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GABAB Receptor Association with the PDZ Scaffold Mupp1 Alters Receptor Stability and Function

2006; Elsevier BV; Volume: 282; Issue: 6 Linguagem: Inglês

10.1074/jbc.m607695200

1083-351X

Srividya Balasubramanian, Sami R. Fam, Randy A. Hall,

Neurobiology and Insect Physiology Research

γ-Aminobutyric acid, type B (GABAB) receptors are heterodimeric G protein-coupled receptors that mediate slow inhibitory synaptic transmission in the central nervous system. To identify novel interacting partners that might regulate GABAB receptor (GABABR) functionality, we screened the GABABR2 carboxyl terminus against a recently created proteomic array of 96 distinct PDZ (PSD-95/Dlg/ZO-1 homology) domains. The screen identified three specific PDZ domains that exhibit interactions with GABABR2: Mupp1 PDZ13, PAPIN PDZ1, and Erbin PDZ. Biochemical analysis confirmed that full-length Mupp1 and PAPIN interact with GABABR2 in cells. Disruption of the GABABR2 interaction with PDZ scaffolds by a point mutation to the carboxyl terminus of the receptor dramatically decreased receptor stability and attenuated the duration of GABAB receptor signaling. The effects of mutating the GABABR2 carboxyl terminus on receptor stability and signaling were mimicked by small interference RNA knockdown of endogenous Mupp1. These findings reveal that GABAB receptor stability and signaling can be modulated via GABABR2 interactions with the PDZ scaffold protein Mupp1, which may contribute to cell-specific regulation of GABAB receptors in the central nervous system. γ-Aminobutyric acid, type B (GABAB) receptors are heterodimeric G protein-coupled receptors that mediate slow inhibitory synaptic transmission in the central nervous system. To identify novel interacting partners that might regulate GABAB receptor (GABABR) functionality, we screened the GABABR2 carboxyl terminus against a recently created proteomic array of 96 distinct PDZ (PSD-95/Dlg/ZO-1 homology) domains. The screen identified three specific PDZ domains that exhibit interactions with GABABR2: Mupp1 PDZ13, PAPIN PDZ1, and Erbin PDZ. Biochemical analysis confirmed that full-length Mupp1 and PAPIN interact with GABABR2 in cells. Disruption of the GABABR2 interaction with PDZ scaffolds by a point mutation to the carboxyl terminus of the receptor dramatically decreased receptor stability and attenuated the duration of GABAB receptor signaling. The effects of mutating the GABABR2 carboxyl terminus on receptor stability and signaling were mimicked by small interference RNA knockdown of endogenous Mupp1. These findings reveal that GABAB receptor stability and signaling can be modulated via GABABR2 interactions with the PDZ scaffold protein Mupp1, which may contribute to cell-specific regulation of GABAB receptors in the central nervous system. GABAB 2The abbreviations used are: GABAB, γ-aminobutyric acid type B; GABAAR, GABA type A receptor; GABABR1, GABAB receptor 1; GABABR2, GABAB receptor 2; GBR1, GABABR1; GBR2, GABABR2; PDZ, PSD-95/Drosophila Discs Large/ZO1 homology; PSD, post synaptic density; ZO, Zona occludens; CREB, cAMP response element-binding protein; ATF, activating transcription factor; GST, glutathione S-transferase; CT, carboxyl terminus; Mupp1, multi PDZ domain protein 1; ERBIN, ErbB2 interacting protein; PAPIN, plakophilin-related armadillo repeat protein-interacting PDZ protein; ERK, extracellular signal-regulated kinase; HtrA, high temperature requirement; 5-HT2C, 5-hydroxytryptamine receptor type 2C; c-Kit, class III transmembrane tyrosine kinase receptor; TAPP1, tandem PH-domain-containing protein-1; NG2, protein new-glue 2 precursor; siRNA, small interference RNA; HA, hemagglutinin; GFP, green fluorescent protein; BSA, bovine serum albumin. 