellular cAMP was measured by using a non-acetylation cAMP enzyme immunoassay kit (Amersham Biosciences). Briefly, cultured cells were transfected with either FLAG-beta 1AR alone or FLAG-beta 1AR/HA-alpha 2AAR 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 1.0 M sulfuric acid. Absorbance was determined in a plate reader at 450 nm, and cAMP levels were determined using standard curves.

Surface Expression Assay-- 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 (Amersham Biosciences) 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).

Ligand Binding Assays-- 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 mM MgCl2, 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 site versus two sites was performed using Prism software (GraphPad, San Diego, CA). Goodness of fit was quantified using F tests, comparing sum-of-squares values for the one-site versus two-site fits.

Immunofluorescence Microscopy-- HEK-293 cells were transiently transfected with pcDNA3/FLAG-beta 1AR and pcDNA3/HA-alpha 2AAR. 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 beta 1AR and alpha 2AAR, 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-beta 1AR 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 beta 1AR and FITC-labeled alpha 2AAR 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.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Co-immunoprecipitation of alpha 2A- and beta -Adrenergic Receptors-- To assess the potential physical association of alpha 2A- and beta -adrenergic receptors, HA-alpha 2AAR was expressed in HEK-293 cells either alone or in combination with FLAG-beta 1AR or FLAG-beta 2AR. As shown in Fig. 1, Western blotting for HA-alpha 2AAR 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 (9). When the FLAG-tagged beta ARs were immunoprecipitated with an anti-FLAG antibody, the co-transfected HA-alpha 2AAR was robustly co-immunoprecipitated. All of the bands of HA-alpha 2AAR immunoreactivity were evident in FLAG-beta AR immunoprecipitates. Somewhat more co-immunoprecipitation was observed with beta 1AR than with beta 2AR, and thus further experiments in this area focused on the beta 1AR/alpha 2AAR 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-alpha 2AAR and FLAG-beta 1AR were transfected separately into different plates of cells, which were harvested, prepared as detergent-solubilized lysates, and then mixed together. Immunoprecipitation of FLAG-beta 1AR in these experiments did not yield any detectable co-immunoprecipitation of HA-alpha 2AAR (data not shown), revealing that the two receptors need to be expressed in the same cell in order to physically associate.


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Fig. 1.   Co-immunoprecipitation of alpha 2A- and beta 1AR and beta 2AR. HEK-293 cells were transfected with empty vector (lane 1), HA-alpha 2AAR alone (lane 2), HA-alpha 2AAR/FLAG-beta 1AR (lane 3), or HA-alpha 2AAR/FLAG-beta 2AR (lane 4). The expression of HA-alpha 2AAR in detergent-solubilized lysates prepared from the transfected cells is shown in the 1st 4 lanes of the Western blot (IB) shown in this figure. Several nonspecific bands were evident in untransfected cell lysates (lane 1), whereas specific immunoreactivity for HA-alpha 2AAR (lanes 2-4) was observed as major bands at ~65 and 120 kDa (arrows). The lysates were incubated with anti-FLAG affinity agarose to immunoprecipitate the FLAG-tagged beta -adrenergic receptors, and the resultant immunoprecipitates (IP) were examined via Western blot for anti-HA immunoreactivity. As shown in the last 2 lanes of this figure, specific co-immunoprecipitation of HA-alpha 2AAR was observed with both FLAG-beta 1AR and FLAG-beta 2AR. The positions of molecular mass standards are indicated on the left side of the figure. This experiment was repeated five times with nearly identical results.

Co-internalization of alpha 2A- and beta 1-Adrenergic Receptors-- As a second method of assessing the physical association between alpha 2AAR and beta 1AR, 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 (19). HA-alpha 2AAR and FLAG-beta 1AR were expressed either separately or together in HEK-293 cells and then stimulated with one of three agonist conditions: the beta AR agonist isoproterenol alone ("Iso"), the alpha 2AR agonist UK 14,034 alone ("UK"), or Iso + UK together. When endocytosis of alpha 2AAR was examined via a quantitative luminometer-based assay (Fig. 2A), 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 beta 1AR was examined (Fig. 2B), ~25-30% receptor internalization was observed in response to Iso. The extent of internalization was not significantly different for beta 1AR expressed alone as compared with beta 1AR expressed in the presence of alpha 2AAR. In response to UK, no significant internalization was observed for beta 1AR expressed alone, which is the expected result because UK does not activate beta 1AR. Strikingly, however, beta 1AR coexpressed with alpha 2AAR exhibited ~15% internalization in response to UK stimulation. These data indicate that stimulation of alpha 2AAR can cause co-internalization of beta 1AR.


