Metabotropic Glutamate Receptor 5/Homer Interactions Underlie Stress Effects on Fear
Natalie C. TronsonYomayra F. GuzmánAnita L. GuedeaKyu Hwan HuhCan GaoMartin K. SchwarzJelena Raduloviç
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A cDNA clone for a new metabotropic glutamate receptor, mGluR5, was isolated through polymerase chain reaction-mediated DNA amplification by using primer sequences conserved among the metabotropic glutamate receptor (mGluR) family and by the subsequent screening of a rat brain cDNA library. The cloned receptor consists of 1171 amino acid residues and exhibits a structural architecture common to the mGluR family, possessing a large extracellular domain preceding the seven putative membrane-spanning segments. mGluR5 shows the highest sequence similarity to mGluR1 among the mGluR members and is coupled to the stimulation of phosphatidylinositol hydrolysis/Ca2+ signal transduction in Chinese hamster ovary cells transfected with the cloned cDNA. This receptor also resembles mGluR1 in its agonist selectivity and antagonist responses; the potency rank order of agonists for mGluR5 was determined to be quisqualate greater than L-glutamate greater than or equal to ibotenate greater than trans-1-aminocyclopentane-1,3-dicarboxylate. Blot and in situ hybridization analyses indicated that mGluR5 mRNA is widely distributed in neuronal cells of the central nervous system and is expressed differently from mGluR1 mRNA in many brain regions. This investigation thus demonstrates that there is an additional mGluR subtype which closely resembles mGluR1 in its signal transduction and pharmacological properties and is expressed in specialized neuronal cells in the central nervous system.
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Abstract Metabotropic glutamate receptors alter the vulnerability of neurons to excitotoxic damage and are reported to display abnormal expression in the central nervous system of ALS patients. Using reverse transcriptase polymerase chain reaction, we investigated the mRNA expression of specific metabotropic glutamate receptor subtypes in T lymphocytes of 20 patients with sporadic ALS, compared with healthy age‐matched control subjects and patients with other neurological disorders. The levels of metabotropic glutamate receptor 2 mRNA were markedly reduced, whereas the expression of other subtypes (1b, 3, 8) was similar to control levels. Our findings may provide a reliable peripheral marker of the glutamatergic dysfunction that characterizes ALS. Ann Neurol 2006
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Group III presynaptic metabotropic glutamate receptors (mGluRs) play a central role in regulating presynaptic activity through G-protein effects on ion channels and signal transducing enzymes. Like all Class C G-protein-coupled receptors, mGluR8 has an extended intracellular C-terminal domain (CTD) presumed to allow for modulation of downstream signaling. In a yeast two-hybrid screen of an adult rat brain cDNA library with the CTDs of mGluR8a and 8b (mGluR8-C) as baits, we identified sumo1 and four different components of the sumoylation cascade (ube2a, Pias1, Piasγ, Piasxβ) as interacting proteins. Binding assays using recombinant GST fusion proteins confirmed that Pias1 interacts not only with mGluR8-C but also with all group III mGluR CTDs. Pias1 binding to mGluR8-C required a region N-terminal to a consensus sumoylation motif and was not affected by arginine substitution of the conserved lysine 882 within this motif. Co-transfection of fluorescently tagged mGluR8a-C, sumo1, and enzymes of the sumoylation cascade into HEK293 cells showed that mGluR8a-C can be sumoylated in vivo. Arginine substitution of lysine 882 within the consensus sumoylation motif, but not other conserved lysines within the CTD, abolished in vivo sumoylation. Our results are consistent with post-translational sumoylation providing a novel mechanism of group III mGluR regulation. Group III presynaptic metabotropic glutamate receptors (mGluRs) play a central role in regulating presynaptic activity through G-protein effects on ion channels and signal transducing enzymes. Like all Class C G-protein-coupled receptors, mGluR8 has an extended intracellular C-terminal domain (CTD) presumed to allow for modulation of downstream signaling. In a yeast two-hybrid screen of an adult rat brain cDNA library with the CTDs of mGluR8a and 8b (mGluR8-C) as baits, we identified sumo1 and four different components of the sumoylation cascade (ube2a, Pias1, Piasγ, Piasxβ) as interacting proteins. Binding assays using recombinant GST fusion proteins confirmed that Pias1 interacts not only with mGluR8-C but also with all group III mGluR CTDs. Pias1 binding to mGluR8-C required a region N-terminal to a consensus sumoylation motif and was not affected by arginine substitution of the conserved lysine 882 within this motif. Co-transfection of fluorescently tagged mGluR8a-C, sumo1, and enzymes of the sumoylation cascade into HEK293 cells showed that mGluR8a-C can be sumoylated in vivo. Arginine substitution of lysine 882 within the consensus sumoylation motif, but not other conserved lysines within the CTD, abolished in vivo sumoylation. Our results are consistent with post-translational sumoylation providing a novel mechanism of group III mGluR regulation. G-protein-coupled metabotropic glutamate receptors (mGluRs) 4The abbreviations used are: mGluR, metabotropic glutamate receptor; CFP, cyan fluorescent protein; CTD, C-terminal domain; GFP, green fluorescent protein; GLUT, glucose transporter; GPCR, G-protein-coupled receptor; GST, glutathione S-transferase; HEK cell, human embryonic kidney cell; l-AP4, l(+)-2-amino-4-phosphonobutyric acid; MBP, maltose-binding protein; Pias1, protein inhibitor of activated STAT1; RanBP2, Ran-binding protein 2; RanGAP1, Ran GTPase-activating protein; SBM, sumobinding motif; SAP domain, SAF-A/B, Acinus, and Pias domain; Siah, seven in absentia homolog; sumo1, small ubiquitin-related modifier 1; ubc9, ubiquitin-conjugating enzyme 9; ubl1, ubiquitin-like 1; YFP, yellow fluorescent protein. have been implicated in the regulation of transmitter release, short and long term modulation of synaptic transmission, neuronal development, and synaptic plasticity (1.Nakanishi S. Masu M. Bessho Y. Nakajima Y. Hayashi Y. Shigemoto R. EXS. 1994; 71: 71-80PubMed Google Scholar, 2.Nakanishi S. Neuron. 1994; 13: 1031-1037Abstract Full Text PDF PubMed Scopus (648) Google Scholar, 3.Pin J.P. Duvoisin R. Neuropharmacology. 1995; 34: 1-26Crossref PubMed Scopus (1234) Google Scholar, 4.Conn P.J. Pin J.P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2729) Google Scholar, 5.Nakanishi S. Nakajima Y. Masu M. Ueda Y. Nakahara K. Watanabe D. Yamaguchi S. Kawabata S. Okada M. Brain Res. Brain Res. Rev. 1998; 26: 230-235Crossref PubMed Scopus (292) Google Scholar). mGluRs are structurally distinct from family A and B G-protein-coupled receptors (GPCRs), as they possess a large extracellular ligand binding domain and an extended intracellular C terminus (4.Conn P.J. Pin J.P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2729) Google Scholar, 6.Dev K.K. Nakanishi S. Henley J.M. Trends Pharmacol. Sci. 2001; 22: 355-361Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). At least eight different mGluR isoforms have been identified and classified into three subgroups based on sequence homology, downstream effectors, and agonist specificity. Group III mGluRs (mGluR4, -6, -7, and -8) are specifically activated by l(+)-2-amino-4-phosphonobutyric acid (l-AP4), negatively coupled to adenylate cyclase and, apart from mGluR6, exclusively localized presynaptically. Aside from mGluR6, which is only found in the retina, group III mGluRs are expressed throughout the brain. Compared with other mGluRs, mGluR8 expression is regionally restricted, with highest mRNA levels detected in olfactory bulb, pontine gray, thalamus, and mamillary body (7.Duvoisin R.M. Zhang C. Ramonell K. J. Neurosci. 