2The abbreviations used are: GABAB, γ-aminobutyric acid type B; GABAAR, GABA type A receptor; GABABR1, GABAB receptor 1; GABABR2, GABAB receptor 2; GBR1, GABABR1; GBR2, GABABR2; PDZ, PSD-95/Drosophila Discs Large/ZO1 homology; PSD, post synaptic density; ZO, Zona occludens; CREB, cAMP response element-binding protein; ATF, activating transcription factor; GST, glutathione S-transferase; CT, carboxyl terminus; Mupp1, multi PDZ domain protein 1; ERBIN, ErbB2 interacting protein; PAPIN, plakophilin-related armadillo repeat protein-interacting PDZ protein; ERK, extracellular signal-regulated kinase; HtrA, high temperature requirement; 5-HT2C, 5-hydroxytryptamine receptor type 2C; c-Kit, class III transmembrane tyrosine kinase receptor; TAPP1, tandem PH-domain-containing protein-1; NG2, protein new-glue 2 precursor; siRNA, small interference RNA; HA, hemagglutinin; GFP, green fluorescent protein; BSA, bovine serum albumin. receptors are G protein-coupled receptors responsible for mediating slow inhibitory synaptic transmission by the neurotransmitter GABA (1Bowery N.G. Bettler B. Froestl W. Gallagher J.P. Marshall F. Raiteri M. Bonner T.I. Enna S.J. Pharmacol. Rev. 2002; 54: 247-264Crossref PubMed Scopus (515) Google Scholar). They belong to G protein-coupled receptor Family C and bear a high degree of homology to other family members such as the metabotropic glutamate receptors, calcium receptor, and vomeronasal receptors. GABAB receptors are believed to be heterodimeric combinations of two G protein-coupled receptors, GABABR1 and GABABR2 (2Kaupmann K. Malitschek B. Schuler V. Heid J. Froestl W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (998) Google Scholar, 3White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (992) Google Scholar, 4Jones K.A. Borowsky B. Tamm J.A. Craig D.A. Durkin M.M. Dai M. Yao W.J. Johnson M. Gunwaldsen C. Huang L.Y. Tang C. Shen Q. 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Chem. 1999; 274: 13362-13369Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), whereas GABABR2 is believed to be the primary G protein contact site (8Duthey B. Caudron S. Perroy J. Bettler B. Fagni L. Pin J.P. Prezeau L. J. Biol. Chem. 2002; 277: 3236-3241Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 9Margeta-Mitrovic M. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14649-14654Crossref PubMed Scopus (153) Google Scholar, 10Robbins M.J. Calver A.R. Filippov A.K. Hirst W.D. Russell R.B. Wood M.D. Nasir S. Couve A. Brown D.A. Moss S.J. Pangalos M.N. J. Neurosci. 2001; 21: 8043-8052Crossref PubMed Google Scholar, 11Havlickova M. Prezeau L. Duthey B. Bettler B. Pin J.P. Blahos J. Mol. Pharmacol. 2002; 62: 343-350Crossref PubMed Scopus (82) Google Scholar). Given that GABAB receptors are important therapeutic targets for a wide variety of diseases, including depression, anxiety, epilepsy, and drug addiction (12Bettler B. Kaupmann K. Bowery N. Curr. Opin. Neurobiol. 1998; 8: 345-350Crossref PubMed Scopus (140) Google Scholar, 13Bowery N.G. Curr. Opin. Pharmacol. 2006; 6: 37-43Crossref PubMed Scopus (200) Google Scholar), understanding GABAB receptor signaling and regulation is of significant clinical interest. The cloning of the GABAB receptors has advanced the study of the GABAB receptors substantially over the past decade. However, some discrepancies between the properties of native GABAB receptors and heterologously expressed recombinant receptors still remain. For example, GABAB receptors in native tissue undergo robust endocytosis and desensitization (14Perroy J. Adam L. Qanbar R. Chenier S. Bouvier M. EMBO J. 2003; 22: 3816-3824Crossref PubMed Scopus (105) Google Scholar), whereas recombinant GABABR1/GABABR2 expressed in most heterologous cells neither internalize nor desensitize (14Perroy J. Adam L. Qanbar R. Chenier S. Bouvier M. EMBO J. 2003; 22: 3816-3824Crossref PubMed Scopus (105) Google Scholar, 15Fairfax B.P. Pitcher J.A. Scott M.G. Calver A.R. Pangalos M.N. Moss S.J. Couve A. J. Biol. Chem. 2004; 279: 12565-12573Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). One possible explanation for such discrepancies is that GABAB receptor signaling and trafficking properties are highly dependent on cellular context. This implies that interaction with differentially expressed cellular proteins might modulate GABAB receptor function. Indeed, we previously reported that association of GABAB