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• W2159672893 abstract "Ca2+/calmodulin (Ca2+/CaM) and the βγ subunits of heterotrimeric G-proteins (Gβγ) have recently been shown to interact in a mutually exclusive fashion with the intracellular C terminus of the presynaptic metabotropic glutamate receptor 7 (mGluR 7). Here, we further characterized the core CaM and Gβγ binding sequences. In contrast to a previous report, we find that the CaM binding motif localized in the N-terminal region of the cytoplasmic tail domain of mGluR 7 is conserved in the related group III mGluRs 4A and 8 and allows these receptors to also bind Ca2+/CaM. Mutational analysis of the Ca2+/CaM binding motif is consistent with group III receptors containing a conventional CaM binding site formed by an amphipathic α-helix. Substitutions adjacent to the core CaM target sequence selectively prevent Gβγ binding, suggesting that the CaM-dependent regulation of signal transduction involves determinants that overlap with but are different from those mediating Gβγ recruitment. In addition, we present evidence that Gβγ uses distinct nonoverlapping interfaces for interaction with the mGluR 7 C-terminal tail and the effector enzyme adenylyl cyclase II, respectively. Although Gβγ-mediated signaling is abolished in receptors lacking the core CaM binding sequence, α subunit activation, as assayed by agonist-dependent GTPγS binding, was not affected. This suggests that Ca2+/CaM may alter the mode of group III mGluR signaling from mono- (α) to bidirectional (α and βγ) activation of downstream effector cascades. Ca2+/calmodulin (Ca2+/CaM) and the βγ subunits of heterotrimeric G-proteins (Gβγ) have recently been shown to interact in a mutually exclusive fashion with the intracellular C terminus of the presynaptic metabotropic glutamate receptor 7 (mGluR 7). Here, we further characterized the core CaM and Gβγ binding sequences. In contrast to a previous report, we find that the CaM binding motif localized in the N-terminal region of the cytoplasmic tail domain of mGluR 7 is conserved in the related group III mGluRs 4A and 8 and allows these receptors to also bind Ca2+/CaM. Mutational analysis of the Ca2+/CaM binding motif is consistent with group III receptors containing a conventional CaM binding site formed by an amphipathic α-helix. Substitutions adjacent to the core CaM target sequence selectively prevent Gβγ binding, suggesting that the CaM-dependent regulation of signal transduction involves determinants that overlap with but are different from those mediating Gβγ recruitment. In addition, we present evidence that Gβγ uses distinct nonoverlapping interfaces for interaction with the mGluR 7 C-terminal tail and the effector enzyme adenylyl cyclase II, respectively. Although Gβγ-mediated signaling is abolished in receptors lacking the core CaM binding sequence, α subunit activation, as assayed by agonist-dependent GTPγS binding, was not affected. This suggests that Ca2+/CaM may alter the mode of group III mGluR signaling from mono- (α) to bidirectional (α and βγ) activation of downstream effector cascades. metabotropic glutamate receptor calmodulin l-amino(+)-2-amino-4-phosphonobutyric acid 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (−)-N 6-3-[125I](iodo-4-hydroxyphenyl-isopropyl)- adenosine guanosine trisphosphate binding protein β/γ subunits glutathione S-transferase guanosine 5′-O-(3′-thiotriphosphate) maltose-binding protein glutathione S-transferase ((RS)-α-Methyl-4-phosphonophenylglycine) Glutamate receptors mediate excitatory neurotransmission