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Fig. 2.   Co-internalization of beta 1AR with alpha 2AAR. HA-alpha 2AAR and FLAG-beta 1AR were expressed either separately (solid bars) or together (striped bars) in HEK-293 cells. The internalization of alpha 2AAR (A) and beta 1AR (B) was examined using a luminometer-based assay following 10-min stimulations with the beta -adrenergic agonist isoproterenol (Iso; 10 µm), the alpha 2-adrenergic agonist UK 14,304 (UK; 10 µM), or a combination of the two agonists together. As shown in A, alpha 2AAR exhibited ~15% internalization in response to UK stimulation but no significant internalization in response to isoproterenol under any condition. Conversely, as shown in B, beta 1AR exhibited ~25-30% internalization in response to isoproterenol but also exhibited ~15% internalization in response to stimulation with UK. This effect was only observed, however, when alpha 2AAR was coexpressed (** indicates significantly different from beta 1AR alone, p < 0.01). These data suggest that beta 1AR can co-internalize with agonist-activated alpha 2AAR. The bars and error bars represent the means ± S.E. for 4-5 independent experiments for each condition, with each experiment being performed in triplicate.

The internalization of alpha 2AAR and beta 1AR was also studied via immunofluorescence confocal microscopy. In cells co-transfected with HA-alpha 2AAR and FLAG-beta 1AR, 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-beta 1AR (Fig. 3D) but had no apparent effect on the pattern of immunostaining for HA-alpha 2AAR (Fig. 3E). In contrast, stimulation with UK resulted in mobilization of both HA-alpha 2AAR and FLAG-beta 1AR inside the cell (Fig. 3, G and H), where the two receptors exhibited significant co-localization (Fig. 3I). These data are consistent with the findings obtained using the luminometer-based assay (Fig. 2) and offer additional evidence that alpha 2AAR and beta 1AR co-internalize from the cell surface following stimulation with alpha 2-adrenergic agonists.


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Fig. 3.   Immunofluorescence confocal microscopy reveals agonist-promoted co-internalization of alpha 2A- and beta 1-adrenergic receptors. HA-alpha 2AAR (red) and FLAG-beta 1AR (green) were co-transfected into HEK-293 cells and visualized using secondary antibodies coupled to rhodamine and FITC, respectively. In the absence of agonist stimulation, immunostaining for both receptors was found predominantly in the plasma membrane (A-C). Stimulation with isoproterenol (Iso) for 10 min induced significant mobilization of beta 1AR inside the cell (D) but had no significant effect on the subcellular distribution of alpha 2AAR (E and F). Stimulation with UK 14,034 (UK), in contrast, resulted in significant internalization of both alpha 2AAR (H) and beta 1AR (G) and marked co-localization of the two receptors in intracellular regions (I, with co-localization indicated in yellow). The specificity of staining was determined in control (Con) experiments using both untransfected and transfected cells incubated in the absence and presence of the relevant primary antibodies. These data are representative of 3-5 experiments for each condition.

