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J. Biol. Chem., Vol. 281, Issue 5, 2820-2827, February 3, 2006

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Proteomic Analysis of ₁-Adrenergic Receptor Interactions with PDZ Scaffold Proteins^*

Junqi He¹, Michele Bellini¹, Hiroyuki Inuzuka, Jianguo Xu, Ying Xiong, Xiaomei Yang, Amanda M. Castleberry, and Randy A. Hall²

From the Department of Biochemistry and Molecular Biology, Capital University of Medical Sciences, Beijing 100054, China and the Department of Pharmacology, Rollins Research Center, Emory University, School of Medicine, Atlanta, Georgia 30322

Received for publication, August 29, 2005 , and in revised form, November 8, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Many G protein-coupled receptors possess carboxyl-terminal motifs ideal for interaction with PDZ scaffold proteins, which can control receptor trafficking and signaling in a cell-specific manner. To gain a panoramic view of ₁-adrenergic receptor ( AR) interactions with PDZ scaffolds, the ₁AR carboxyl terminus was screened against a newly developed proteomic array of PDZ domains. These screens confirmed ₁AR associations with several previously identified PDZ partners, such as PSD-95, MAGI-2, GIPC, and CAL. Moreover, two novel ₁AR-interacting proteins, SAP97 and MAGI-3, were also identified. The ₁AR carboxyl terminus was found to bind specifically to the first PDZ domain of MAGI-3, with the last four amino acids (E-S-K-V) of ₁AR being the key determinants of the interaction. Full-length ₁AR robustly associated with full-length MAGI-3 in cells, and this association was abolished by mutation of the ₁AR terminal valine residue to alanine (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. MAGI-3 co-expression with ₁AR profoundly impaired ₁AR-mediated ERK1/2 activation but had no apparent effect on ₁AR-mediated cyclic AMP generation or agonist-promoted ₁AR internalization. These findings revealed that the interaction of MAGI-3 with ₁AR can selectively regulate specific aspects of receptor signaling. Moreover, the screens of the PDZ domain proteomic array provide a comprehensive view of ₁AR interactions with PDZ scaffolds, thereby shedding light on the molecular mechanisms by which ₁ AR signaling and trafficking can be regulated in a cell-specific manner.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cellular responses to a given hormone or neurotransmitter can vary markedly between different types of cells. In many cases, such cellular differences are because of differential expression of receptor subtypes. However, even cells expressing exactly the same receptor subtypes can exhibit distinct responses to a given ligand, because receptors often behave quite differently in distinct cellular environments. The trafficking and signaling properties of G protein-coupled receptors (GPCRs),³ which comprise the largest family of cell surface receptors, are known to be especially influenced by cellular context. The activity of a GPCR depends not only on the complement of downstream effectors expressed in a given cell but also on the set of expressed G proteins, kinases, and scaffold proteins that directly interact with the receptor to shape its signaling capability.

PDZ scaffolds comprise a key class of GPCR-interacting proteins that can strongly influence receptor trafficking and signaling. The term PDZ is derived from the names of the first three proteins in which these domains were first identified: the post-synaptic density protein PSD-95, the Drosophila protein Discs-large, and the tight junction protein ZO-1. PDZ domains are 90 amino acids in length and bind to specific carboxyl-terminal motifs on their target proteins. There are three general classes of PDZ domain: Class I domains, which recognize the carboxyl-terminal motif S/T-X- (where "" indicates a hydrophobic amino acid and "X" indicates any amino acid), Class II domains, which recognize the carboxyl-terminal motif -X-, and Class III domains, which recognize D/E-X- as their preferred carboxyl-terminal motif (1).