at most synapses in the central nervous system. Ionotropic glutamate receptors, such as AMPA, kainate, and NMDA receptors are involved in fast neurotransmission. In contrast, G protein-coupled metabotropic glutamate receptors (mGluRs)1have been implicated in the short and long term modulation of synaptic transmission within many pathways of the brain. In particular, activation of mGluRs may regulate neuronal development and survival, transmitter release, electrical excitability, synaptic plasticity, and memory formation (1Nakanishi S. Neuron. 1994; 13: 1031-1037Abstract Full Text PDF PubMed Scopus (648) Google Scholar, 2Pin J.P. Duvoisin R. Neuropharmacology. 1995; 34: 1-26Crossref PubMed Scopus (1234) Google Scholar, 3Conn P.J. Pin J.P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2728) Google Scholar, 4Nakanishi S. Nakajima Y. Masu M. Ueda Y. Nakahar K. Watanabe D. Yamaguchi S. Kawabata S. Okada M. Brain Res. Rev. 1998; 26: 230-235Crossref PubMed Scopus (292) Google Scholar). At least eight different mGluR subtypes have been identified by molecular cloning. Based on sequence homology, pharmacology, and signal transduction mechanisms in heterologous expression systems, these receptors are classified into three groups. Group I receptors (mGluRs 1 and 5) are linked to phospholipase C, whereas members of the groups II (mGluRs 2 and 3) and III (mGluRs 4, 6, 7, and 8) couple to the cAMP signaling pathway. However, this effector coupling may not be stringent, since in cerebellar neurons mGluR 7-mediated inhibition of P/Q type calcium channels involves phospholipase C (5Perroy J. Prezeau L. de Ward M. Shigemoto R. Bockaert J. Fagni L. J. Neurosci. 2000; 20: 7896-7904Crossref PubMed Google Scholar). The different groups of mGluRs not only use distinct signaling pathways but also display different subcellular localizations. Group I mGluRs are predominantly postsynaptic receptors located at perisynaptic membrane areas. Group II receptors can be found pre- as well as postsynaptically and appear to be distributed over the surface of axons and dendrites. Members of group III mGluRs are primarily presynaptic and concentrated at or near active zones (6Pin J.P. Bockaert J. Curr. Opin. Neurobiol. 1995; 5: 342-349Crossref PubMed Scopus (128) Google Scholar, 7Shigemoto R. Kulik A. Roberts J.D. Ohishi H. Nusser Z. Kaneko T. Somogyi P. Nature. 1996; 381: 523-525Crossref PubMed Scopus (337) Google Scholar, 8Cartmell J. Schoepp D.D,. J. Neurochem. 2000; 75: 889-907Crossref PubMed Scopus (777) Google Scholar). This selective localization of mGluRs to discrete subcellular domains has an important impact on receptor signaling. First, restricting expression to the pre- or postsynaptic compartment will define the sites at which mGluRs regulate synaptic transmission. Second, receptor distance from release sites will determine how readily the receptors are confronted by activating concentrations of glutamate. Finally, their precise subcellular localization will regulate the receptors' proximity to target ion channels, the transmitter release machinery, and/or enzyme cascades that regulate synaptic function. The relevance of such precise topological arrangements for the synaptic function of mGluRs is highlighted by the group III receptor, mGluR 7. This low affinity receptor is selectively targeted to presynaptic transmitter release sites, enabling it to sense high levels of released glutamate. This in turn allows for rapid autoinhibition of the neighboring Ca2+ channels, whose opening triggers neurotransmitter release (7Shigemoto R. Kulik A. Roberts J.D. Ohishi H. Nusser Z. Kaneko T. Somogyi P. Nature. 