Ligand Binding and Signaling of Coexpressed alpha 2A- and beta 1-Adrenergic Receptors-- Because coexpressed alpha 2AAR was able to regulate beta 1AR internalization, we next examined if coexpression with alpha 2AAR was able to regulate beta 1AR pharmacological properties. The binding of the beta AR-selective antagonist [3H]DHA to lysed membranes derived from cells transfected with either beta 1AR alone or beta 1AR/alpha 2AAR was examined. Saturation binding studies revealed that [3H]DHA bound with comparable affinity to beta 1AR expressed in the absence and presence of alpha 2AAR coexpression (KD = 2.8 ± 0.5 nM, Bmax = 39.3 ± 7.7 pmol/mg for beta 1AR alone; KD = 2.3 ± 0.4 nM, Bmax = 33.0 ± 6.5 pmol/mg for beta 1AR/alpha 2AAR). However, studies examining the displacement of [3H]DHA binding by a variety of beta AR-selective ligands revealed that many of these compounds exhibited altered affinity for beta 1AR coexpressed with alpha 2AAR relative to beta 1AR expressed alone. Inhibition curves for displacement of [3H]DHA binding to membranes expressing beta 1AR 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 beta 1AR/alpha 2AAR 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 beta 1AR/alpha 2AAR heterodimers than to beta 1AR alone. On the other hand, norepinephrine, an endogenous agonist for both alpha - and beta -adrenergic receptors, exhibited slightly enhanced affinity for binding to the beta 1AR in the presence of alpha 2AAR coexpression as compared with beta 1AR expressed alone, whereas epinephrine, which is also an endogenous agonist for both receptors, exhibited no significant change in its apparent affinity for beta 1AR alone versus beta 1AR/alpha 2AAR. In control experiments, membranes derived from cells expressing beta 1AR alone and alpha 2AAR 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 beta 1AR were observed for any of the ligands examined (data not shown), suggesting that beta 1AR and alpha 2AAR must be expressed in the same cell for the modulation of beta 1AR pharmacological properties to occur. Furthermore, the effects of alpha 2AAR coexpression on beta 1AR 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 alpha 2AAR. In related experiments, the binding of various alpha 2AR-selective ligands to membranes expressing alpha 2AAR alone versus beta 1AR/alpha 2AAR was examined. No differences in the binding properties of the alpha 2AR-selective agonist UK 14,034, the alpha 2AR-selective partial agonist clonidine, or the alpha 2AR-selective partial agonist guanfacine were observed (Table II), indicating that although beta 1AR possesses altered pharmacological properties when expressed in the presence of alpha 2AAR, it does not seem to reciprocally be the case that alpha 2AAR possesses altered pharmacological properties when expressed in the presence of beta 1AR.


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Fig. 4.   Coexpression with alpha 2AAR alters beta 1AR pharmacological properties. HEK-293 cells were transfected with either FLAG-beta 1AR alone (filled squares, solid line) or HA-alpha 2AAR/FLAG-beta 1AR (open triangles, dashed line). Membranes were prepared, and the binding of the beta -adrenergic antagonist [3H]DHA was studied in the presence of increasing concentrations of the beta -adrenergic antagonist bisoprolol. The apparent binding affinity of bisoprolol decreased in the presence of alpha 2AAR coexpression (please see Table I for a summary of other ligands examined in these experiments). Notably, the curve for bisoprolol inhibition of [3H]DHA binding was well fit by assuming a single binding site in the case of beta 1AR alone but was a significantly better fit by a two-site analysis in the case of the beta 1AR/alpha 2AAR co-transfected samples. The points and error bars shown are the means ± S.E. for 4 independent determinations each.


                              
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Table I
Ligand binding properties of beta 1AR expressed in the absence and presence of alpha 2AAR
The binding of [3H]DHA to lysed membranes was studied in the presence of increasing concentrations of various adrenergic receptor ligands. The estimated Ki 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 beta 1AR/alpha 2AAR 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 beta 1AR alone were not significantly better fit in any case by two-site fits relative to one-site fits. Hence, two Ki values (KH for the high affinity component and KL for the low affinity component) are provided for binding to beta 1AR/alpha 2AAR for most of the ligands, whereas only a single Ki value is provided for binding to beta 1AR expressed alone. Note that for all of the two-site fits, the KH value for binding to beta 1AR/alpha 2AAR is similar to the single Ki value for binding to alpha 2AAR alone, suggesting that the majority of binding sites in the membranes expressing beta 1AR/alpha 2AAR possess binding properties similar to the binding sites in membranes expressing beta 1AR alone. However, membranes expressing beta 1AR/alpha 2AAR also exhibit, in most cases, a small low affinity component (KL), 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.