1995; 15: 3075-3083Crossref PubMed Google Scholar, 8.Saugstad J.A. Kinzie J.M. Shinohara M.M. Segerson T.P. Westbrook G.L. Mol. Pharmacol. 1997; 51: 119-125Crossref PubMed Scopus (181) Google Scholar). Alternative splicing and out-of-frame insertion generates two splice variants of mGluR8, named mGluR8a and 8b, which differ in the last 16 amino acids of the cytoplasmic C-terminal domain (CTD) (9.Corti C. Restituito S. Rimland J.M. Brabet I. Corsi M. Pin J.P. Ferraguti F. Eur. J. Neurosci. 1998; 10: 3629-3641Crossref PubMed Scopus (113) Google Scholar). Both splice variants share similar pharmacological profiles and expression levels throughout the brain, except for the lateral reticular and ambiguous nuclei where only mGluR8a mRNA is found (9.Corti C. Restituito S. Rimland J.M. Brabet I. Corsi M. Pin J.P. Ferraguti F. Eur. J. Neurosci. 1998; 10: 3629-3641Crossref PubMed Scopus (113) Google Scholar). Pronounced mGluR8 expression in the dentate gyrus and CA3 region of the hippocampus (10.Kinoshita A. Ohishi H. Neki A. Nomura S. Shigemoto R. Takada M. Nakanishi S. Mizuno N. Neurosci. Lett. 1996; 207: 61-64Crossref PubMed Scopus (53) Google Scholar, 11.Shigemoto R. Kinoshita A. Wada E. Nomura S. Ohishi H. Takada M. Flor P.J. Neki A. Abe T. Nakanishi S. Mizuno N. J. Neurosci. 1997; 17: 7503-7522Crossref PubMed Google Scholar) and performance deficits of mice lacking mGluR8 in learning tasks (12.Gerlai R. Adams B. Fitch T. Chaney S. Baez M. Neuropharmacology. 2002; 43: 235-249Crossref PubMed Scopus (59) Google Scholar) suggest that this receptor plays a role in memory formation. In stress-related behavioral tests, mGluR8-deficient mice show increased anxiety, indicating that mGluR8 may alter neurotransmission at synapses that regulate adaptation to novel stressful environments (13.Linden A.M. Johnson B.G. Peters S.C. Shannon H.E. Tian M. Wang Y. Yu J.L. Koster A. Baez M. Schoepp D.D. Neuropharmacology. 2002; 43: 251-259Crossref PubMed Scopus (92) Google Scholar). Sumo1 (small ubiqitin-related modifier 1) is a 101-amino acid protein that can be covalently linked to the ϵ-amino group of lysine side chains of target proteins. This post-translational modification is catalyzed by an enzymatic cascade termed "sumoylation pathway," which requires an activation enzyme (E1), conjugase (E2), and for most substrates, ligase (E3) (14.Schwartz D.C. Hochstrasser M. Trends Biochem. Sci. 2003; 28: 321-328Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 15.Melchior F. Schergaut M. Pichler A. Trends Biochem. Sci. 2003; 28: 612-618Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 16.Muller S. Hoege C. Pyrowolakis G. Jentsch S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 202-210Crossref PubMed Scopus (650) Google Scholar). This pathway is mechanistically but not functionally related to ubiquitination. Whereas ubiquitination destines target proteins to internalization and degradation, sumoylation can have such diverse effects as promoting transport between the cytoplasm and the nucleus, protection from ubiquitination (15.Melchior F. Schergaut M. Pichler A. Trends Biochem. Sci. 2003; 28: 612-618Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar), and regulation of protein-protein interactions (17.Song J. Durrin L.K. Wilkinson T.A. Krontiris T.G. Chen Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14373-14378Crossref PubMed Scopus (470) Google Scholar). Members of the Pias (protein inhibitor of activated STAT) family promote the conjugation of sumo1 to different proteins and have been classified as E3 protein ligases (18.Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1385) Google Scholar). They have been found to interact with several targets, among them nuclear proteins and steroid receptors (19.Kotaja N. Karvonen U. Janne O.A. Palvimo J.J. Mol. Cell. Biol. 2002; 22: 5222-5234Crossref PubMed Scopus (355) Google Scholar). Pias proteins contain an N-terminal SAP (SAF-A/B, Acinus, and Pias) domain consisting of a four helix bundle that is responsible for binding to A/T-rich DNA oligomers and p53 tumor suppressor protein (20.Okubo S. Hara F. Tsuchida Y. Shimotakahara S. Suzuki S. Hatanaka H. Yokoyama S. Tanaka H. Yasuda H. Shindo H. J. Biol. Chem. 2004; 279: 31455-31461Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The sumo ligase activity of Pias family proteins (but not the Ran-binding protein 2 (RanBP2) sumo1 E3 ligase) requires a conserved C-terminal zinc finger domain, which is related to the essential ring finger motif of many ubiquitin ligases (21.Schmidt D. Muller S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2872-2877Crossref PubMed Scopus (371) Google Scholar). Known sumo targets are predominantly nuclear proteins, like transcription factors, nuclear body proteins, and viral and nuclear pore complex components, although soluble signal transduction proteins (MAPK kinase 1 (Mek1), calmodulin kinase II (CaMKII)) and other cytoplasmic proteins (e.g. yeast septins) have also been identified (22.Seeler J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Crossref PubMed Scopus (578) Google Scholar). Subcellularly, the largest fraction of sumo conjugates localizes to nuclear speckles and the nuclear envelope (23.Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (653) Google Scholar). Sumoylation of plasma membrane proteins has rarely been reported and can have bimodal effects on trafficking within the same protein family: overexpression of the E2 conjugase ubc9 results in a 4-fold increase of GLUT4 at the membrane, whereas levels of GLUT1 are decreased (24.Lalioti V.S. Vergarajauregui S. Pulido D. Sandoval I.V. J. Biol. Chem. 2002; 277: 19783-19791Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 25.Giorgino F. de Robertis O. Laviola L. Montrone C. Perrini S. McCowen K.C. Smith R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1125-1130Crossref PubMed Scopus (141) Google Scholar). Recently, sumoylation has been found to silence the plasma membrane leak potassium channel K2P1 (26.Rajan S. Plant L.D. Rabin M.L. Butler M.H. Goldstein S.A. Cell. 2005; 121: 37-47Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). To our knowledge, no plasma membrane receptors have yet been identified as targets of the sumoylation machinery. Here we report an interaction of family C GPCRs, namely group III mGluRs, with the E3 ligase Pias1. In addition, we provide evidence that the CTD of mGluR8a can undergo in vivo sumoylation on a consensus site in HEK cells. Yeast Two-hybrid Screen and Yeast Mating—Yeast two-hybrid screening was carried out using the DupLEX-A system (OriGene Tech. Inc., Rockville, MD) as described previously (27.El Far O. Airas J. Wischmeyer E. Nehring R.B. Karschin A. Betz H. Eur. J. Neurosci. 2000; 12: 4215-4221PubMed Google Scholar). Briefly, the cDNA fragments encoding the CTDs of mouse mGluR8a (mGluR8a-C) and mGluR8b (mGluR8b-C) were generated by PCR, cloned into pGilda and used as baits to screen an adult rat brain cDNA library cloned into pJG4–5, according to the user's manual (Version 1.2). Yeast strain EGY48 was transformed using lithium acetate, and protein expression was induced by 1.5% (w/v) galactose. For screening with the mGluR8a and mGluR8b baits, 3 × 106 and 1.25 × 106 independent recombinants were examined, and 1,385 and 934 clones, respectively, were identified to be positive by Leu– auxotrophy and β-galactosidase expression. Yeast mating was performed with yeast strains EGY48 and RFY206, using the same selection conditions as in the two-hybrid screen. After examining insert sizes and HaeIII digestion patterns, ∼100 candidate clones identified with each bait were selected for sequencing. Expression Constructs—Glutathione S-transferase (GST) fusion proteins of the CTDs of mGluR4, -6, -7a, -7b, -8a, and -8b, and the mGluR7a truncations GST-7a-N38 and GST-7a-C27 have been described previously (28.El Far O. Bofill-Cardona E. Airas J.M. O'Connor V. Boehm S. Freissmuth M. Nanoff C. Betz H. J. Biol. Chem. 2001; 276: 30662-30669Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The EcoRI- and SalI-flanked truncated mGluR8a tail