Many GPCRs possess carboxyl-terminal motifs appropriate for interaction with PDZ domains. One subfamily of GPCRs for which there are already several reports of PDZ interactions is the subfamily of -adrenergic receptors (ARs). This subfamily includes the closely related subtypes ₁AR, ₂AR, and ₃AR and mediates physiological responses to epinephrine and norepinephrine (2). It has been shown that several Class I PDZ proteins associate with the carboxyl terminus (CT) of ₁AR, including PSD-95 (postsynaptic density-95) (3-5), MAGI-2 (membrane-associated guanylate kinase inverted-2) (4), CNrasGEF (cAMP-dependent guanine nucleotide exchange factor, also known as PDZ-GEF1) (6), GIPC (GAIP-interacting protein, carboxyl terminus) (7), and CAL (cystic fibrosis transmembrane conductance regulator-associated ligand, also known as GOPC or FIG) (8). MAGI-2 and PSD-95 are structurally related PDZ proteins of the membrane-associated guanylate kinase-like (MAGUK) family, but nonetheless they exhibit diametrically opposing effects on agonist-induced ₁AR internalization; MAGI-2 strongly promotes ₁AR internalization (4), whereas PSD-95 markedly inhibits it (3, 4). In contrast, CNrasGEF and GIPC have no obvious effects on ₁AR endocytosis but rather regulate various aspects of ₁AR signaling (6, 7), whereas the Golgi-associated protein CAL directs ₁AR anterograde trafficking through the endoplasmic reticulum-Golgi complex to the plasma membrane (8). Interestingly, despite the high degree of overall sequence similarity between ₁AR and ₂AR, none of the aforementioned ₁AR-associated PDZ proteins interact with ₂AR. Instead, ₂AR has been shown to interact with the Na⁺/H⁺-exchanger regulatory factors NHERF-1 (also known as EBP50) and NHERF-2 (also known as E3KARP) (9-11). These interactions mediate regulation of Na⁺/H⁺ exchange by ₂AR (9) and also dictate ₂AR postendocytic sorting following agonist-promoted internalization (11).

It has been known for many years that ₁AR exhibits differential behavior in distinct cellular environments, with tissue-specific differences in the rate of receptor endocytosis being particularly pronounced (12-15). Such differences in ₁AR behavior in distinct cells may be explained in large part by the differential expression of ₁AR-interacting proteins such as PDZ scaffolds, which as described above can strongly regulate ₁AR trafficking and activity. Thus, it is a point of clear physiological interest to determine the full set of PDZ scaffolds that can associate with ₁AR. There are 440 PDZ domains in the human proteome, with 25% of these being probable Class I PDZ domains (1). We have recently prepared the majority of known or probable Class I PDZ domains as hexahistidine-tagged fusion proteins and arranged them on a proteomic array to facilitate the rapid analysis of PDZ domain-mediated interactions (16). Here, we screened this PDZ domain array with the ₁AR-CT resulting in the confirmation of several known ₁AR/PDZ interactions as well as the elucidation of several novel interactions. Furthermore, one of the novel interactions, the association between ₁AR and MAGI-3, was found to occur in a cellular context and to also regulate ₁AR signaling activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Preparation of Plasmids and Fusion Proteins—The ₁AR and ₂AR carboxyl termini (the last 30 amino acids of the human ₁- and ₂-adrenergic receptors) were amplified via PCR and subcloned into pGEX-4T-1 for expression as GST fusion proteins. Point mutations of both ₁AR-CT and full-length Flag-₁AR construct (V477A) were introduced via PCR and verified by ABI sequencing. The full-length V5/His-MAGI-3 construct in pcDNA3 was kindly provided by Rich Laura (Genentech). The individual PDZ domains of MAGI-3, and all of the other PDZ domains utilized on the PDZ array, were amplified via PCR and inserted into pET30A for expression as His/S-tagged fusion proteins, as described (16).

PDZ Array Overlay Assay—Gridded nylon membranes were spotted with His/S-tagged PDZ domain fusion proteins (1 µg/bin), as described (16). The membranes were placed at room temperature for 24 h to dry and then stored at 4 °C for later use. Prior to experiments, membranes were blocked in blot buffer for 30 min at room temperature then overlaid with 100 nM GST fusion proteins in blot buffer for 1 h at room temperature. The arrays were washed three times with blot buffer, and incubated with an horseradish peroxidase-conjugated anti-GST antibody (1:3000, Amersham Biosciences). Interactions of the GST fusion proteins with the various PDZ domains were then visualized via chemiluminescence using the ECL kit (Pierce).

