COMMUNICATION:
A Novel Interaction between Adrenergic Receptors and the alpha -Subunit of Eukaryotic Initiation Factor 2B*

(Received for publication, March 25, 1997, and in revised form, May 2, 1997)

Uwe Klein Dagger §par , M. Teresa Ramirez Dagger §**, Brian K. Kobilka Dagger Dagger Dagger and Mark von Zastrow §§§

From the § Nina Ireland Laboratory, Departments of Psychiatry and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-0984 and the Dagger Dagger  Howard Hughes Medical Institute and Dagger  Division of Cardiovascular Medicine, Stanford University Medical School, Stanford, California 94305

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The alpha -subunit of eukaryotic initiation factor 2B (eIF-2B), a guanine nucleotide exchange protein that functions in regulation of translation, was observed to associate with the carboxyl-terminal cytoplasmic domains of the alpha 2A- and alpha 2B-adrenergic receptors in a yeast two-hybrid screen of a cDNA library prepared from 293 cells. This protein association was confirmed in vitro by affinity chromatography and was shown to be specific for a subset of G protein-coupled receptors, including the alpha 2A-, alpha 2B-, alpha 2C-, and beta 2-adrenergic receptors, but not the vasopressin (V2) receptor. Association of these proteins in vivo was confirmed by specific co-immunoprecipitation of eIF-2Balpha with full-length beta 2-adrenergic receptors expressed in transfected 293 cells and by fluorescence microscopy showing co-localization of these proteins in intact cells. Remarkably, eIF-2Balpha co-localized with receptors exclusively in regions of the plasma membrane that are in contact with the extracellular medium, but failed to associate with membranes making cell-cell contacts. Overexpression of eIF-2Balpha in 293 cells caused a small (~15%) but significant enhancement of beta 2-adrenergic receptor-mediated activation of adenylyl cyclase, without affecting forskolin or V2 receptor-mediated activation. These observations suggest a new role for a previously identified guanine nucleotide exchange protein in membrane biology and cell signaling.


INTRODUCTION

G protein-coupled receptors interact with several classes of cytoplasmic proteins, including heterotrimeric G proteins, kinases, phosphatases, and arrestins (1-4). Specific roles of these protein associations in receptor signaling and regulation are now well established. These protein interactions were first inferred from their functional effects on receptor signaling and desensitization before direct physical associations of these proteins with receptors were observed biochemically (5-8). This observation raises the possibility that receptors may interact with additional cellular proteins that could play unanticipated roles in determining the efficacy or specificity of receptor-G protein coupling.

We have investigated this possibility by searching for novel protein interactions with adrenergic receptors, focusing on the carboxyl-terminal cytoplasmic domain because mutations within this domain have pleiotropic effects on receptor physiology (9-12).1 Using interaction cloning and biochemical techniques, we have observed that several subtypes of adrenergic receptors associate specifically with eIF-2Balpha ,2 the smallest subunit of a cytoplasmic guanine nucleotide exchange factor. While this protein has a well defined role in regulation of translation, it has never been shown previously to interact with any membrane receptor. Immunocytochemical studies suggest that receptors associate with eIF-2Balpha only in restricted regions of the plasma membrane, and functional studies suggest that this protein interaction may play a role in the regulation of receptor-mediated signaling.


EXPERIMENTAL PROCEDURES

Yeast Two-hybrid Cloning

The MATCHMAKER II two-hybrid system (Clontech) was used to screen a 293-cell cDNA library (complexity ~2.5 × 106 total recombinants) constructed in pACT2 (Clontech) with the COOH-terminal cytoplasmic domains of both the murine alpha 2A- and alpha 2B-adrenergic receptors (13, 14). Bait plasmids were constructed in pAS2-1 (Clontech) using the carboxyl-terminal cytoplasmic domains from the alpha 2A and alpha 2B receptors, starting from the "NPXXY-motif" in the seventh transmembrane domain (e.g. amino acids 422-450 of the alpha 2B receptor), which were amplified using the polymerase chain reaction. Screening of ~1 × 106 transformants yielded two independent clones (B8 and B10), which interacted with both the alpha 2A and alpha 2B receptor tails, but not with the lamin C control (Clontech protocol). The coding sequences of both clones were identical to human eIF-2Balpha (EBI data base, accession number X95648).