                              
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Table II
Ligand binding properties of alpha 2AAR expressed in the absence and presence of beta 1AR
The binding of [3H]RX821002 to lysed membranes was studied in saturation binding studies, and no significant differences were found for alpha 2AAR/beta 1AR relative to alpha 2AAR alone (KD = 6.0 ± 2.1 nM, Bmax = 7.7 ± 1.8 pmol/mg for alpha 2AAR alone; KD = 4.6 ± 1.5 nM, Bmax = 6.8 ± 1.6 pmol/mg for alpha 2AAR/beta 1AR). The binding of [3H]RX821002 to lysed membranes was also studied in the presence of increasing concentrations of several other alpha -adrenergic receptor ligands. The estimated Ki values (in nM) are shown for each ligand. The levels of significance of differences in ligand binding to alpha 2AAR/beta 1AR relative to alpha 2AAR alone were assessed via t tests, and no significant differences were found between any of the matched sets. Moreover, one-site versus two-site fits were performed as described under "Materials and Methods" and in no cases were two-site fits significantly better than one-site fits. Thus, alpha 2AAR ligand binding properties showed no obvious differences when alpha 2AAR was examined in the absence and presence of beta 1AR coexpression. The data for these inhibition curves were derived from three independent determinations for each ligand.

We next examined the ability of alpha 2AAR to modulate beta 1AR signaling. We utilized a transfection-based approach to study beta 1AR stimulation of cAMP production in the absence and presence of alpha 2AAR coexpression in HEK-293 cells. These studies revealed that isoproterenol was significantly less potent at stimulating cAMP production when beta 1AR was expressed in the presence of alpha 2AAR than when beta 1AR was expressed alone (Fig. 5). The maximal extent of cAMP production, however, was comparable in both cases, and the expression level of beta 1AR was unaltered by coexpression of alpha 2AAR. Moreover, the effect of alpha 2AAR coexpression on isoproterenol-induced cAMP production was not attributable to constitutive coupling of alpha 2AAR to Gi, because the effect was not blocked by pertussis toxin treatment (data not shown). These data reveal that isoproterenol has a higher potency at beta 1AR expressed alone versus beta 1AR coexpressed with alpha 2AAR. These findings are consistent with the ligand binding data presented in Table I, which indicate that coexpression with alpha 2AAR results in reduced beta 1AR affinity for isoproterenol and other beta AR-selective ligands.


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Fig. 5.   Coexpression of beta 1AR with alpha 2AAR alters the potency of isoproterenol-induced stimulation of adenylyl cyclase. HEK-293 cells were transfected with either beta 1AR alone (filled squares, solid line) or beta 1AR/alpha 2AAR (open triangles, dotted line). Expression levels of the beta 1AR were identical for the two transfection conditions, as assessed by Western blot. The cells were stimulated with increasing concentrations of isoproterenol, and agonist-induced rises in cellular cyclic AMP were quantified. The maximal extent of cyclic AMP produced in the beta 1AR/alpha 2AAR cells was 106 ± 8% of that produced in the cells transfected with only beta 1AR. The EC50 for isoproterenol stimulation of beta 1AR alone was 0.16 ± 0.02 nM, as compared with 0.68 ± 0.17 for beta 1AR/alpha 2AAR (significantly different from beta 1AR alone, p < 0.01). The points and error bars represent the means and S.E. values for four independent determinations.

Regulation of alpha 2A-AR/beta 1AR Heterodimerization by Receptor Glycosylation-- Glycosylation of G protein-coupled receptors can have variable effects on receptor trafficking and signaling (20). The beta 1AR contains one consensus site for N-linked glycosylation on its extracellular amino terminus (Asn-15). We mutated this site to alanine, creating a mutant receptor (N15A) that exhibited a significant decrease in apparent size on SDS-PAGE (Fig. 6A). Enzymatic deglycosylation with N-glycosidase F also decreased the apparent size of the wild-type beta 1AR on SDS-PAGE but had no effect on the apparent size of the N15A mutant receptor suggesting that Asn-15 is the sole site of beta 1AR N-linked glycosylation (21). We examined the capacity of the N15A mutant receptor for heterodimerization with the alpha 2AAR. HA-alpha 2AAR was coexpressed with either FLAG-beta 1AR wild-type or FLAG-beta 1AR N15A, which exhibited equivalent levels of total cellular expression as shown in Fig. 6A. The FLAG-tagged beta 1ARs were immunoprecipitated with an anti-FLAG antibody, and the amount of co-immunoprecipitated HA-alpha 2AAR was examined via Western blot (Fig. 6B) and quantified (as shown in Fig. 6C). Strikingly, alpha 2AAR was co-immunoprecipitated much more efficiently with the N15A mutant beta 1AR than with the wild-type beta 1AR. These data reveal that blockade of beta 1AR glycosylation enhances beta 1AR heterodimerization with alpha 2AAR.