Blot Overlay Assay—The binding of receptor carboxyl terminus-GST fusion proteins to His/S-tagged PDZ domain fusion proteins was assayed via a blot overlay technique. The receptor CT-GST fusion proteins (1 µg/lane) were run on 4-20% SDS-polyacrylamide gels (Novex, San Diego, CA), blotted, and overlaid with the His/S-tagged PDZ domains (50 nM final concentration) in blot buffer for 1 h at room temperature. The blots were then washed three times with blot buffer and incubated with S-protein horseradish peroxidase conjugate (Novagen, 1:4000) for 1 h at room temperature and were visualized via chemiluminescence as described above.

GST Fusion Protein Pull-down Assay—GST fusion proteins were purified from bacteria using glutathione-Sepharose 4B beads (Sigma) according to the manufacturer's protocol and resuspended in PBS containing 0.5% Nonidet P-40 and protease inhibitors. Equal amounts of GST fusion proteins (conjugated on beads) were incubated with 1 ml of clarified whole cell extracts from COS-7 cells transfected with His/V5-MAGI-3. After incubation at 4 °C with gentle rotation for 4 h, the beads were extensively washed with ice-cold PBS containing 0.5% Nonidet P-40. Proteins were eluted from the beads with SDS-PAGE sample buffer, resolved on SDS-polyacrylamide gels, and transferred to nitrocellulose. MAGI-3 was then detected via Western blotting with the monoclonal anti-V5 antibody followed by a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:2000).

Cell Culture and Transfection—All tissue culture medium and related reagents were purchased from Invitrogen. HEK-293 and COS-7 cells were maintained in complete medium (Dulbecco's modified eagle medium plus 10% fetal bovine serum and 1% penicillin/streptomycin) in a 37 °C/5% CO₂ incubator. For transfections, 1 µg of total DNA was mixed with Lipofectamine 2000 (15 µl) (Invitrogen) and added to 5 ml of complete medium in 10-cm tissue culture plates containing cells at 50-80% confluency. Following a 4-h incubation, fetal bovine serum was added to the medium to 10%, and cells were then harvested after 48 h.

Western Blotting and Antibodies—Samples were run on 4-20% SDS-polyacrylamide gels (Invitrogen) for 1 h at 150 V and then transferred to nitrocellulose. The blots were blocked in "blot buffer" (2% nonfat 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 blots were then washed three times with 10 ml of blot buffer each and incubated for 30 min 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 each and visualized via enzyme-linked chemiluminescence as described above. Horseradish peroxidase-conjugated anti-GST, polyclonal anti-His, and horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from Amersham Biosciences. Monoclonal anti-V5 was from Invitrogen, and monoclonal anti-hemagglutinin (HA) 12CA5 antibody was from Roche Molecular Biochemicals. Polyclonal anti-MAGI-3, anti-FLAG M2 monoclonal antibody, and anti-FLAG M2 affinity gel were from Sigma. Polyclonal anti-₁AR (V-19) was purchased from Santa Cruz.

Immunoprecipitation—Cells were harvested in ice-cold solubilization buffer (10 mM Hepes, 50 mM NaCl, 5 mM EDTA, 1 mM benzamidine, 0.5% Triton X-100) and incubated end-over-end at 4 °C for 1 h to achieve solubilization. Particulate matter was removed via centrifugation for 15 min at 15,000 x g (4 °C). The solubilizates were then incubated with 30 µl of anti-FLAG M2 affinity gel (for transfected cells) or 30 µl of protein A/G-agarose plus 5 µl of polyclonal anti-₁AR antibody (for brain lysates) for 2 h with end-over-end rotation at 4 °C. After five washes with 1.0 ml of ice-cold solubilization buffer, the immunoprecipitated proteins were eluted from the beads via incubation with SDS-PAGE sample buffer and visualized via Western blot using appropriate antibodies.

Cell Surface Expression Assay—Receptor expression in the plasma membrane was quantified as described (17). Briefly, cells were grown in 35-mm dishes and incubated in the absence and presence of agonist. The cells were then rinsed in PBS and fixed with 4% paraformaldehyde in PBS for 30 min and then rinsed three more times in PBS and blocked with blocking buffer (2% nonfat dry milk in PBS) for at least 30 min. The fixed cells were 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 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 incubated with 2 ml of ECL reagent for exactly 15 s. The luminescence, which corresponds to the amount of receptor on the cell surface, was determined by using a TD 20/20 luminometer.