constructs GST-8a-N24 and GST-8a-C44 were generated by standard PCR and inserted into pGEX-5X-1 (Amersham Biosciences). A site-directed mutagenesis kit (Stratagene, Amsterdam, Netherlands) was used to introduce lysine to arginine substitutions. Flanking EcoRI and SalI sites were used to shuttle inserts between pGEX-5X-1 and pEGFP-C2 to generate bacterial or mammalian fusion proteins. MBP-ube2a and MBP-Piasγ were generated by transferring in-frame inserts between EcoRI and XhoI sites from the identified clones to pMAL-c2 (New England Biolabs, Frankfurt, Germany). The ube2a fragment contained the full-length coding sequence including 69 bp of the 5′-untranslated region. The Piasγ fragment encoded the 157 C-terminal amino acids of rat Piasγ. MBP-Pias1, GFP-Pias1, and CFP-Pias1 were constructed by transferring the full-length Pias1 cDNA from pCMV5-FLAG-Pias1 (gift from Dr. Shuai Ke, University of California, Los Angeles) into pMAL-c2, pEGFP-C2, and pECFP-C1 (Clontech, Heidelberg, Germany), respectively. BamHI and HindIII flanked full-length cDNAs of ube2a and sumo1 were generated by PCR and subcloned into the BglII and Hind III sites of pEYFP-C1 (Clontech) and pECFP-C1, respectively. Full-length His6-aos1 and uba2 expression constructs were similarly generated by PCR using pET28a-His6-aos1 and pET11d-uba2 (gifts from Dr. Frauke Melchior, University of Goettingen, Germany), respectively, as templates, and subcloned between the EcoRI and XbaI sites of pcDNA3 (Invitrogen), and the EcoRI and XhoI sites of pcDNA3.1, respectively. Protein Expression and Binding Studies—Expression of GST and MBP fusion proteins was performed in Escherichia coli BL21 (Stratagene) as described (28.El Far O. Bofill-Cardona E. Airas J.M. O'Connor V. Boehm S. Freissmuth M. Nanoff C. Betz H. J. Biol. Chem. 2001; 276: 30662-30669Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Bacteria were lysed in phosphate-buffered saline containing the protease inhibitor mixture CompleteTM (Roche Diagnostics) via passage through a French press. The supernatant was collected after centrifugation at 100,000 × g for 45 min at 4 °C. GFP, CFP, or YFP fusion proteins were expressed in HEK293 cells and processed as described (27.El Far O. Airas J. Wischmeyer E. Nehring R.B. Karschin A. Betz H. Eur. J. Neurosci. 2000; 12: 4215-4221PubMed Google Scholar). Protein expression in the bacterial lysates and cell homogenates was confirmed by Western blotting with anti-GST, anti-MBP, and anti-GFP antibodies. For pull-down assays, the lysates were incubated with 25 μl of glutathione-agarose beads (Amersham Biosciences) preloaded with GST-mGluR-C in incubation buffer (PBS containing 0.1% (v/v) Triton X-100, 2 mm EDTA, 2 mm EGTA, 2 mm dithiothreitol, and protease inhibitor mixture). After 2 h of rotary agitation, beads were collected by centrifugation and washed three times with incubation buffer. After elution with SDS sample buffer, eluted proteins were resolved by SDS-PAGE, followed by Western blotting with monoclonal anti-MBP (New England Biolabs) or polyclonal rabbit anti-GFP (Clontech). About one-fifth of the glutathione-agarose beads preloaded with GST-mGluR-C were loaded onto another gel and used to evaluate the amount of GST fusion protein bound. After transfer to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany), proteins were stained for 4 min with 2% (w/v) Ponceau in 3% (w/v) trichloroacetic acid. Sumo1 Conjugation in Vivo—pEGFP-mGluR8a-C, or mutants thereof, was co-transfected with empty vector pEGFP-C2, or with different combinations of pECFP-sumo1, pcDNA3-His6-aos1, pcDNA3.1-uba2, pEYFP-ube2a, and pEGFP-Pias1 into 60% confluent HEK293 cells by calcium phosphate precipitation (27.El Far O. Airas J. Wischmeyer E. Nehring R.B. Karschin A. Betz H. Eur. J. Neurosci. 