receptors with the GABAA receptor γ2S subunit confers agonist-mediated endocytosis on GABAB receptors expressed in heterologous cells (16Balasubramanian S. Teissere J.A. Raju D.V. Hall R.A. J. Biol. Chem. 2004; 279: 18840-18850Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Furthermore, GABAB receptors have also been shown to be regulated by interactions with several other protein partners, including the transcription factors cAMP-response element-binding protein 2 and ATF4 (17Nehring R.B. Horikawa H.P. El Far O. Kneussel M. Brandstatter J.H. Stamm S. Wischmeyer E. Betz H. Karschin A. J. Biol. Chem. 2000; 275: 35185-35191Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 18White J.H. McIllhinney R.A. Wise A. Ciruela F. Chan W.Y. Emson P.C. Billinton A. Marshall F.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13967-13972Crossref PubMed Scopus (155) Google Scholar), the adaptor protein 14-3-3 (19Couve A. Kittler J.T. Uren J.M. Calver A.R. Pangalos M.N. Walsh F.S. Moss S.J. Mol. Cell Neurosci. 2001; 17: 317-328Crossref PubMed Scopus (104) Google Scholar), the RNA-binding protein Marlin-1 (20Couve A. Restituito S. Brandon J.M. Charles K.J. Bawagan H. Freeman K.B. Pangalos M.N. Calver A.R. Moss S.J. J. Biol. Chem. 2004; 279: 13934-13943Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and the coat protein I (21Brock C. Boudier L. Maurel D. Blahos J. Pin J.P. Mol. Biol. Cell. 2005; 16: 5572-5578Crossref PubMed Scopus (62) Google Scholar). GABABR2 possesses a carboxyl-terminal motif (VSGL) that has the potential to interact with PDZ-domain containing scaffold proteins. PDZ (PSD-95/Discs-large/ZO-1) domains are 90-amino acid protein-protein interaction modules that recognize and bind to specialized motifs in the distal carboxyl termini of target proteins such as G protein-coupled receptors and ion channels (22Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1029) Google Scholar). Multiple PDZ domains on the same PDZ protein can allow these proteins to act as scaffolds for the assembly of large protein complexes at the cell surface. In addition, PDZ proteins can play crucial roles in regulating the sorting, clustering, trafficking, signaling, and stability of proteins in multicellular organisms (23Harris B.Z. Lim W.A. J. Cell Sci. 2001; 114: 3219-3231Crossref PubMed Google Scholar). More than 440 PDZ domains are predicted to exist in the human genome, of which more than a quarter are likely to be Class I PDZ domains based on the amino acid requirement for their binding partners. Class I PDZ proteins bind to the motif (S/T)XΦ, where Φ represents a hydrophobic residue at the carboxyl terminus and X represents any amino acid. The GABABR2 carboxyl-terminal motif of VSGL thus conforms to the preferred binding motif for Class I PDZ domains and may therefore interact with PDZ proteins that could potentially regulate GABAB receptor function. In this study, we screened a proteomic array consisting of known or putative Class I PDZ domains to identify PDZ proteins that might interact with GABABR2. We identified three PDZ proteins that interact with the GABABR2 carboxyl terminus: Mupp1, Erbin, and PAPIN. We further studied the interactions of these proteins with GABABR2 in cells and examined the roles of these interactions in regulating GABAB receptor signaling, trafficking, and stability. Construction of the PDZ Domain Proteomic Array—PDZ protein cDNA constructs were kindly donated by a large number of colleagues (24Fam S.R. Paquet M. Castleberry A.M. Oller H. Lee C.J. Traynelis S.F. Smith Y. Yun C.C. Hall R.