Immunofluorescence Microscopy—HEK-293 cells were transiently transfected with combinations of Flag-₁AR, Flag-V477A, and/or V5/His-MAGI-3. Forty-eight hours after transfection, cells were washed three times with Dulbecco's PBS and then fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. To visualize the sub-cellular localization of ₁AR and MAGI-3, 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 mouse monoclonal anti-FLAG and rabbit polyclonal anti-His antibodies for 1 h at room temperature. After three washes (1 min) with saponin buffer, the cells were incubated with a rhodamine red-conjugated anti-mouse IgG at 1:200 dilution and fluorescein isothiocyanate-conjugated anti-rabbit 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 ₁AR and fluorescein isothiocyanate-labeled MAGI-3 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.

Phospho-ERK Assay—Twenty-four hours after transfection, cells were split into 6-well dishes and incubated in serum-free medium overnight prior to experiments. Agonist stimulation was performed at 37 °C in serum-free media for 5 min. The medium was removed, and cells were then harvested in SDS-PAGE sample buffer. The samples were sonicated briefly and analyzed via SDS-PAGE. The levels of p42/44 ERK phosphorylation were visualized by Western blot using an anti-phospho-ERK1/2 antibody (Cell Signaling Technology), whereas the levels of total ERK in the same lysates were assessed using an anti-ERK antibody (Cell Signaling Technology). Immunoreactive bands were visualized via chemiluminescence and quantified using NIH Image 1.62. For each sample, the level of phospho-ERK immunoreactivity was normalized to total ERK immunoreactivity. Variation in total ERK immunoreactivity between samples was typically very small (<10%), so only a small amount of normalization was required in each experiment.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Proteomic Analysis of ₁AR/PDZ Interactions—To obtain a panoramic view of the set of PDZ domains that bind to ₁AR, a GST fusion protein corresponding to the ₁AR-CT was purified and subsequently used in overlay experiments. Individual His/S-tagged fusion proteins of 96 distinct PDZ domains were purified and spotted on a gridded nylon membrane (1 µg of fusion protein/bin) and then overlaid with purified ₁AR-CT-GST (100 nM). As shown in Fig. 1, ₁AR-CT did not bind to the majority of PDZ domains on the array but rather bound strongly to a handful of specific PDZ domains. These included MAGI-1 PDZ1 (bin A1), MAGI-2 PDZ1 and PDZ2 (A5-A6), PSD-95 PDZ3 (B8), CAL PDZ (B10), and GIPC PDZ (D8), which are PDZ domains that have previously been reported to bind to ₁AR-CT (3, 4, 7, 8). The overlays of the PDZ array also revealed ₁AR-CT interactions with MAGI-3 PDZ1 (A10) and SAP97 PDZ3 (C3), which have not previously been shown to interact with ₁AR. In matching control membranes where GST alone was overlaid onto the array, no binding of GST was detectable (data not shown), demonstrating the low amount of nonspecific binding in these assays. Because the interaction of ₁AR-CT with MAGI-3 PDZ1 was consistently the most robust association observed, we decided to characterize this interaction further and assess its functional significance.

Structural Determinants of the ₁AR-CT Interaction with MAGI-3 PDZ1—The binding of MAGI-3 PDZ1 to various point-mutated versions of the ₁AR-CT was examined in overlay experiments (Fig. 2A, lanes 1-6). The wild-type amino acid sequence of ₁AR is SESKV. The mutation to Ala of the terminal Val residue, as well as Ser at the -2 position, completely blocked binding to MAGI-3 PDZ1. In addition, the mutation of Glu at the -3 position also impaired the interaction. However, mutation to Ala at the -1 and -4 positions had little effect. To further characterize the structural determinants of this interaction, we mutated the wild-type SESKV to SESRV, SETKV, and STSKV and examined the binding of these mutant fusion proteins to MAGI-3 PDZ1 (Fig. 2A, lanes 7-9). The mutation to Thr at the -3 position and Arg at -1 strongly inhibited binding, but the mutation to Thr at the -2 position had little effect on the interaction. These findings revealed a selective inhibition of binding to MAGI-3 PDZ1 by specific point mutations to three of the last five amino acids of the ₁AR-CT.