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Fig. 6.   Heterodimerization of alpha 2AAR and beta 1AR is enhanced when receptor glycosylation is blocked. HEK-293 cells were transfected with HA-alpha 2AAR and FLAG-beta 1AR wild-type (lane 1), HA-alpha 2AAR wild-type and the FLAG-beta 1AR N15A mutant (lane 2), or empty vectors (lane 3). The total expression levels of all the transfected proteins are shown in A. For the anti-FLAG-beta 1AR samples (right), note the decrease in the apparent size of the band for the N15A mutant, corresponding to the decreased glycosylation of this mutant receptor. For both wild-type and N15A mutant beta 1AR, several higher order immunoreactive bands were evident in transfected cell lysates, but only the lowest molecular weight band (~54 kDa) is shown here to demonstrate the comparable levels of expression of the wild-type and mutant receptors. The transfected cells were harvested, solubilized, and incubated with anti-FLAG affinity resin to immunoprecipitate FLAG-beta 1AR. The resultant immunoprecipitates (IP) were run on 4-20% SDS-PAGE gels and probed on Western blots (IB) to detect both anti-HA (B, left blot) and anti-FLAG (B, right blot) immunoreactivity. The FLAG-tagged wild-type and N15A mutant beta 1-adrenergic receptors were immunoprecipitated equally (B, right blot). The amount of HA-alpha 2AAR that was co-immunoprecipitated (B, left blot) differed markedly, however, with the N15A mutant pulling down an average of nearly 3-fold more HA-alpha 2AAR than the wild-type beta 1AR. These data reveal that beta 1AR/alpha 2AAR heterodimerization is enhanced for the N15A mutant beta 1AR relative to wild-type beta 1AR. Quantification of all of these data is shown in C. Similar experiments were performed in the experiments illustrated in D-F, except that the three lanes that are shown correspond to HA-alpha 2AAR and FLAG-beta 1AR wild-type (lane 1), HA-alpha 2AAR N10A/N14A mutant and FLAG-beta 1AR wild-type (lane 2), and empty vectors (lane 3). Blockade of alpha 2AAR glycosylation, like blockade of beta 1AR glycosylation, resulted in enhanced heterodimerization between the two receptors. The bars and error bars shown in C and F represent the means ± S.E. for 4 independent determinations for each condition. WT, wild type.

Because alpha 2AAR/beta 1AR heterodimerization was more efficient for the N15A mutant, we examined whether or not alpha 2AAR might exert a more robust regulation of N15A-beta 1AR relative to wild-type beta 1AR. However, we found that agonist-promoted beta 1AR internalization following a 10-min stimulation with UK 14,304 was not significantly different for wild-type beta 1AR versus the N15A mutant (wild type = 16.8 ± 3.5%; N15A = 18.0 ± 4.4% in matched plates examined side-by-side; n = 3). Similarly, the changes in ligand binding properties induced by coexpression with alpha 2AAR were comparable for the wild-type beta 1AR and the N15A mutant (data not shown). Thus, although blockade of beta 1AR glycosylation results in a clear enhancement of alpha 2AAR/beta 1AR heterodimerization, it may not lead to an enhancement in heterodimerization of functional receptors on the cell surface. This observation may be related to the fact that the N15A mutant beta 1AR is deficient in its ability to traffic to the cell surface relative to the wild-type receptor (21).