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8042-8047Crossref PubMed Scopus (82) Google Scholar, 25He J. Bellini M. Inuzuka H. Xu J. Xiong Y. Yang X. Castleberry A.M. Hall R.A. J. Biol. Chem. 2006; 281: 2820-2827Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). These cDNAs were used as templates to amplify by means of PCR the regions encoding various PDZ domains, which were ultimately subcloned into pET30A for fusion protein expression. PDZ domains were expressed as His- and S-tagged fusion proteins by using the vector pET30A (Novagen) and purified using ProBond nickel resin (Invitrogen). Plasmids—Epitope-tagged (HA-, FLAG-, Myc-, and Histagged) versions of human GABABR1b and GABABR2 in the mammalian expression vector pcDNA3.1 were kindly provided by Fiona Marshall (GlaxoSmithKline). Myc-Mupp1 was kindly provided by Dr. Yoko Hamazaki (Kyoto University). GFPPAPIN was kindly provided by Dr. Yutaka Hata (Tokyo Medical and Dental University). Myc-Erbin was kindly provided by Dr. Amy Lee (Emory University). FLAG-GABABR2V938A, FLAG-GABABR2S939A, and FLAG-GABABR2L941A mutants were generated using a site-directed mutagenesis kit from Stratagene. Overlay Assays—To assess the binding of receptor carboxyl-terminal GST fusion proteins to the PDZ domain array, the purified PDZ domain fusion proteins were spotted at 1 μg per bin onto Nytran SuperCharge 96-grid nylon membranes (Schleicher & Schuell). The membranes were allowed to dry overnight and then blocked in “blot buffer” (2% nonfat dry milk/0.1% Tween 20/50 mm NaCl/10 mm HEPES, pH 7.4) for 1 h at room temperature. GST-GABABR2 carboxyl terminus (CT) was prepared via PCR amplification of the region encoding the last 35 amino acids of rat GABABR2 and subcloned into the pGEX-4T1 vector (Amersham Biosciences) using EcoR1 and XhoI restriction enzymes. Overlays with GABABR2-CT fusion protein (100 nm in blot buffer) were then performed by using a previously described technique (24Fam S.R. Paquet M. Castleberry A.M. Oller H. Lee C.J. Traynelis S.F. Smith Y. Yun C.C. Hall R.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8042-8047Crossref PubMed Scopus (82) Google Scholar, 25He J. Bellini M. Inuzuka H. Xu J. Xiong Y. Yang X. Castleberry A.M. Hall R.A. J. Biol. Chem. 2006; 281: 2820-2827Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Fusion Protein Pull-down Assays—Hexahistidine-tagged PDZ domain fusion proteins were grown in Escherichia coli and purified on ProBond nickel resin (Invitrogen). Aliquots of the fusion protein on beads were blocked for 30 min with 1 ml of a 3% “BSA buffer” (10 mm HEPES, 50 mm NaCl, 0.1% Tween 20, 3% BSA) at 4 °C. Solubilized lysates from transfected COS-7 cells were then incubated with the beads end-over-end at 4 °C for 2 h. Following three washes with 1 ml of BSA buffer, the proteins were eluted off of the beads with sample buffer, resolved via SDS-PAGE, and analyzed via Western blot using appropriate antibodies. Cell Culture and Transfection—All tissue culture media and related reagents were purchased from Invitrogen. COS-7 and 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, 2-4 μg of cDNA was mixed with 15 μl of Lipofectamine 2000 (Invitrogen) and added to 5 ml of serum-free medium in 10-cm tissue cultures plates containing cells at 80-90% confluency. Following overnight incubation, the medium was replaced with 12 ml of complete media, and the cells were harvested 24 h later. Cerebellar Granule Neuron Culture—Primary cultures of cerebellar granule neurons were obtained from 7-day-old Sprague-Dawley rats. Isolated cerebella were stripped of meninges, minced by mild trituration with a Pasteur pipette, and treated with trypsin for 15 min at 37 °C. Granule cells were then dissociated by three successive trituration and sedimentation steps in DNase-containing Neurobasal media, centrifuged, and resuspended in Neurobasal medium containing 10% heatinactivated fetal bovine serum, B-27 serum-free supplement, 0.5 mm glutamine, 25 μm glutamic acid, and 25 mm KCl. The neurons were plated onto poly-d-lysine-coated culture slides (Fisher) at a density of ∼0.25 × 106 cells/well and incubated at 37 °C in a 5% CO2/95% humidity atmosphere. Cytosine arabinoside (10 μm) was added after 18-24 h to inhibit replication of non-neuronal cells. Immunoprecipitation, Surface Expression Assay, and Western Blotting—Co-immunoprecipitation of full-length proteins from COS-7 cells was performed using appropriate primary antibodies and methods described previously (16Balasubramanian S. Teissere J.A. Raju D.V. Hall R.A. J. Biol. Chem. 2004; 279: 18840-18850Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Monoclonal