1996; 381: 523-525Crossref PubMed Scopus (337) Google Scholar, 9O'Connor V. El Far O. Bofill-Cardona E. Nanoff C. Freissmuth M. Karschin A. Airas J.M. Betz H. Boehm S. Science. 1999; 286: 1180-1184Crossref PubMed Scopus (135) Google Scholar). Axon targeting of mGluR 7 has been shown to involve the cytoplasmic C terminus of the receptor (10Stowell J.N. Craig A.M. Neuron. 1999; 22: 525-536Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Furthermore, the formation of presynaptic clusters of mGluR 7 is also mediated by this C-terminal tail, which provides a binding motif for PICK1 (protein interacting with Ckinase; Refs. 11Boudin H. Doan A. Xia J. Shigemoto R. Huganir R.L. Worley P. Craig A.M. Neuron. 2000; 28: 485-497Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 12El Far O. Airas J.M. Wischmeyer E. Nehring R.B. Karschin A. Betz H. Eur. J. Neurosci. 2000; 12: 4215-4221PubMed Google Scholar, 13Dev K.K. Nakajima Y. Kitano J. Braithwaite S.P. Henley J.M. Nakanishi S. J. Neurosci. 2000; 20: 7252-7257Crossref PubMed Google Scholar). Although the interaction with PICK1 was found to be essential for clustering, but not for axonal targeting, of mGluR 7 (11Boudin H. Doan A. Xia J. Shigemoto R. Huganir R.L. Worley P. Craig A.M. Neuron. 2000; 28: 485-497Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), PICK1 binding did not influence effector regulation via this receptor (12El Far O. Airas J.M. Wischmeyer E. Nehring R.B. Karschin A. Betz H. Eur. J. Neurosci. 2000; 12: 4215-4221PubMed Google Scholar). Different lines of evidence indicate that the C-terminal portion of mGluR 7 is essential for signal transduction. First, this region binds Gβγ subunits, which mediate Ca2+ channel closure and inhibition of neurotransmitter release (14Herlitze S. Garcia D.E. Mackie K. Hille B. Scheuer T. Catterall W.A. Nature. 1996; 380: 258-262Crossref PubMed Scopus (702) Google Scholar, 15Ikeda S.R. Nature. 1996; 380: 255-258Crossref PubMed Scopus (709) Google Scholar). Second, Ca2+/CaM binding to this region of the receptor promotes G protein-mediated signaling by displacing Gβγ subunits from the C-terminal tail (9O'Connor V. El Far O. Bofill-Cardona E. Nanoff C. Freissmuth M. Karschin A. Airas J.M. Betz H. Boehm S. Science. 1999; 286: 1180-1184Crossref PubMed Scopus (135) Google Scholar). Third, binding of Ca2+/CaM prevents phosphorylation of the C terminus by protein kinase C (16Nakajima Y. Yamamoto T. Nakayama T. Nakanishi S. J. Biol. Chem. 1999; 274: 27573-27577Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), a reaction that may underlie receptor desensitization (17Macek T.A. Schaffhauser H. Conn P.J. J. Neurosci. 1998; 18: 6138-6146Crossref PubMed Google Scholar). In addition, protein kinase C-dependent phosphorylation of mGluR 7 may also be hindered by PICK1 binding (13Dev K.K. Nakajima Y. Kitano J. Braithwaite S.P. Henley J.M. Nakanishi S. J. Neurosci. 2000; 20: 7252-7257Crossref PubMed Google Scholar). To further elucidate how the C terminus of mGluR 7 interacts with the various signaling components mentioned above, we investigated the determinants that mediate binding of the mGluR 7 C-tail to both Ca2+/CaM and Gβγ subunits. Previously, we and others have found that PICK1 interacts with the last three amino acids of the mGluR C-tail (11Boudin H. Doan A. Xia J. Shigemoto R. Huganir R.L. Worley P. Craig A.M. Neuron. 2000; 28: 485-497Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 12El Far O. Airas J.M. Wischmeyer E. Nehring R.B. Karschin A. Betz H. Eur. J. Neurosci. 