In Vitro Transcription/Translation

The full-length coding sequence of eIF-2Balpha was subcloned into the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA). This construct, referred to as the M1 form, begins with the first methionine in the predicted coding sequence. Amino-terminal truncations were constructed at each of two downstream ATG codons to generate versions beginning at methionine 14 or 20 relative to the first predicted methionine and are referred to as M14 and M20, respectively. Mutant constructs included an engineered HindIII site followed by a Kozak consensus sequence to promote initiation of translation at the selected residue (15, 16). Constructs were generated using PCR amplification and subcloned into pcDNA3, and sequences were verified using dideoxynucleotide sequencing (Sequenase, U. S. Biochemical Corp.). In vitro translation of these constructs was performed in the presence of 35S-labeled methionine (Amersham Corp.) using the T7 RNA polymerase promoter and a coupled in vitro transcription/translation system (Promega, Madison, WI).

GST-fusion Protein Affinity Chromatography

Cytoplasmic receptor tails were amplified by PCR and cloned into a pGEX vector derived from pGEX-KG and expressed in Escherichia coli (17). GST fusion proteins were prepared as described (18). The GST-fusion protein load of individual resins was normalized by densitometric scanning of SDS-polyacrylamide gels stained with Coomassie Blue. For affinity chromatography of in vitro translated eIF-2Balpha on the different receptor tails, 30 µl of the GST-fusion protein loaded resins (50% (v/v) suspensions) were preblocked in binding buffer (20 mM Hepes, pH 7.4, 100 mM KCl, 5 mM MgCl2, 0.1% Triton X-100) with 10 mg/ml ovalbumin for 15 min at room temperature. In vitro translated, [35S]methionine-labeled proteins were incubated with the GST-fusion protein resins in binding buffer for 1 h at room temperature. Resins were washed four times with binding buffer and eluted with SDS-PAGE sample buffer for analysis by SDS-PAGE and fluorography.

Co-immunoprecipitation

Carboxyl-terminally HA epitope-tagged versions of the full-length (M1) and amino-terminally truncated (M20) forms of eIF-2Balpha were constructed by PCR and subcloned into pcDNA3. These constructs were expressed in human embryonal kidney 293 cells (ATCC), or in 293 cells stably transfected with FLAG-tagged human beta 2-adrenergic receptors (19), by transient transfection using calcium phosphate precipitation. Cells were harvested 48-72 h after transfection and lysed in 20 mM Hepes, pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 0.1% Triton X-100. Receptors were immunoprecipitated with M1 monoclonal antibody, recognizing the FLAG epitope (Eastman Kodak Co.) and protein A-Sepharose (Pharmacia Biotech Inc.). Samples were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with anti-HA monoclonal antibody (HA.11, Berkeley Antibody Co., Richmond, CA). Epitope-tagged eIF-2Balpha was detected using horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch, West Grove, PA) and enzyme-linked chemiluminescence (ECL, Amersham).

Immunofluorescence Microscopy

HA epitope-tagged versions of eIF-2Balpha were transiently transfected into a 293-cell line stably expressing the NH2-terminally FLAG epitope-tagged beta 2-adrenergic receptor. Cells were grown on glass coverslips, fixed, and permeabilized as described (20). HA epitope-tagged eIF-2Balpha was detected using the monoclonal mouse antibody HA.11 (Babco, Richmond, CA), and beta 2-adrenergic receptor was detected using receptor-specific rabbit antiserum (21).