anti-FLAG M2 antibody resin (Sigma) was the primary antibody used to immunoprecipitate epitope-tagged proteins. Surface expression of GABAB receptors was verified using a luminometer-based surface expression assay as described previously (16Balasubramanian S. Teissere J.A. Raju D.V. Hall R.A. J. Biol. Chem. 2004; 279: 18840-18850Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Purified proteins, cell extracts, and/or immunoprecipitated samples were separated by SDS-PAGE, blotted onto nitrocellulose, and detected with appropriate antibodies as described previously (16Balasubramanian S. Teissere J.A. Raju D.V. Hall R.A. J. Biol. Chem. 2004; 279: 18840-18850Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Antibodies—The primary antibodies utilized were M2 monoclonal anti-FLAG antibody (Sigma), horseradish peroxidase-coupled 12CA5 anti-HA antibody (Roche Applied Science), monoclonal anti-c-myc 9E10 antibody (Sigma), anti-GABABR1 antibody, anti-GABABR2 antibody (Chemicon), anti-Mupp1 antibody (Upstate Biotechnology), and anti-GFP antibody (BD Biosciences). Double Immunofluorescence Microscopy—Cerebellar granule neurons or transfected COS-7 cells were plated in culture slides, fixed with 4% paraformaldehyde, and permeabilized with buffer containing 2% bovine serum albumin and 1% Triton X-100 in phosphate-buffered saline for 30 min at room temperature. The cells were then incubated with anti-GABABR2 antibody (Chemicon) plus either monoclonal anti-Mupp1 (Upstate) or anti-c-myc 9E10 antibody (Sigma) for 1 h at room temperature. After three washes (5 min) with buffer, the cells were incubated with a Rhodamine Red-conjugated anti-mouse IgG and fluorescein isothiocyanate-conjugated anti-guinea pig IgG (Jackson ImmunoResearch Laboratories) for 30 min at room temperature. After three washes (5 min) with buffer, 4′,6-diamidino-2-phenylindole was used to label the nucleus. After one wash with phosphate-buffered saline, coverslips were mounted, and Rhodamine Red-labeled Mupp1 and fluorescein isothiocyanate-labeled GABABR2 were visualized with a Zeiss LSM-510 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 cells was specific. Pulse-Chase Analysis—Transiently transfected COS-7 cells were split into 60-mm tissue culture plates. Approximately 40 h after transfection, the cells were washed with sterile phosphate-buffered saline and incubated for 30 min in methionine-free Dulbecco's modified Eagle's medium (BIOSOURCE). 60 μCi of Redivue l-[35S]methionine (Amersham Biosciences) was added to each plate and incubated for another 30 min. The radioactive media was removed; the cells were washed with sterile phosphate-buffered saline and then chased with Dulbecco's modified Eagle's medium supplemented with 3 mm cold l-methionine (Sigma) for various time periods. Cells were harvested at 0-, 1-, 2-, 4-, 8-, 12-, 24-, and 48-h time points and frozen at -80 °C. The cells were solubilized, adjusted for protein concentration, and immunoprecipitated using anti-FLAG resin. The immunoprecipitates were run on an SDS-PAGE gel, dried, and exposed to a phosphor screen for 1 week. The autoradiographic images were obtained with a phosphorimaging device (Typhoon) and analyzed with ImageQuaNT and GraphPad prism software. Within each experiment, the values of GABABR2 expression at the zero time point were considered as 100%, and then other time point values were normalized as a percentage of this starting value. The averaged data were subjected to nonlinear regression curve fitting (one phase exponential decay) to determine the protein half-life values. ERK Activation Assay—Transfected HEK-293 cells were plated in 35-mm tissue culture plates at 80% confluency and serum starved overnight the day before the assay. The cells were stimulated with 200 μm baclofen for specified time periods, rinsed with ice-cold phosphate-buffered saline/Ca2+, and lysed in 80 μl of sample buffer. The cell lysates were run on SDS-PAGE gels and then analyzed via Western blotting with anti-phospho-p44/42 MAPK and anti-p44/42 MAPK antibodies (Cell Signaling). Calcium