2000; 12: 4215-4221PubMed Google Scholar, 13Dev K.K. Nakajima Y. Kitano J. Braithwaite S.P. Henley J.M. Nakanishi S. J. Neurosci. 2000; 20: 7252-7257Crossref PubMed Google Scholar). Here, we show that Ca2+/CaM and Gβγ bind to partially overlapping domains located at the N-terminal part of the mGluR 7 C-tail. This region is conserved in the related group III receptors, mGluR 4A and mGluR 8A and 8B, which are also shown to interact with Ca2+/CaM. Mutations that prevent CaM binding selectively inhibit mGluR 7 signaling through Gβγ subunits but do not affect trimeric G-protein recruitment to the receptor. Our data are consistent with Ca2+/CaM causing group III mGluRs to switch from mono- (Gα) to bidirectional (Gα and Gβγ) signaling. EcoRI andSalI site flanked cDNA fragments encoding the C-terminal regions of mGluR 4A, 7A, 7B, 8A, and 8B, respectively, were generated by standard polymerase chain reactions on mouse brain cDNA using procedures similar to those described in Ref. 9O'Connor V. El Far O. Bofill-Cardona E. Nanoff C. Freissmuth M. Karschin A. Airas J.M. Betz H. Boehm S. Science. 1999; 286: 1180-1184Crossref PubMed Scopus (135) Google Scholar. The polymerase chain reaction products were inserted in pGEX-5 × 1 (Amersham Pharmacia Biotech). The mGluR 4B and 6 C-terminal tail regions were amplified using mGluR 4 and mGluR 6 full-length cDNAs (kindly provided by Dr. S. Nakanishi, University of Kyoto, Japan). Due to insolubility of mGluR 4B as a GST fusion protein, the cDNA sequences encoding the mGluR 7A and mGluR 4B C-terminal tails were introduced into the pMAL-c2 vector (New England Biolabs) to produce soluble maltose-binding protein (MBP) fusion tails. The following truncated and mutated tail regions of mGluR 7A were also generated by polymerase chain reaction and fused to GST: mGluR 7A-N38 (GST-7A-N38), mGluR 7A-C27 (GST-7A-C27), mGluR 7A-N25 (GST-7A-N25), mGluR 7A-N25-Q857A (GST-N25-Q857A), mGluR 7A-R859E (GST-7A-R859E), mGluR 7A-R861P (GST-7A-R861P), mGluR 7A-F863A (GST-7A-F863A), mGluR 7A-M872A (GST-7A-M872A), mGluR 7A-M872E (GST-7A-M872E), mGluR 7A-N25-K860E (GST-N25-K860E), mGluR 7A-N25-S862E (GST-N25-S862E), mGluR 7A-N25-S862A (GST-N25-S862A), mGluR 7A-N25-K864E/A865E/V866E (GST-N25–3E), mGluR 7A-N25-F863E/K864E/A865E/V866E (GST-N25–4E), mGluR 7A-N25-S862E/T868E/T871E/S873E (GST-N25–4X), and mGluR 7A-ΔCaM (GST-7A-ΔCaM), where residues 864–876 (KAVVTAATMSSRL) were deleted. All amplification products were verified by automated DNA sequencing. Expression of GST fusion proteins in Escherichia coli BL21 (Stratagene) was induced by adding 0.5 mm isopropyl β-d-thiogalactopyranoside for 3–5 h. After passage through a French press, the 100,000 × g supernatants containing the soluble fusion proteins were kept frozen (−76 °C) until use. GST fusion protein binding to CaM-agarose (Sigma) was performed in phosphate-buffered saline containing 0.1% (w/v) Triton X-100 in the presence of 2 mm CaCl2 or 5 mm EGTA, respectively. For every binding reaction, 20 µl of CaM-agarose were preequilibrated in the corresponding binding buffer (+CaCl2 or +EGTA) and incubated in the presence of either CaCl2 or EGTA with 0.5 ml of 100,000 × gsupernatant prepared from bacterial extracts containing ∼0.2 mg/ml GST fusion protein. After 2 h of rotary agitation at 4 °C, the beads were collected by centrifugation and washed three times with 1 ml each of the corresponding binding buffer prior to elution with SDS sample buffer. Binding of purified CaM to GST fusion proteins immobilized on glutathione-Sepharose 4B beads was performed as described (9O'Connor V. El Far O. Bofill-Cardona E. Nanoff C. Freissmuth M. Karschin A. Airas J.M. Betz H. Boehm S. Science. 