2000; 12: 4215-4221PubMed Google Scholar), using 5 μg of each DNA per 10-cm dish. Two days after transfection, cells were washed with PBS and harvested in 2× sample buffer (20 mm N-ethylmaleimide (Sigma-Aldrich), 0.1% (w/v) SDS, 20% (v/v) glycerol, 0.2% (w/v) bromphenol blue, 4% (v/v) β-mercaptoethanol in 0.125 m Tris-Cl, pH 6.8). Western blotting was performed after 8% SDS-PAGE using guinea pig anti-mGluR8a-C (11.Shigemoto R. Kinoshita A. Wada E. Nomura S. Ohishi H. Takada M. Flor P.J. Neki A. Abe T. Nakanishi S. Mizuno N. J. Neurosci. 1997; 17: 7503-7522Crossref PubMed Google Scholar) or monoclonal mouse anti-sumo1 (anti-Sentrin1, Zytomed, Berlin, Germany). Yeast Two-hybrid Screen and Yeast Mating—To identify mGluR8-C-interacting proteins, we performed a yeast two-hybrid screen of an adult rat brain cDNA library, using the entire CTD coding regions of the mGluR8a and mGluR8b cDNAs as baits. Twelve and thirteen clones encoding the C-terminal region of protein inhibitor of activated STAT1 (Pias1) were identified with mGluR8a-C and 8b-C, respectively (TABLE ONE). The shortest cDNA fragment isolated for Pias1 comprised only the coding region for amino acids 514–721, which corresponds to the extreme C terminus not including the zinc finger domain. Additionally, 12 (mGluR8a) and 9 (mGluR8b) clones were found for the Msx-interacting zinc finger (Miz1), which encodes the C terminus of mammalian Piasxβ. Nine (mGluR8a) and three (mGluR8b) clones contained the entire coding sequence for the ubiquitin-conjugating enzyme E2a (ube2a), the rat homologue of the yeast sumo-specific E2-conjugating enzyme (ubc9). All clones were in-frame with the B42 activation domain, and interactions were confirmed in yeast mating assays using the respective CTDs of mGluR8 (TABLE ONE). For mGluR8b, we isolated three additional clones, two encoding ubiquitin-like 1 (sumo1), and one encoding a C-terminal fragment of Piasγ. These clones were tested in yeast mating experiments and gave positive signals also with mGluR8a-C (TABLE ONE).TABLE ONEcDNAs identified by yeast two-hybrid screening using mGluR8a-C and mGluR8b-C as baitsPrey (GenBank™ no.)Predicted no. aa of full-length proteinFragments identifiedNumber of clones identified 8a/8bYeast mating 8a/8baaPias1 (62653796)1-721209-72112/13+/+283-721319-721412-721514-721Piasxβ (Miz1) (16758049)1-572312-57212/9+/+Ube2a (ubc9) (4079642)1-1581-1589/3+/+Sumol (ubl1, sentrin) (57528278)1-1016-1010/2+/+Piasγ (62651709)1-507351-5070/1+/+ Open table in a new tab GST-mGluR8-C Interacts with MBP-Pias1—To biochemically confirm the results of the yeast two-hybrid screen, we expressed candidate-interacting proteins, except Piasxβ, in bacteria as MBP fusion proteins and used them with GST-mGluR8-C fusion proteins in pull-down assays (Fig. 1). Batch adsorption onto glutathione-agarose, followed by SDS-PAGE and Western blotting with an antibody against MBP revealed that the MBP-Pias1 could be affinity-purified on GST-mGluR8b-C and, to a lesser extent, on GST-mGluR8a-C, but not on GST alone. Normalization of the GST fusion protein levels retained on the beads by Ponceau staining of the nitrocellulose membrane (data not shown) revealed that the amount of GST-mGluR8a-C adsorbed onto the agarose beads was only about 25% of that of mGluR8b-C. A comparatively weak specific interaction was also detected for the MBP fusion of the C-terminal region of Piasγ, which similarly bound to both mGluR8-C isoforms. GST alone failed to bind MBP and all MBP fusion proteins tested. MBP-ube2a did not exhibit detectable binding to any of the GST-mGluR8-C termini. It is, however, important to note that rather stringent washing conditions had to be used in these pull-down assays because of high unspecific binding of MBP (data not shown). GFP-Pias1 Interacts with All Group III mGluRs—Based on its frequent detection in the yeast two-hybrid screen, we next focused on Pias1. To examine whether binding of Pias1 is shared by other group III or even group II mGluR members, we performed binary yeast two-hybrid assays with the C-terminal coding fragment of the Pias1 cDNA and group II/III mGluR-CTDs (except mGluR7b-C and mGluR8b-C; Fig. 2A). In these assays, mGluR8a-C showed the strongest interaction, followed by mGluR7a-C and then mGluR6-C/mGluR4-C. In contrast, neither mGluR2-C nor mGluR3-C interacted with Pias1. Presuming that Pias1 binds in its function as E3 ligase in sumoylation, we compared group II (29.Tanabe Y. Masu M. Ishii T. Shigemoto R. Nakanishi S. Neuron. 