Imaging—The Ca2+-sensitive fluorophore fura-2AM (Molecular Probes) was used for ratiometric Ca2+ imaging in COS-7 cells. All fluorescence measurements were made from subconfluent areas of the dishes so that individual cells could be readily identified. After transfection in 100-mm plates, cells were split onto coverslips immersed in 0.5 ml of media in 24-well plates and grown for 1-2 days. Before imaging, coverslips were incubated at room temperature for 30 min in extracellular recording solution composed of 150 mm NaCl/10 mm Hepes/3 mm KCl/2 mm CaCl2/2 mm MgCl2/5.5 mm glucose, pH 7.3, 325 mosm. Extracellular recording solution was supplemented with pluronic acid (0.001%) and fura-2 AM (2 μm). Subsequently, coverslips were thoroughly rinsed with extracellular solution lacking fura-2AM and BSA and mounted onto the microscope stage for imaging. Intensity images of 510 nm emission wavelengths were taken at 340 and 380 nm excitation wavelengths, and the two resulting images were taken from individual cells for ratio calculations. Imaging work-bench 2.2.1 (Axon Instruments, Union City, CA) was used for acquisition of intensity images and conversion to ratios. Baclofen (100 μm) was dissolved in extracellular recording solution and applied by bath perfusion. Mupp1 siRNA—A Mupp1 siRNA construct (identification no. 107246) was purchased from Ambion along with control siRNA. Approximately 6 h after transfection with appropriate plasmids, cells were transfected for 36-48 h with 100 nm of either Mupp1 siRNA or control siRNA using TransIT-Quest transfection reagent from Mirus. Screening of a PDZ Proteomic Array with the GABABR2 Carboxyl Terminus and Elucidation of the Structural Determinants of GABABR2 Binding to PDZ Proteins—To identify PDZ domain-containing proteins that might associate with the GABABR2 carboxyl terminus (GABABR2-CT), we first created a GST fusion protein corresponding to the last 35 amino acids of GABABR2, which possesses the putative PDZ binding motif VSGL. We next screened a previously reported (24Fam S.R. Paquet M. Castleberry A.M. Oller H. Lee C.J. Traynelis S.F. Smith Y. Yun C.C. Hall R.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8042-8047Crossref PubMed Scopus (82) Google Scholar, 25He J. Bellini M. Inuzuka H. Xu J. Xiong Y. Yang X. Castleberry A.M. Hall R.A. J. Biol. Chem. 2006; 281: 2820-2827Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) proteomic array containing 96 distinct Class I PDZ domains for interactions with the GABABR2-CT-GST fusion protein. GABABR2-CT did not detectably associate with the vast majority of PDZ domains on the array but did specifically interact with three PDZ domains: Mupp1-PDZ13, PAPIN-PDZ1, and Erbin-PDZ (data not shown). The results from the proteomic array screens were confirmed via a second independent technique in pull-down experiments examining PDZ interactions with both wild-type full-length GABABR2 and various full-length GABABR2 carboxyl terminus mutants. The amino acids Val-938, Ser-939, and Leu-941 of the GABABR2 carboxyl-terminal motif (VSGL) were sequentially mutated to alanine. Lysates from COS-7 cells transfected with wild-type GABABR2 or one of the three GABABR2 mutants were separately incubated with the three PDZ domains (Mupp1-PDZ13, Erbin-PDZ, and Papin-PDZ1) expressed as hexahistidine-tagged fusion proteins and adsorbed to nickel resin. A robust association of all the three PDZ fusion proteins with the wild-type GABABR2 was observed (Fig. 1). Alanine mutations at the Ser-939 and Val-938 positions of the GABABR2 PDZ-binding motif partially inhibited GABABR2 binding with all the three PDZ proteins. Strikingly, mutation of the GABABR2 terminal leucine (Leu-941) to alanine strongly reduced the interaction with the PDZ proteins. These results confirm that full-length GABABR2 associates with PDZ domains from Mupp1, PAPIN, and Erbin and also elucidate key residues on GABABR2 that mediate the interaction with PDZ proteins. Mupp1 and PAPIN, but Not Erbin, Associate with GABAB Receptors in Cells—We next examined whether GABABR2 can interact with full-length