1999; 286: 1180-1184Crossref PubMed Scopus (135) Google Scholar). Eluates were resolved by SDS-polyacrylamide gel electrophoresis using 12 or 15% gels and examined after Coomassie Blue staining. Native mGluR 7 (or mGluR 4A) was solubilized from rat hippocampal (or cerebellar) synaptosomal membranes that were prewashed with binding buffer (20 mm HEPES/NaOH, pH 7.4, 100 mmKCI, protease inhibitor mixture Complete (Roche Diagnostics)), containing a 1 mm concentration each of EDTA and EGTA. Stripped membranes were centrifuged at 30,000 × g and solubilized in binding buffer containing 1.5% (w/v) Triton X-100. After centrifugation at 100,000 × g, the extract was supplemented with 1 mm CaCl2 and incubated for 2 h at 4 °C with 1.5 mg of CaM-agarose. After removing unbound material, the beads were divided and washed three times with binding buffer containing 0.5% (w/v) Triton X-100 and either 5 mmEGTA or 1 mm CaCl2. Bound material was eluted with SDS sample buffer, and fractions were processed for Western blotting with mGluR 7 antibody, a generous gift from Dr. R. Shigemoto (National Institute for Physiological Sciences, Okazaki, Japan), or mGluR 4A antibody (Upstate Biotechnology, Inc., Lake Placid, NY), respectively. Fluorescence spectra were recorded in a Hitachi F4500 fluorescence spectrophotometer under continuous stirring at room temperature in a final volume of 0.2 to 0.25 ml. Dansyl-CaM (Molecular Probes, Leiden, The Netherlands) was diluted in 50 mm Tris/HCl buffer, pH 8.0, 1 mmEDTA, 150 mm NaCl, and 2 mm CaCl2to final concentrations between 0.1 and 0.8 µm (where indicated, nominally Ca2+-free solution was used). The excitation wavelength was 350 nm; the emission monochromator was changed at a rate of 200 nm/s between 400 and 600 nm. Subsequently, GST-7A, GST-7A-N25, or an equivalent concentration of GST were added in 2–5-µl aliquots to generate cumulative concentration-response curves in the concentration range from 0.1 to 4 µm. Data were fitted to the Hill equation by curvilinear regression to estimate EC50 values for GST-7A and for GST-7A-N25. At each dansyl-CaM concentration, three independent concentration-response curves were obtained using different preparations of both recombinant fusion proteins, and dansyl-CaM. EC50 values are given as means ± S.D. G proteins were solubilized from porcine brain membranes using the zwitterionic detergent CHAPS; βγ dimers were chromatographically resolved from the α-subunits (18Nanoff C. Waldhoer M. Roka F. Freissmuth M. Neuropharmacol. 1997; 36: 1211-1219Crossref PubMed Scopus (23) Google Scholar). GST fusion proteins or GST (20 µg/reaction) were immobilized on glutathione-Sepharose 4B beads (50 µl of preequilibrated 1:1 slurry) in buffer containing 50 mm Tris/HCl, pH 8.0, 1 mm EDTA, 2 mm MgCl2, 120 mm NaCl, and 8 mm CHAPS. After three washes, the beads were incubated with purified G protein βγ-subunits (80 pmol/reaction) for 2 h at 4 °C in 100 µl of buffer. Where indicated, either 2 mm CaCl2 or 2 mm CaCl2 plus 10 µm CaM was added to the mixture. After three washes, bound proteins were eluted with 40 µl of buffer containing 15 mm glutathione (pH adjusted to 8.0 with NaOH), subjected to 12% SDS-polyacrylamide gel electrophoresis, and analyzed by Western blotting with rabbit antiserum 7 that recognizes the predominant forms Gβ1 and Gβ2 (19Nanoff C. Mitterauer T. Roka F. Hohenegger M. Freissmuth M. Mol. Pharmacol. 