The alpha 2AAR is also known to be glycosylated on its amino terminus, on residues Asn-10 and Asn-14 (22). We therefore prepared a mutant version of the alpha 2AAR (N10A/N14A) that cannot be glycosylated. Transfection of this mutant construct into HEK-293 cells resulted in the expression of receptors with significantly decreased apparent size on SDS-PAGE gels relative to wild-type alpha 2AAR (Fig. 6D), as reported previously (22). The heterodimerization of the HA-tagged N10A/N14A mutant with wild-type FLAG-beta 1AR was assessed in side-by-side experiments in comparison to the heterodimerization of wild-type alpha 2AAR with wild-type FLAG-beta 1AR (Fig. 6E). The N10A/N14A mutant alpha 2AAR exhibited an ~3-fold enhancement over wild-type alpha 2AAR in heterodimerization with beta 1AR (Fig. 6F). Taken together with the experiments described above examining the N15A beta 1AR, these findings indicate that blockade of glycosylation of both alpha 2AAR and beta 1AR results in enhanced alpha 2AAR/beta 1AR heterodimerization.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our findings reveal a functionally important heterodimerization between alpha 2A- and beta 1-adrenergic receptors. The evidence for the physical association of these two receptor subtypes is derived from both co-immunoprecipitation and co-internalization assays. The co-internalization experiments not only represent evidence for the physical association of the two receptors, they also represent a specific mechanism by which alpha 2AAR stimulation may influence beta 1AR function. Although alpha 2ARs and beta ARs couple primarily to G proteins with opposing cellular effects on cAMP production, it is known that agonist activation of alpha 2ARs can in some cases paradoxically sensitize beta AR signaling in brain tissue (2-4). Because internalization of GPCRs is known to play a key role in promoting GPCR resensitization (19), our observation that alpha 2AAR stimulation can promote beta 1AR internalization provides a specific molecular mechanism that could potentially account for the previously reported ability of alpha 2AR stimulation to sensitize beta AR-mediated responses in native tissues.

Coexpression of alpha 2AAR and beta 1AR in our studies not only allowed for alpha 2AAR regulation of beta 1AR internalization, it also resulted in altered beta 1AR pharmacological properties. We found that the curves for displacement of [3H]DHA binding to membranes expressing beta 1AR alone were fit well by one-site analyses for all ligands examined. Whereas it is true that analyses of agonist binding to beta -adrenergic receptors often require resolution into two sites, which correspond to G protein-coupled (high affinity) versus uncoupled (low affinity) states (23), our analyses of agonist binding to beta 1AR alone were fit well by assuming a single site. This is probably due to the fact that the transfected beta 1AR was expressed in our cells at much higher levels than the endogenous G proteins, meaning that the G protein-coupled (high affinity) component of agonist binding to the receptors in our assays represented only a tiny and unresolvable component of the inhibition curves. In any case, coexpression of beta 1AR with alpha 2AAR resulted in the appearance of a significant low affinity component of binding for many of the beta AR-selective ligands. Our interpretation of these data is that a proportion of the beta 1AR in the cells assembled with alpha 2AAR to form heterodimers that exhibited unaltered affinity for some ligands (such as DHA and propranolol), substantially reduced affinity for other ligands (such as metoprolol, labetalol, bisoprolol, isoproterenol and dobutamine), and slightly increased affinity for yet other ligands (such as the endogenous agonist norepinephrine). Many of the ligands examined share significant structural similarity, and it is therefore uncertain why the binding properties of the various ligands should be differentially altered by beta 1AR coexpression with alpha 2AAR. Moreover, the affinity constant values derived for the low affinity component of binding in these studies must be considered as rough estimates, because it is difficult to derive accurate affinity constant estimates from curves where the size of the low affinity component (10-25%) represents such a small minority of the total population of binding sites.

A question of interest is why coexpression of beta 1AR with alpha 2AAR should result in ligand binding curves that are best fit by two sites, rather than simply resulting in a single population of novel binding sites as might be expected if every beta 1AR were to heterodimerize with an alpha 2AAR to form receptors with novel pharmacological properties. The most likely explanation for the observed mixed population of binding sites is that, regardless of how efficiently alpha 2AAR heterodimerizes with beta 1AR, it is unlikely that all beta 1AR in a given cell will form heteromeric complexes with coexpressed alpha 2AAR. A large proportion of cellular beta 1-adrenergic receptors are likely to exist either as monomers or homodimers, with only a fraction of the total beta 1AR population assembling with other receptors such as alpha 2AAR. Thus, studies examining the binding of ligands to co-transfected alpha 2AAR/beta 1AR are almost certainly studying mixed populations of receptors, complicating attempts to estimate the true changes in beta 1AR pharmacological properties induced by heterodimerization with alpha 2AAR. This is a general problem shared by all studies examining pharmacological changes induced by heterodimerization of GPCRs.