1992; 8: 169-179Abstract Full Text PDF PubMed Scopus (891) Google Scholar) and group III (28.El Far O. Bofill-Cardona E. Airas J.M. O'Connor V. Boehm S. Freissmuth M. Nanoff C. Betz H. J. Biol. Chem. 2001; 276: 30662-30669Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) CTD sequences for the presence of candidate acceptor lysine residues (Fig. 2A). Notably, the group II mGluR C termini contain only a single lysine (mGluR2, Lys823; mGluR3, Lys832), just C-terminal of the end of the transmembrane domain 7. Group III mGluRs do not carry a lysine at this position but display multiple lysine side chains throughout their CTDs. Next, we investigated whether GFP-Pias1 generated in mammalian cells binds other group III mGluR C termini and tested its interaction with the respective GST fusion proteins (Fig. 2B). Whereas GST failed to bind GFP-Pias1 and conversely GFP did not interact with GST-mGluR7a-C (data not shown), all group III mGluR C termini showed some interaction. Strongest binding was seen with mGluR7a-C, mGluR4-C, and mGluR6-C. The weak band recovered with GST-mGluR8a could be attributed to substantially less GST fusion protein being retained on the agarose beads; a sequential Western blot with anti-GST antibody produced only a signal corresponding to ∼25% of the other GST fusion proteins used in this experiment (data not shown). In conclusion, all group III mGluR-CTDs were able to bind GFP-Pias1. The same assay was also used to examine whether the different components of the sumoylation pathway found in the yeast two-hybrid screen can directly interact with GST-mGluR8a-C. For these experiments, we now used cDNA constructs encoding full-length Pias1, ube2a, and sumo1. CFP-sumo1, YFP-ube2a, and GFP-Pias1 all were expressed in HEK293 cells, and Triton X-100 extracts of the transfected cells were used in binding assays (Fig. 3). Interaction could be confirmed for mGluR8a-C and GFP-Pias1, whereas only very little or no YFP-ube2a was recovered in the bound protein fraction. GFP alone did not bind to GST-mGluR8a-C. In contrast to the results obtained in the original two-hybrid screen, GST-mGluR8a failed to bind CFP-sumo1 (molecular mass ∼40 kDa) under our assay conditions but enriched two high molecular mass (≥90 kDa) sumo-conjugated proteins from the HEK cell lysates. We did not try to disclose the identity of these proteins but an unbiased mass spectrometry-based analysis of sumo-conjugated HEK cell proteins has identified several candidates in the respective molecular mass range (30.Zhao Y. Kwon S.W. Anselmo A. Kaur K. White M.A. J. Biol. Chem. 2004; 279: 20999-21002Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Together these results are consistent with Pias1 representing the primary group III mGluR binding partner of the sumoylation machinery. Mapping of the Pias1 Interaction Domain of mGluR7a-C and mGluR8a-C—To determine which domains of mGluR7a-C and mGluR8a-C interact with Pias1, we tested binding of mammalian expressed CFP-Pias1 to respective truncated GST fusion proteins (Fig. 4B). A schematic drawing of the truncation mutants used is shown in Fig. 4A. The mGluR7a-C truncation constructs did not overlap whereas those for mGluR8a-C overlapped by three amino acids. Also, the positions of the truncations were different in the respective CTDs: for mGluR7a-C, GST-mGluR7a-N38 ends, and GST-mGluR7a-C27 starts, just before the conserved lysine. For mGluR8a-C, GST-mGluR8a-N24 only included the proximal signal transduction domain with the G-protein βγ and Ca2+/calmodulin binding sites (28.El Far O. Bofill-Cardona E. Airas J.M. O'Connor V. Boehm S. Freissmuth M. Nanoff C. Betz H. J. Biol. Chem. 2001; 276: 30662-30669Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), whereas GST-mGluR8a-C44 contained all conserved lysines outside of this