1995; 48: 806-817PubMed Google Scholar) followed by a horseradish peroxidase-coupled anti-rabbit secondary antibody (Amersham Pharmacia Biotech). The short splice variant of recombinant Gαs (rGαs-s) was expressed in E. coli (BL21DE3) and purified from bacterial lysates. Where applicable, rGαs-s (10 µm) was preactivated for 60 min at 30 °C in buffer containing 50 mm HEPES/NaOH, pH 7.6, 1 mm EDTA, 10 mm MgSO4, 100 µm GTPγS, and 0.025% (w/v) Lubrol; the nucleotide was subsequently removed by gel filtration (20Freissmuth M. Gilman A.G. J. Biol. Chem. 1989; 264: 21907-21914Abstract Full Text PDF PubMed Google Scholar). Sf9 cell membranes expressing mammalian adenylyl cyclase type II (21Weitmann S. Wursig N. Navarro J.M. Kleuss C. Biochemistry. 1999; 38: 3409-3413Crossref PubMed Scopus (23) Google Scholar) were a generous gift of C. Kleuss (Freie Universität, Berlin). For the determination of adenylyl cyclase activity, Sf9 membranes (10 µg/assay) were incubated in a final volume of 50 µl containing 50 mm HEPES/NaOH, pH 7.5, 10 mm MgCl2, 0.5 mm[α-32P]ATP (PerkinElmer Life Sciences; specific activity 20–40 cpm/pmol), 10 mm creatine phosphate, 1 mg/ml creatine kinase, and 0.1 mm RO201724 (Sigma). The carry-over from the preactivation of rGαs-s and the dilution of βγ dimers resulted in the presence of 1 mmMgSO4 and 0.01% (w/v) Lubrol, which both have no appreciable effect on catalysis (22Kudlacek O. Mitterauer T. Nanoff C. Hohenegger M. Tang W.-J. Freissmuth M. Kleuss C. J. Biol. Chem. 2001; 276: 3010-3016Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Where indicated, the final concentrations of rGαs-s, βγ dimers, and GST-7A-N25 (or GST) were 0.3 µm, 25 nm, and 100 µm, respectively. The reaction was allowed to proceed at 25 °C for 20 min; [32P]cAMP formed during the incubation was quantified according to Ref. 23Johnson R.A. Salomon Y. Methods Enzymol. 1991; 195: 3-21Crossref PubMed Scopus (122) Google Scholar. The synthesis of the agonist radioligand [125I]HPIA, the generation of HEK293 cell lines stably expressing the human A1-adenosine receptor, and the assay conditions for A1-adenosine receptor binding have been described previously (24Waldhoer M. Bofill-Cardona E. Milligan G. Freissmuth M. Nanoff C. Mol. Pharmacol. 1998; 53: 808-818PubMed Google Scholar). In brief, EGTA-stripped HEK293 cell membranes (9–15 µg of protein/assay) were incubated in 40 µl of buffer containing 50 mm Tris/HCl, pH 8.0, 1 mmEDTA, 5 mm MgCl2, 8 µg/ml adenosine desaminase, and [125I]HPIA (final concentration 0.8–1.2 nm). The binding reaction was allowed to proceed for 1.5 h at 20 °C in the presence of GST, GST-7A, GST-7A-N25, GST-N25-Q857A, or GST-N25-S862E, at concentrations of 10–70 µm. Incubations were performed in the absence and presence of 2 mm Ca2+ or of 2 mmCa2+ and 20 µm CaM, respectively. Control experiments with the antagonist 3H-labeled 8-cyclopentyl-1,3-dipropylxanthine showed that none of our experimental conditions affected antagonist binding. All assays were performed in duplicate and repeated 3–4 times. HEK293 cells were co-transfected with plasmids driving the expression of wild-type mGluR 7A or mGluR-7A-ΔCaM and the human D2-dopamine receptor (25Bofill-Cardona E. Kudlacek O. Yang Q. Ahorn H. Freissmuth M. Nanoff C. J. Biol. Chem. 2000; 275: 32672-32680Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). FLAG epitopes and c-Myc epitopes were present at the N termini of mGluR 7A (or mGluR-7A-ΔCaM) and of the D2-dopamine receptor, respectively, to verify cell surface expression of the receptors. Agonist-stimulated [35S]GTPγS binding was determined as described (19Nanoff C. Mitterauer T. Roka F. Hohenegger M. Freissmuth M. Mol. Pharmacol. 