Over the past several years, heterodimerization of a number of different types of GPCRs has been reported. Examples where heterodimerization is required for the formation of functional receptors include the GABA receptors GABABR1 and GABABR2 (10) and the taste receptor combinations T1R1/T1R3 and T1R2/T1R3, which have been reported to form receptors for umami and sweet stimuli, respectively (24-26). Examples where heterodimerization allows for cross-regulation between receptors but is not required for receptor function include the following: beta 1- and beta 2-adrenergic receptors (15); delta  and kappa  opioid receptors (27); delta  and µ opioid receptors (28, 29); delta  opioid and beta 2-adrenergic receptors (30, 31); muscarinic acetylcholine M2 and M3 receptors (32); angiotensin AT1 and bradykinin B2 receptors (33); dopamine D1 and adenosine A1 receptors (34); dopamine D2 and somatostatin SSTR5 receptors (35); dopamine D2 and adenosine A2 receptors (36); mGluR1 glutamate and A1 adenosine receptors (37); SSTR1 and SSTR5 somatostatin receptors (38); SSTR2A and SSTR3 somatostatin receptors (39); and µ opioid and SSTR2A somatostatin receptors (40). Many of these receptor/receptor interactions have been found to result in altered pharmacological properties for one or both receptors (27-29, 32, 35, 38, 39), similar to what we have found for the alpha 2AAR/beta 1AR interaction. Additionally, several of the previously reported (30, 36, 40) receptor-receptor interactions have been found to facilitate receptor co-internalization, similar to our observation that stimulation of alpha 2AAR can lead to co-internalization of beta 1AR.

In both the luminometer-based surface expression assays and the immunofluorescence microscopy experiments performed on alpha 2AAR/beta 1AR co-transfected cells, we observed that alpha 2AAR stimulation resulted in internalization of both alpha 2AAR and beta 1AR, whereas stimulation of beta 1AR resulted in internalization of only beta 1AR. The reason for this difference is not clear. It may be case that the alpha 2AAR/beta 1AR heterodimer has internalization properties that are distinct from either of the two individual receptors. Alternatively, alpha 2AAR/beta 1AR heterodimerization may be impaired by beta -adrenergic agonist stimulation, allowing beta 1AR to temporarily internalize in the absence of alpha 2AAR co-internalization. Our co-immunoprecipitation studies, however, did not reveal any consistent effects of agonist stimulation on the amount of HA-alpha 2AAR co-immunoprecipitated with FLAG-beta 1AR. It is uncertain, however, whether or not this technique is sensitive enough to detect changes in alpha 2AAR/beta 1AR co-immunoprecipitation in the range of 10-20%, as might be expected if receptor internalization were correlated with a temporary release from heterodimerization. One thing that is interesting to note is the similarity between our findings for the effects of alpha 2AAR/beta 1AR heterodimerization on receptor ligand binding properties versus internalization; we found that assembly with alpha 2AAR influenced beta 1AR pharmacological properties, whereas conversely assembly with alpha 2AAR did not result in any evident change in the ligand binding properties of alpha 2AAR. Similarly, we found that alpha 2AAR stimulation led to beta 1AR internalization, but conversely beta 1AR stimulation did not lead to any evident internalization of alpha 2AAR. Thus, for both ligand binding and internalization, alpha 2AAR was able to influence beta 1AR, but beta 1AR was not able to influence alpha 2AAR.

If beta 1-adrenergic receptors can form beta 1AR/beta 1AR homodimers (14), beta 1AR/beta 2AR heterodimers (15), and beta 1AR/alpha 2AAR heterodimers, as the present data reveal, it is a point of significant interest to understand the factors that regulate the proportion of cellular homodimers versus heterodimers. Studies on the dimerization of other GPCRs have provided evidence that agonist stimulation can regulate dimerization (9). However, as mentioned above, our co-immunoprecipitation experiments did not reveal any consistent effects of agonist stimulation on alpha 2AAR/beta 1AR heterodimerization. Association with cytoplasmic scaffold proteins is another factor that might potentially regulate heterodimer formation. The beta 1-adrenergic receptor is known to associate with PSD-95/Discs-large/ZO-1 homology domain-containing scaffold proteins such as PSD-95 (14, 41, 42) and MAGI-2 (14). However, we have not observed any significant effects of PSD-95 or MAGI-2 coexpression on the extent of either beta 1AR/beta 1AR homodimerization (14) or alpha 2AAR/beta 1AR heterodimerization (data not shown). The alpha 2A-adrenergic receptor is known to associate with cytoplasmic proteins such as 14-3-3 (43) and spinophilin (44), but we have not examined the effects of these interactions on alpha 2AAR/beta 1AR heterodimerization.