signaling domain. GST fusion protein levels were normalized and tested semiquantitatively for the amount retained on beads (Ponceau stain on nitrocellulose membrane, Fig. 4B, lower panel). Lower protein levels were seen particularly for GST-mGluR7a-N38, GST-mGluR7a-K889R, GST-mGluR8a-C, and mGluR8a-K882R. Binding of CFP-Pias1 was found with GST-mGluR7a-N38 and mGluR8a-C44, whereas the complementary truncations GST-mGluR7a-C27 and GST-mGluR8a-N24 failed to interact. In sequence alignments (Figs. 2A and 4A), mGluR7a-N38 and mGluR8a-C44 overlap by 17 amino acids but are only identical in the last six residues preceding the consensus sumoylation motif (sequence DRPNGE; see amino acids 875–880 of mGluR8a). We therefore deduce that these residues are important for Pias1 recruitment to group III mGluRs. In Vivo Sumoylation of mGluR8a-C Requires Lys882—To demonstrate that mGluR8a-C is sumoylated in vivo, GFP-mGluR8a-C and the following tagged components of the sumoylation pathway were co-expressed in HEK293 cells: CFP-sumo1, E1 components aos1/uba2, YFP-ube2a, and GFP-Pias1. After detergent extraction of the transfected cells in the presence of protease inhibitors and 20 mm N-ethylmaleimide, which blocks sumo1-deconjugating enzymes (31.Suzuki T. Ichiyama A. Saitoh H. Kawakami T. Omata M. Chung C.H. Kimura M. Shimbara N. Tanaka K. J. Biol. Chem. 1999; 274: 31131-31134Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), the extracts were separated by SDS-PAGE and Western-blotted with an antibody directed against the mGluR8 CTD. In the transfected, but not in untransfected cells, a significant fraction of mGluR8a-C immunoreactivity displayed a size shift to ∼70 kDa, consistent with the addition of a single CFP-sumo1 molecule (Fig. 5A). Parallel Western blotting with an anti-sumo antibody confirmed that the 70-kDa band was indeed sumoylated (Fig. 5B). We also examined whether co-transfection of all six cDNAs was necessary for this sumoylation and found that only ube2a was not endogenously expressed at levels high enough to yield visible sumoylation of the overexpressed mGluR8a-C (data not shown). Inclusion of the E1 enzyme (aos1/uba2) proved to be second most important, whereas overexpression of Pias1 appeared not to be required and hence was omitted from subsequent transfections. Substitutions of target lysines by equally charged arginines are commonly used to identify motifs for sumoylation, whereas corresponding alanine substitutions have been shown to result in reduced binding of E2 to substrate proteins like Ran GTPase-activating protein (RanGAP1) (32.Sampson D.A. Wang M. Matunis M.J. J. Biol. Chem. 2001; 276: 21664-21669Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Here, sumoylation of mGluR8a-C was also abolished upon replacing specific lysines by arginines within the CTD (for positions of lysine substitutions, see Fig. 4A). Substitutions were selected based on two criteria: location within C44 of mGluR8a and conservation in both, mGluR8a and 8b. We found that single or triple arginine substitutions, including Lys882, abolished sumo-conjugation, whereas single or combined substitution of Lys868 and Lys872 did not interfere with this modification (Fig. 5A). Notably, sumo-conjugation did not on occur the Lys868 neighboring lysines or Lys872 when the consensus sumoylation lysine 882 had been substituted. Also, K882R substitution or the triple mutation K868R/K872R/K882R did not lead to sumoylation of one of the remaining four lysines in the CTD of mGluR8a (Fig. 5A). Thus, in transfected cells, sumoylation of mGluR8a-C occurs specifically at lysine 882 located within the conserved consensus sumoylation motif. Notably, arginine substitution of Lys882 in mGluR8a-C and of the homologous lysine Lys889 in mGluR7a-C did not affect binding of CFP-Pias1 in the GST pull-down assay (Fig. 4B). This further confirms that the interaction of Pias1 with mGluRs does not depend on an intact sumoylation consensus motif in the CTD. In th
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