Adenylyl Cyclase Assay and Receptor Quantification

293 cells were transiently transfected with carboxyl-terminally HA epitope-tagged eIF-2Balpha and/or FLAG-epitope tagged beta 2-adrenergic receptor or human vasopressin V2 receptor constructs (22) using the LipofectAMINE protocol (Life Technologies, Inc.). Transfected cells plated in 12-well plates were labeled for 18-24 h with 4 µCi/ml [2,8-3H]adenine (NEN Life Science Products) and incubated in Hepes-buffered medium containing 1 mM 3-isobutyl-1-methylxanthine with or without the indicated drugs for 30 min at 37 °C. Reactions were terminated by adding 0.5 ml of 5% trichloroacetic acid containing 1 mM ATP and 1 mM cAMP to each well. Intracellular [3H]ATP and [3H]cAMP were separated subsequently on ion-exchange and alumina columns as described (23). cAMP accumulation was expressed as the ratio cAMP/(cAMP + ATP). For receptor quantification, cells were lysed in 5 mM Tris/HCl, 2 mM EDTA, pH 7.4, using a glass homogenizer. Crude membranes were pelleted by centrifugation, washed, and resuspended in the same buffer. For assessment of receptor expression levels, 10-µg membrane protein aliquots were incubated with 125I-cyanopindolol (250 pM) in 75 mM Tris/HCl, 12.5 mM MgCl2, 1 mM EDTA, pH 7.4, at room temperature for 3 h. Binding reactions were terminated by rapid filtration over Whatman glass fiber filters. Specific binding was defined as the amount of 125I-cyanopindolol binding inhibited by 1 µM (-)-alprenolol. Nonspecific binding represented <1% of the total binding measured.


RESULTS AND DISCUSSION

The yeast two-hybrid system (24, 25) was used to identify candidate proteins that interact with the carboxyl-terminal cytoplasmic tails of the alpha 2A- and alpha 2B-adrenergic receptors. Screening of approximately 1 × 106 transformants resulted in the isolation of two independent clones that interacted specifically with both the alpha 2A- and the alpha 2B-tails. Both of these clones encoded the same polypeptide, a full-length form of the alpha -subunit of eIF-2B. eIF-2Balpha is a subunit of a heteropentameric guanine nucleotide exchange factor, which has a well established function in regulating the initiation of protein translation by mediating GTP exchange on eIF-2 (26-29).

Two products were observed by in vitro translation of this coding sequence, with apparent molecular masses of 34 and 31 kDa (Fig. 1A, first lane). Sequence analysis revealed two favorable Kozak translation initiation sequences (15, 16) in the cloned cDNA (corresponding to methionines in positions 1 (M1) and 20 (M20) of the predicted amino acid sequence) as noted previously in studies of rat eIF-2Balpha (30). The mobility of the two translation products is in agreement with the molecular masses predicted for the M1 (33.7 kDa) and M20 (31.4 kDa) forms by sequence analysis. This was further confirmed by comparing the SDS-PAGE mobility of the translation products with those produced from amino-terminally truncated sequences (Fig. 1A).


Fig. 1. Specific binding of eIF-2Balpha to adrenergic receptors. A, in vitro transcription/translation of eIF-2Balpha . The coding sequence of full-length eIF-2Balpha (M1) was subcloned into pcDNA3 and in vitro translated. The [35S]methionine-labeled translation product of the full-length coding sequence migrates as two bands in the SDS-PAGE, with apparent molecular masses of 34 and 31 kDa (first lane). To investigate on the identity of the translation products, two 5'-truncated versions of the coding sequence starting with either the second or third ATG codons (referred to as M14 and M20, respectively) were in vitro translated. Translation of M14 (lane 2) yields two smaller translation products migrating as a broad doublet, and no detectable 34-kDa polypeptide. Translation of M20 (lane 3) results in a single product co-migrating with the 31-kDa polypeptide observed by in vitro translation of the full-length coding sequence. B, binding of in vitro translated eIF-2Balpha to different receptor tails expressed as GST fusion proteins. Affinity chromatography of in vitro translated full-length cDNA (corresponding to lane 1 in A) on immobilized fusion proteins was performed as described under "Experimental Procedures." The percentage of in vitro translated eIF-2Balpha bound to the immobilized fusion proteins was estimated by liquid scintillation counting of the eluted fractions (GST, 1%; alpha 2A-AR, 7%; alpha 2B-AR, 10%; alpha 2C-AR, 8%; beta 2-AR, 12%; V2R, 1%; LDLR, 1%). C, co-imunoprecipitation of eIF-2Balpha with the beta 2-adrenergic receptor. 293 cells or a 293-cell line stably expressing the FLAG-tagged beta 2-adrenergic receptor were transfected with COOH-terminally HA epitope-tagged versions of either full-length (M1-HA) or NH2-terminally truncated (M20-HA) forms of eIF-2Balpha . Cells were lysed and receptor complexes were immunoprecipitated as described under "Experimental Procedures." Whole cell extracts (lanes 1 and 2) or immunoprecipitates (lanes 3-7) were subjected to SDS-PAGE, blotted, and probed for the presence of the HA epitope.
[View Larger Version of this Image (51K GIF file)]