To examine the binding of the ₁AR-CT to full-length MAGI-3 rather than simply the isolated PDZ1 domain, HEK-293 cells were transfected with full-length V5/His-tagged MAGI-3, and pull-down assays were performed using purified GST fusion proteins corresponding to ₁AR-CT with the terminal Val residue mutated to either Ala, Ile, Met, or Leu (Fig. 2B). Each of these mutations completely abolished the interaction between MAGI-3 and ₁AR-CT, revealing a critical importance of the terminal Val residue in mediating the ₁AR/MAGI-3 interaction.

₁AR and MAGI-3 Associate in Cells—We next examined the interaction between full-length ₁AR and full-length MAGI-3 in a cellular context. HEK-293 cells were transfected with V5/His-MAGI-3 and either Flag-₁AR or Flag-V477A, a mutant receptor with the terminal Val changed to Ala. Co-immunoprecipitation experiments were performed in the presence and absence of stimulation with the AR-selective agonist isoproterenol (Fig. 3A). MAGI-3 co-immunoprecipitated with ₁AR, and the amount of co-immunoprecipitation did not significantly change when ₁AR was stimulated with agonist for 15 min. No co-immunoprecipitation was observed, however, between the V477A mutant receptor and MAGI-3, revealing the importance of the terminal Val residue for the cellular interaction of ₁AR and MAGI-3.

In addition to the transfection experiments, we also studied the interactions of full-length endogenous ₁AR and MAGI-3 in various native tissues, including brain, heart, and kidney. Solubilized lysates derived from these tissues were incubated with an anti-₁AR antibody linked to protein A/G-agarose beads, and the resultant immunoprecipitates were probed via Western blot for MAGI-3 co-immunoprecipitation using an anti-MAGI-3 antibody. Robust co-immunoprecipitation of MAGI-3 with ₁AR was observed from all three tissues (Fig. 3B). These data reveal the existence of a physical complex between ₁AR and MAGI-3 in native tissues where neither protein is overexpressed.

The potential co-localization of ₁AR and MAGI-3 in cultured cells was examined via immunofluorescence microscopy (Fig. 4). HEK-293 cells were transfected with V5/His-MAGI-3 and/or Flag-₁AR or Flag-V477A. When expressed alone, ₁AR and the V477A mutant receptor were both predominantly localized to the plasma membrane (Fig. 4A), whereas MAGI-3 expressed alone was found predominantly in the nucleus, with light cytoplasmic staining also evident (Fig. 4B). Strikingly, when co-expressed with wild-type ₁AR, MAGI-3 was found almost exclusively at the plasma membrane where it co-localized well with ₁AR (Fig. 4, C-E). In contrast, no co-localization was observed when MAGI-3 was co-expressed with V477A (Fig. 4, F-H), as MAGI-3 remained predominantly targeted to the nucleus and exhibited little if any co-localization with the mutant receptor. Stimulation of the cells with isoproterenol (15 min) had no significant effect of the extent of ₁AR/MAGI-3 co-localization (data not shown). These data confirm the findings from the co-immunoprecipitation experiments that ₁AR and MAGI-3 can interact in cells and, furthermore, reveal that this interaction results in a striking redistribution of MAGI-3 from the nucleus to the plasma membrane.