1995; 48: 806-817PubMed Google Scholar). In brief, membranes (15–20 µg of protein/assay) prepared from the transiently transfected cells were preincubated for 15 min in 40 µl of 20 mm HEPES/NaOH, pH 7.5, containing 1 mm EDTA and 1.5 mmMgCl2 at 20 °C in the presence of either the group III mGluR agonist l-AP4 (300 µm), or the antagonist MPPG (100 µm); quinpirol (1 µm) and sulpirid (10 µm) were used for the D2-dopamine receptor. Thereafter, the reaction was started by adding 10 µl of buffer containing 50 µm GDP and 100 nm [35S]GTPγS (PerkinElmer Life Sciences; specific activity ∼1600 cpm/fmol) and quenched at the indicated time points by adding 1 ml of ice-cold stop buffer (10 mmTris/HCl, pH 8.0, 20 mm MgCl2, 100 mm NaCl, 0.1 mm GTP). Bound and free nucleotides were separated by filtration over glass fiber filters, and bound radioactivity was determined by scintillation counting. Data represent means ± S.D. from three experiments carried out in duplicate. We have previously shown that Ca2+/CaM and Gβγ bind in a mutually exclusive manner to the C-terminal tail region of mGluR 7 (9O'Connor V. El Far O. Bofill-Cardona E. Nanoff C. Freissmuth M. Karschin A. Airas J.M. Betz H. Boehm S. Science. 1999; 286: 1180-1184Crossref PubMed Scopus (135) Google Scholar). Since both known splice variants of mGluR 7, 7A and 7B, bound Ca2+/CaM and since these variants differ only in their C-terminal half (Fig. 1 A), we assigned the CaM binding site to the N-terminal portion of the tail regions. To confirm this idea and to exclude a contribution of the divergent sequences at the extreme C terminus, we used binary interaction assays with truncated receptor tail GST fusion constructs. Pull-down assays employing CaM immobilized on agarose beads proved superior to the previously used binding assay with purified CaM and GST fusion proteins immobilized on glutathione-Sepharose. Fig.1 B shows that both of the N-terminal region constructs, GST-7A-N38 and GST-7A-N25, bound CaM-agarose in the presence of Ca2+ but not EGTA, whereas the C-terminal GST-7A-C27 fusion protein failed to bind CaM even in Ca2+-containing buffer. Ca2+/CaM binding to mGluR 7 sequences was not a fusion protein artifact, since native mGluR 7 solubilized from hippocampal membranes with Triton X-100 also bound CaM-agarose in a Ca2+-dependent manner (Fig. 1 C). The C-terminal tail of mGluR 7A also contains a binding site for Gβγ, and Ca2+/CaM is known to inhibit binding of Gβγ to GST-7A (9O'Connor V. El Far O. Bofill-Cardona E. Nanoff C. Freissmuth M. Karschin A. Airas J.M. Betz H. Boehm S. Science. 1999; 286: 1180-1184Crossref PubMed Scopus (135) Google Scholar). We therefore examined whether the binding site for Gβγ also resides in the N-terminal region of mGluR 7A by incubating GST-7A-N38, GST-7A-N25, and GST-7A-C27 with purified Gβγ. GST-7A-C27 did not retain Gβγ, while comparable amounts of Gβγ were recovered with both GST-7A-N38 and GST-7A-N25 (Fig.1 D, top panel). When directly compared with GST-7A, GST-7A-N25 was as effective in retaining Gβγ as the full-length tail region (see Fig. 5 A). Finally, binding of Gβγ to GST-7A-N25 was abolished in the presence of CaM (Fig.1 D, lower panel). We therefore conclude that the first 25 residues in the N-terminal region of the mGluR 7 tail domain suffice to support both Ca2+/CaM and Gβγ binding. In order to obtain an estimate of the affinity of the C-terminal tail of mGluR 7A for Ca2+/CaM, we incubated dansylated CaM (dansyl-CaM) with GST, GST-7A, or GST-7A-N25 fusion