To examine the biochemical specificity of the interaction between eIF-2Balpha and receptor domains, eIF-2Balpha prepared by in vitro translation was tested for specific binding to various cytoplasmic receptor tails fused to GST. Specific binding of eIF-2Balpha was observed to the cytoplasmic tails of the alpha 2A-, alpha 2B-, alpha 2C-, and beta 2-adrenergic receptor, while nonspecific binding to GST alone was negligible (Fig. 1B). eIF-2Balpha did not bind to the carboxyl-terminal cytoplasmic domain of the low density lipoprotein receptor, indicating that eIF-2Balpha interacts specifically with a subset of plasma membrane receptors. No binding was observed to the carboxyl-terminal cytoplasmic domain of the vasopressin (V2) receptor, suggesting further that eIF-2Balpha associates specifically with a limited subset of G protein-coupled receptors. Alignment of the carboxyl-terminal cytoplasmic domains of the alpha 2A-, alpha 2B-, alpha 2C-, and beta 2-adrenergic receptors revealed five identical amino acids (Fig. 2). However, only one of those five residues was found to be identical in the vasopressin V2 receptor. This suggests a potential role of the proximal portion of the carboxyl terminus of the adrenergic receptors in binding of eIF-2Balpha .


Fig. 2. Sequence comparison of the carboxyl termini of the alpha 2-adrenergic receptors, the beta 2-adrenergic receptor and the vasopressin V2 receptor. Sequences are aligned starting from the NPXXY-motif in the seventh transmembrane domain (TM VII). Residues identical in all four of the shown adrenergic receptors are marked. For the beta 2-adrenergic receptor and the vasopressin V2 receptor only the NH2-terminal 22 residues of the cytoplasmic tails are shown.
[View Larger Version of this Image (33K GIF file)]

Interestingly, while both the M1 and M20 forms of eIF-2Balpha were observed to bind to adrenergic receptor tails, examination of SDS-PAGE fluorographs indicated that the M20 form bound considerably more strongly (compare Fig. 1, A and B). These observations suggest that the amino-terminal domain of eIF-2Balpha plays a role in mediating or regulating receptor binding. Since both the M1 and M20 forms of eIF-2Balpha can be produced in vivo from the same transcript (see below), it is possible that these polypeptides may serve different physiological functions according to their different affinities for receptors. An example of this type of specificity is the Oskar protein in Drosophila, where different protein forms produced by alternative start codon usage serve distinct functions in oogenesis (31).

The ability of eIF-2Balpha to bind to full-length beta 2-adrenergic receptors in vivo was examined by co-immunoprecipitation from transfected 293 cells. The beta 2-adrenergic receptor was tagged in the amino-terminal extracellular domain with a FLAG-epitope to facilitate specific immunoprecipitation of receptors using an antibody that does not interfere with the cytoplasmic tail. eIF-2Balpha was HA-tagged in the carboxyl terminus to allow detection of proteins originating from alternate start codon usage. Two protein products, corresponding to the M1 and M20 forms of eIF-2Balpha , were observed in transfected cells. The M1 form was by far the predominant product (Fig. 1C, lanes 1 and 2). Both forms of eIF-2Balpha were co-immunoprecipitated specifically from cell lysates in association with the beta 2-adrenergic receptor (Fig. 1C, lanes 6 and 7). Neither form of eIF-2Balpha was detected in control immunoprecipitates, including those prepared from cells expressing eIF-2Balpha without FLAG-tagged receptors (Fig. 1C, lanes 3-5), confirming the specificity of this protein association in vivo. Interestingly, the M20 form of eIF-2Balpha preferentially associated with immunoprecipitated receptors, even though the M1 form was expressed in significant excess (Fig. 1C, compare lanes 1 and 6). These data further confirm the specificity of the co-immunoprecipitation and suggest that individual forms of eIF-2Balpha display similar binding selectivity for full-length receptors in vivo as they do for isolated carboxyl-terminal cytoplasmic domains in vitro.