View larger version (59K): [in this window] [in a new window]   FIGURE 1. The 1AR-CT binds selectively to specific PDZ domains. Equal amounts (1 µg) of purified His-tagged fusion proteins corresponding to 96 distinct PDZ domains were spotted on nylon membranes. An overlay with 1AR-CT-GST (100 nM) revealed strong and specific binding to several PDZ domains, including PSD-95 PDZ3, CAL PDZ, MAGI-1 PDZ1, MAGI-2 PDZ1, MAGI-2 PDZ2, MAGI-3 PDZ1, SAP97 PDZ3, and GIPC PDZ. The data shown here are representative of eight independent experiments.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. MAGI-3 associates with 1AR-CT via the ESKV motif of the receptor. A, mutations of the ESKV motif at the 1AR-CT abolishes binding to MAGI-3 PDZ1. Purified GST fusion proteins (2µg) corresponding to either the wild-type 1AR-CT (denoted by its last four amino acids, ESKV) or point-mutated versions of the 1AR-CT (denoted by sequential replacement of each of the last four amino acids with alanine) were run on SDS-polyacrylamide gels, transferred to nitrocellulose, and overlaid with His/S-tagged MAGI-3 PDZ1 (100 nM). B, mutation of the last amino acid of the 1AR-CT ESKV motif abolishes association with full-length MAGI-3. Purified fusion proteins (2 µg) corresponding to either wild-type 1AR-CT or mutant versions of 1AR-CT with substitutions of the last amino acid were adsorbed to glutathione-agarose beads and used to pull down full-length MAGI-3-V5/His from transfected HEK-293 cell lysates. The pulled down MAGI-3 was detected with an anti-V5 antibody via Western blot. The data shown in the two panels of this figure are representative of 3-4 independent experiments. Molecular mass standards (in kDa) are shown on the left.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Cellular association of full-length MAGI-3 and full-length 1AR. HEK-293 cells were transfected with V5/His-MAGI-3 in conjunction with either Flag-1AR wild-type or Flag-1AR V477A. Cells were incubated before harvesting in the absence or presence of the 1AR-selective agonist, isoproterenol (Iso, 10 µM, 15 min), as indicated in the figure. Co-immunoprecipitation of MAGI-3 (top panel) was apparent under basal conditions (lane 3) as well as after the cells had been stimulated with isoproterenol (lane 4). Co-immunoprecipitation was not evident, however, when MAGI-3 was co-expressed with the V477A mutant receptor (lane 5), revealing the importance of the valine residue at the carboxyl terminus of 1AR. Empty vector (lane 1) and MAGI-3-V5/His alone (lane 2) were transfected as negative controls. The lower panel reveals the relatively equivalent expression of Flag-1AR and Flag-V477A. These data are representative of five independent experiments. IP, immunoprecipitation; IB, immunoblot. B, 1AR and MAGI-3 interact in native tissues. Solubilized lysates from homogenized rat tissues (heart, brain, and kidney) were subjected to immunoprecipitation with a specific anti-1AR antibody. The immunoprecipitated complexes were then probed with an anti-MAGI-3 antibody via Western blot. MAGI-3 was found to robustly co-immunoprecipitate with 1AR from all three tissues (lanes 2-4; n = 3 for each tissue). In contrast, MAGI-3 was not detected in samples that were immunoprecipitated using an irrelevant antibody (first lane). Molecular mass standards (in kDa) are shown on the left.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Co-localization of MAGI-3 and wild-type 1AR but not the V477A mutant receptor in cells. HEK-293 cells were transiently transfected with FLAG-tagged 1AR alone (A), MAGI-3-V5/His alone (B), Flag-1AR wild-type plus MAGI-3-V5/His (C-E), or Flag-1AR mutant (V477A) plus MAGI-3-V5/His (F-H). After fixation and permeabilization, cells were stained with a mouse anti-FLAG M2 antibody followed by a Texas Red-conjugated anti-mouse IgG and/or stained with a rabbit anti-His antibody followed by a fluorescein isothiocyanate-conjugated anti-rabbit IgG. Co-localization of wild-type 1AR with MAGI-3 is revealed in yellow following merging of the two individual images, as shown in E. These data are representative of 4-6 independent experiments for each condition.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. MAGI-3 specifically inhibits 1AR-stimulated ERK activation. COS-7 cells were transiently transfected with Flag-1AR, Flag-V477A mutant receptor, and/or V5/His-MAGI-3. Twenty-four to forty-eight hours after transfection, cells were treated with serum-free medium overnight. Serum-starved cells were stimulated with 10 µM isoproterenol (Iso) for 5 min at 37 °C. ERK activation was assayed as described under "Experimental Procedures." Expression of MAGI-3 was found to dramatically inhibit 1AR-mediated ERK activation (A) but have little effect on ERK activation by the V477A mutant receptor (B). C, quantification of the effect of MAGI-3 on 1AR- and V477A-mediated ERK activation. The amount of phospho-ERK immunoreactivity observed following isoproterenol stimulation was expressed as a fold of the phospho-ERK immunoreactivity in the absence of stimulation. D, time course of the effect of MAGI-3 on 1AR-mediated ERK activation. The results from cells transfected with 1AR alone are indicated by the squares and solid line, whereas the results from cells co-transfected with 1AR/MAGI-3 are indicated by the triangles and dashed line. For both C and D, the data and error bars represent means ± S.E. for three independent experiments. The asterisks indicate a significant decrease (p < 0.05) relative to phospho-ERK immunoreactivity in the absence of transfected MAGI-3. WT, wild-type.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In addition to demonstrating regulation of ₁AR by MAGI-3, our studies also revealed a converse regulation of MAGI-3 by ₁AR. Specifically, coexpression with ₁AR resulted in a striking translocation of MAGI-3 to the plasma membrane, whereas MAGI-3 expressed alone exhibited a predominantly nuclear localization. MAGI-3 was first identified only several years ago (19), and little is known at present about the set of cellular proteins with which MAGI-3 can interact. Given the fact that we and others (20) have observed strong MAGI-3 expression in the nuclei of certain cell types, it is tempting to speculate that MAGI-3 may bind to various partners in the nucleus and play a role in transcriptional regulation, as has been shown to be the case for other PDZ scaffolds (21-24). If MAGI-3 does have a physiological role in the nucleus, then the control of MAGI-3 localization via association with transmembrane proteins such as ₁AR may represent a novel and specific mechanism by which such PDZ-interacting transmembrane proteins can influence nuclear function.