versions of the various PDZ proteins in a cellular environment. Myc-tagged Mupp1 was expressed alone or in the presence of either wild-type FLAG-tagged GABABR2 or FLAG-tagged GABABR2 L941A mutant in COS-7 cells (Fig. 2A). When FLAG-tagged GABABR2 was immunoprecipitated, robust co-immunoprecipitation of Mupp1 was observed from the cell lysates expressing wild-type GABABR2 and Mupp1. However, Mupp1 co-immunoprecipitation from cell lysates expressing the GABABR2 L941A mutant and Mupp1 was much weaker. Similarly, GFP-tagged PAPIN co-immunoprecipitated with GABABR2 from cells expressing GABABR2 or GABABR2/GABABR1 (Fig. 2B). We also expressed either Myc-tagged Erbin or Myc-tagged Erbin lacking the PDZ domain (Myc-ErbinΔPDZ) in the presence or absence of FLAG-tagged GABABR2 in COS-7 cells (Fig. 2C). However, immunoprecipitation of GABABR2 from these cell lysates did not yield any detectable co-immunoprecipitation of Erbin. These results demonstrate that full-length Mupp1 and PAPIN, but not Erbin, physically associate with GABAB receptors in transfected COS-7 cells. Mupp1 Co-localizes with GABABR2 in Neurons and Transfected Cells—GABABR2, Mupp1, and PAPIN have been reported to exhibit overlapping distributions in various regions of the brain (26Deguchi M. Iizuka T. Hata Y. Nishimura W. Hirao K. Yao I. Kawabe H. Takai Y. J. Biol. Chem. 2000; 275: 29875-29880Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 27Margeta-Mitrovic M. Mitrovic I. Riley R.C. Jan L.Y. Basbaum A.I. J. Comp. Neurol. 1999; 405: 299-321Crossref PubMed Scopus (302) Google Scholar, 28Sitek B. Poschmann G. Schmidtke K. Ullmer C. Maskri L. Andriske M. Stichel C.C. Zhu X.R. Luebbert H. Brain. Res. 2003; 970: 178-187Crossref PubMed Scopus (24) Google Scholar), but it is not known if these proteins are expressed in the same cells. Therefore, we examined the subcellular distributions of GABABR2 and Mupp1 in cultured cerebellar granule neurons (Fig. 3, A-C) and cortical neurons (data not shown) via immunohistochemistry using specific GABABR2 and Mupp1 primary antibodies and differentially tagged fluorescent secondary antibodies. We observed a significant overlap in the distribution patterns of GABABR2 and Mupp1 on the plasma membrane of the cell bodies and processes of these neurons, suggesting that these two proteins are present together in the same subcellular domains of the same cells. Comparable studies examining PAPIN were not possible due to the lack of a specific anti-PAPIN antibody. Next, we used fluorescence immunohistochemistry to study the subcellular distribution of GABABR2 and Mupp1 in transfected cells. COS-7 cells were transfected with either Myc-tagged Mupp1 alone or Myc-tagged Mupp1 plus FLAG-tagged GABABR2. Double immunofluorescence was performed with monoclonal anti-Myc and polyclonal anti-GABABR2 antibodies. In contrast to the prominent expression in the plasma membrane that was seen in neurons, Mupp1 expressed alone in COS-7 cells was distributed diffusely throughout the cytoplasm with little or no plasma membrane localization (Fig. 3, D-F). Interestingly, upon co-expression with GABABR2 in COS-7 cells, Mupp1 displayed a predominantly plasma membrane localization similar to its native subcellular distribution in neurons (Fig. 3, G-I). These findings suggest that association with GABABR2 can alter the subcellular distribution of Mupp1. Mutation of the PDZ-binding Motif Decreases GABAB Receptor Stability—Transfection of the GABABR2 L941A mutant into COS-7 cells resulted in consistently low expression of this mutant compared with wild-type GABABR2 as assessed by Western blot (Fig. 4A). Additionally, quantification of the plasma membrane expression of GABAB receptors using a luminometer-based cell surface expression assay yielded evidence for a striking decrease in surface expression of the L941A mutant relative to wild-type GABABR2 (Fig. 4B). Thus, we postulated that the stability of GABABR2 might be affected by the L941A mutation that disrupts the PDZ-binding motif. To test this hypothesis, we compared the halflives of wild-type GABABR2 and the GABA