The subcellular distribution of epitope-tagged eIF-2Balpha was next examined in transfected cells by fluorescence microscopy. Epitope-tagged eIF-2Balpha was visualized in a diffuse distribution, with increased staining intensity near the cell periphery and no detectable staining at regions of cell-cell contact (Fig. 3A), while beta 2-adrenergic receptors were localized throughout the plasma membrane (Fig. 3B). Immunoblotting of extensively washed membrane fractions prepared from transfected cells confirmed that a significant fraction of eIF-2Balpha was membrane-associated (not shown). Optical sectioning of antibody-labeled cells by confocal microscopy revealed eIF-2Balpha distributed throughout the cytoplasm and excluded from the nucleus, consistent with the known role of this protein in regulating ribosome function in the cytoplasm. In addition, confocal microscopy confirmed that eIF-2Balpha was also closely associated with limited regions of the peripheral plasma membrane (Fig. 3C) which contained relatively high concentrations of beta 2-adrenergic receptor (Fig. 3D). Marked co-localization of eIF-2Balpha (green) with beta 2-adrenergic receptors (red) in these regions of plasma membrane was emphasized by the yellow staining in the merged image (Fig. 3E). This close co-localization, which was observed even at high magnification (Fig. 3F), suggests that these membrane microdomains may be sites of interaction between eIF-2Balpha and receptors. A similar plasma membrane localization pattern for epitope-tagged eIF-2Balpha was observed in cells transfected with eIF-2Balpha alone or in cells co-transfected with eIF-2Balpha and the vasopressin V2 receptor (not shown). These observations and the lack of co-localization of eIF-2Balpha with adrenergic receptors at regions of cell-cell contact suggest that the membrane localization of eIF-2Balpha may be influenced by additional protein interactions. Nevertheless, the co-immunoprecipitation of eIF-2Balpha with beta 2-adrenergic receptors from intact cells (Fig. 1C) suggests that these proteins physically interact at regions of co-localization.


Fig. 3. Immunocytochemical co-localization of eIF-2Balpha and beta 2-adrenergic receptor in specialized regions of the cell membrane. A 293-cell line stably expressing the FLAG-tagged beta 2-adrenergic receptor was transiently transfected with HA epitope-tagged versions of full-length eIF-2Balpha . Immunostaining and fluorescence microscopy were carried out as described under "Experimental Procedures." A and B, in addition to its cytoplasmic distribution, eIF-2Balpha (A) localizes to the plasma membrane, but is excluded from membranes making cell-cell contacts. In contrast, staining for the beta 2-adrenergic receptor (B) is distributed evenly over all membranes. C-F, transfected and immunostained cells were imaged by dual-color confocal fluorescence microscopy. eIF-2Balpha (green channel) (C) localizes both in the cytoplasm and is observed in association with the plasma membrane, where it is concentrated in specialized regions. beta 2-Adrenergic receptor (red channel) (D) co-localizes with eIF-2Balpha in these regions as shown in yellow in the two-color merged image (E). Another example of this co-localization is shown at higher magnification in F.
[View Larger Version of this Image (108K GIF file)]

While eIF-2B has a well established role in regulating initiation of translation in the cytoplasm (27, 29), the association of this protein with receptors in the plasma membrane suggested that eIF-2Balpha may have additional physiological role(s). Several proteins that associate with the carboxyl-terminal cytoplasmic tail of G protein-coupled receptors regulate receptor-mediated signaling. The carboxyl-terminal cytoplasmic domain of the beta 2-adrenergic receptor interacts with G protein-coupled receptor kinases and arrestins, causing functional desensitization by reducing the efficacy and potency of agonist-dependent signaling (1-4). To examine the possibility that eIF-2Balpha may also desensitize receptors, the effect of eIF-2Balpha overexpression on agonist-dependent activation of adenylyl cyclase was examined in cells expressing beta 2-adrenergic receptors. No inhibition of signaling was observed over a wide range of agonist concentrations, suggesting that eIF-2Balpha does not increase receptor desensitization in these cells. In contrast, overexpression of eIF-2Balpha actually had the opposite effect on cell signaling. Overexpression of eIF-2Balpha caused a modest enhancement of receptor-mediated activation of adenylyl cyclase at high concentrations of agonist (not shown). In multiple experiments, activation of adenylyl cyclase caused by the beta agonist isoproterenol (10 µM) was enhanced by an average of 15% in cells co-transfected with HA-tagged eIF-2Balpha and beta 2-adrenergic receptors, when compared with cells examined in parallel that were transfected with beta 2-adrenergic receptors and control plasmid (p < 0.01, n = 11). Radioligand binding assays using 125I-cyanopindolol indicated that eIF-2Balpha overexpression had no effect on the number of beta 2-adrenergic receptors expressed in transfected cells (n = 4). Furthermore, no effect of eIF-2Balpha overexpression was observed under the same conditions on vasopressin V2 receptor-mediated activation of adenylyl cyclase (10 µM arginine vasopressin, n = 4), on basal adenylyl cyclase activity, or on direct activation of adenylyl cyclase by forskolin (10 µM, n = 4). Taken together, these observations are consistent with the hypothesis that eIF-2Balpha specifically enhances signaling by interacting directly with beta 2-adrenergic receptors, rather than by influencing receptor expression or downstream signaling components. Furthermore, the specificity of this enhancement for beta 2-adrenergic receptors compared with V2 receptors is consistent with the biochemical specificity of eIF-2Balpha association with isolated receptor tails observed in vitro.