Our screens of the PDZ array revealed that ₂AR interacts with a narrow set of PDZ partners entirely distinct from the set of PDZ domains bound by ₁AR. These findings are consistent with the fact that ₂AR has been previously reported to associate with NHERF-1 (9-11) and NHERF-2 (10) but not with any other PDZ partners. Interestingly, mutation of the last amino acid of the ₁AR-CT to match the last amino acid of ₂AR-CT resulted in a striking change in ₁AR/PDZ interactions, with the ₁AR-CT now exhibiting strong binding to the NHERF PDZ domains instead of its normal set of PDZ partners. This observation emphasizes the specificity of interactions between GPCRs and their associated PDZ scaffolds. ₁AR and ₂AR are known to bind the same endogenous ligands and couple to the same G proteins but yet are also known to exert significantly different effects on cellular physiology in certain cell types (2, 25-32). The association of the two receptors with unique sets of PDZ scaffolds is likely a key factor that underlies the distinct cellular actions of ₁AR and ₂AR.

View larger version (76K): [in this window] [in a new window]   FIGURE 6. Switch of 1AR-CT binding specificity to a 2AR-like profile via mutation of the terminal amino acid. Equal amounts (1 µg) of purified His-tagged fusion proteins corresponding to 96 distinct PDZ domains were spotted on nylon membranes. Overlay with GST fusion proteins (100 nM) corresponding to either the 2AR-CT (A) or a point-mutated version of the 1AR-CT (denoted in B by replacement of the terminal valine residue with leucine, ESKL) resulted in a switch of the 1AR-CT binding specificity to a profile more similar to that of the 2AR-CT. These data are representative of 3-5 independent experiments for each condition.

	FOOTNOTES

 
^* This work was supported by grants from the National Institutes of Health and a Distinguished Young Scholar in Medical Research award from the W. M. Keck Foundation (to R. A. H.) and by Grants 30572183 and 30371643 from the National Natural Science Foundation of the People's Republic of China (to J. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental information.

¹ Both authors contributed equally to this work.

² 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{at}pharm.emory.edu.

³ The abbreviations used are: GPCR, G protein-coupled receptor; PDZ, PSD-95/Discs-large/ZO-1 homology; PSD-95, postsynaptic density protein of 95 kDa; AR, -adrenergic receptor; MAGI, membrane-associated guanylate kinase-like protein with an inverted domain structure; CNrasGEF, cAMP-dependent guanine nucleotide exchange factor; GIPC, GAIP-interacting protein, carboxyl terminus; CAL, cystic fibrosis transmembrane conductance regulator-associated ligand; NHERF, Na⁺/H⁺ exchanger regulatory factor; GST, glutathione S-transferase; CT, carboxyl terminus; HA, hemagglutinin; HEK, human embryonic kidney; PBS, phosphate-buffered saline; ERK, extracellular signal-related kinase.

	ACKNOWLEDGMENTS

 
We thank all of the investigators, listed in the supplementary materials, who contributed PDZ constructs toward the development of the PDZ domain array.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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