One additional way that receptor heterodimerization might be regulated is via post-translational receptor modifications. A post-translational modification common to many GPCRs is receptor glycosylation (20). We have found that the beta 1AR is N-glycosylated on a single residue, Asn-15, and that alpha 2AAR/beta 1AR heterodimerization is markedly enhanced via mutation of Asn-15 to an amino acid that cannot be glycosylated. Moreover, we have also found that alpha 2AAR/beta 1AR heterodimerization is enhanced by blocking glycosylation of the alpha 2AAR. These data could indicate that lack of glycosylation alters the conformations of the alpha 2AAR and beta 1AR such that the efficiency of their heterodimerization is increased. Alternatively, it is possible that the enhanced heterodimerization of the N10A/N14A alpha 2AAR and N15A beta 1AR is the result of a more global alteration in the trafficking and processing of the mutant receptors. Interestingly, we have found previously (21) that beta 1AR homodimerization is impaired for the beta 1AR-N15A mutant relative to the wild-type receptor. Thus, our data indicate that blockade of glycosylation has differential effects on beta 1AR homo- versus heterodimerization. In any case, it is known that the glycosylation state of transmembrane receptors can vary significantly in different tissue types (45, 46). The glycosylation state of the beta 1AR in particular is known to be regulated via polymorphic variation (47). Thus, the extent of alpha 2AAR/beta 1AR heterodimerization may be regulated differentially between tissues and between individuals via differences in beta 1AR glycosylation state.

In summary, we have found a physical association between alpha 2A- and beta 1-adrenergic receptors. This heterodimerization alters beta 1AR pharmacological properties and facilitates cross-internalization of beta 1AR following alpha 2AAR agonist stimulation. Both alpha 2AAR and beta 1AR are abundantly expressed in the brain, and heterodimerization of these two receptors might therefore underlie previously reported functional cross-talk between endogenous alpha 2- and beta -adrenergic receptors in brain tissue (2-8). Therapeutic drugs acting on alpha 2ARs (such as clonidine) and beta -adrenergic receptors (such as propranolol and metoprolol) are commonly utilized in the treatment of hypertension and are known to exhibit significant clinical interactions (48, 49). The heterodimerization of alpha 2AAR and beta 1AR described here may help to provide new insights into both physiological cross-talk between alpha 2- and beta -adrenergic receptors and clinical interactions between therapeutic drugs acting on these receptor subtypes.

    ACKNOWLEDGEMENTS

We thank Robert Lefkowitz (Duke University) for providing the FLAG-beta 1AR construct, Hitoshi Kurose (University of Tokyo) for providing the HA-beta 1AR construct, Lee Limbird (Vanderbilt University) for providing the HA-alpha 2AAR construct, and Ken Minneman (Emory University) for helpful discussion. We also thank Asha Shah for excellent technical assistance with the creation of the N10A/N14A alpha 2AAR mutant.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants RO1-GM60982 and RO1-HL64713 (to R. A. H.) and by a Distinguished Young Investigator in Medical Sciences award from the W. M. Keck Foundation (to R. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, Emory University School of Medicine, 5113 Rollins Research Center, 1510 Clifton Rd., Atlanta, GA 30322. Tel.: 404-727-3699; Fax: 404-727-0365; E-mail: rhall@pharm.emory.edu.

Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M207968200

    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; AR, adrenergic receptor; GABA, gamma -aminobutyric acid; PSD-95, post-synaptic density protein of 95 kDa; MAGI, membrane-associated guanylate kinase-like protein with an inverted domain structure; HA, hemagglutinin; HEK, human embryonic kidney; DHA, dihydroalprenolol; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate.

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ABSTRACT
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RESULTS
DISCUSSION
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