While eIF-2Balpha caused a significant and reproducible enhancement of adenylyl cyclase activation by beta 2-adrenergic receptors, the magnitude of this effect was relatively small (15% on average) in all experiments. It is possible that endogenous levels of eIF-2Balpha in 293 cells may be sufficient to promote receptor signaling, so a relatively small enhancement is observed by overexpression of this protein. Alternatively, the effect of eIF-2Balpha on receptor signaling may depend on associations with other proteins (such as other subunits of the eIF-2B heteropentamer) that are present in limiting amounts relative to overexpressed eIF-2Balpha .

Additional studies will be necessary to elucidate precisely how eIF-2Balpha associates with the plasma membrane and enhances receptor signaling. Interestingly, recent studies suggest that additional, as yet unidentified, membrane-associated proteins influence the propagation of agonist-induced signals of G protein-coupled receptors in isolated plasma membranes (32, 33). In principle, it is also possible that eIF-2Balpha could enhance receptor-mediated signaling by inhibiting a known mechanism of receptor desensitization (e.g. phosphorylation or arrestin binding). While the present studies have focused exclusively on the functional effects of eIF-2Balpha on receptor signaling, it is also possible that this protein interaction may play an additional role in regulation of translation. This possibility is consistent with the well established role of eIF-2B in the regulation of initiation of translation and with recent studies indicating that various extracellular stimuli can control eIF-2B activity (34-36).

In conclusion, we have shown that eIF-2Balpha interacts specifically with the carboxyl-terminal cytoplasmic domain of a subset of G protein-coupled receptors. These results identify a novel protein interaction with adrenergic receptors, suggesting a new role for a previously identified guanine nucleotide exchange protein in receptor biology.


FOOTNOTES

*   This work was supported by the Howard Hughes Medical Institute (to B. K. K.), National Institutes of Health Grant DA00218 (to M. v. Z.), and a grant-in-aid from the American Heart Association (to M. v. Z.).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.
   These authors contributed equally to this work
par    Recipient of a postdoctoral fellowship from the American Heart Association, California Affiliate and of a research fellowship from the Deutsche Forschungsgemeinschaft.
**   Supported by Postdoctoral Training Grant HL07740-05 from the National Institutes of Health.
§§   To whom correspondence should be addressed: Nina Ireland Laboratory, Dept. of Psychiatry, Box 0984-IRE, University of California, San Francisco, CA 94143-0984. Tel.: 415-476-7855; Fax: 415-476-7884; E-mail: zastrow{at}itsa.ucsf.edu.
1   D. Daunt and B. Kobilka, unpublished observations.
2   The abbreviations used are: eIF, eukaryotic initiation factor; bp, base pair(s); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; HA, hemagglutinin.

ACKNOWLEDGEMENTS

We are grateful to Yoram Altschuler for his help in establishing the yeast two-hybrid system. We thank Peter Chu for expert technical assistance, Candace Chi for valuable contributions to the in vitro biochemical studies, and Susan Service for advice and assistance on the statistical analysis. We thank Jane Gitschier and Bruce Conklin for providing cDNAs encoding vasopressin V2 receptor and Henry Bourne, Robert Matts, and Peter Walter for valuable discussion.


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