proteins and measured the changes in fluorescence emission induced by its interaction with the binding motif. In the absence of Ca2+, fluorescence emission by dansyl-CaM was modest (Fig.2 A, trace 1). Binding of Ca2+ induces a conformational change in CaM, which in dansyl-CaM reduces the exposure of the fluorophore to the aqueous environment and could be detected as an enhancement and a blue shift in the emitted fluorescence (Fig.2 A, trace 2). The addition of GST had no effect on the spectrum of dansyl-CaM in both the presence (Fig.2 A, trace 3) or absence of Ca2+ (data not shown). In contrast, GST-7A (trace 4 in Fig. 2 A) and GST-7A-N25 (trace 5 in Fig. 2 A) caused substantial fluorescence enhancement; this increase in light emission probably reflects shielding of the fluorophore by binding of the C-terminal peptide. Because of an excellent signal/noise ratio, the concentration-dependent effects of GST-7A (and of GST-N25) were readily detected (Fig. 2 B and data not shown). Notably, however, the apparent EC50 values of GST-7A depended on the concentration of dansyl-CaM (see Fig. 2 B); this was most readily evident when data were normalized to maximal fluorescence enhancement (Fig. 2 C). This concentration dependence can be attributed to the depletion of free GST-7A, which is most pronounced at low concentrations of GST-7A. Depletion is most easily corrected for by determining EC50 values over a reasonably large range of dansyl-CaM concentrations; for infinitely low concentrations of dansyl-CaM, depletion should be nonexistent, and the EC50value should hence be a true affinity estimate. Fig. 2 Dshows that the EC50 estimates for both GST-7A or of GST-7A-N25 indeed depended in a linear manner on the dansyl-CaM concentration employed. The slope of the regression line was close to unity, indicating a 1:1 stoichiometry; the true affinity value calculated from the y intercept was in the range of 70 nm. Fig. 2 D also indicates that the EC50 values for GST- 7A (closed symbols) and for GST-7A-N25 (open symbols) fell onto the same line. We therefore conclude that GST-7A-N25 comprises all residues required for high affinity binding of CaM. To unravel whether other members of group III mGluRs also might interact with Ca2+/CaM, we tested the CaM binding of fusion proteins of all known group III mGluR tail sequences to CaM-agarose. Fig. 3 A shows that GST fusion proteins encoding the C-terminal tails of mGluR 4A, 8A, and 8B (GST-4A, -8A, -8B) bound CaM-agarose in the presence of Ca2+, but not of EGTA. Notably, GST-6 failed to interact with CaM agarose even in the presence of Ca2+ (not shown). To confirm this observation by a different approach, we immobilized GST-6 on glutathione-Sepharose and incubated the resulting matrix with purified CaM. Again, no CaM binding was detected (Fig. 3 C). This result is consistent with several substitutions in the putative CaM binding region of mGluR 6 (Fig. 1 A). A high intrinsic insolubility of GST-4B precluded assaying its potential CaM binding potential (data not shown). To overcome this problem, we generated an MBP fusion construct encoding the C-terminal tail of mGluR 4B. The resulting fusion protein (MBP-4B) showed improved solubility upon expression in bacterial cells. As a positive control, the mGluR 7A tail region was also fused to MBP. Fig. 3 A demonstrates that MBP-7A but neither MBP nor MBP-4B interacted with CaM-agarose in the presence of Ca2+. This again is consistent with the predicted CaM binding consensus sequence highlighted in Fig.1 A that is conserved in all group III mGluRs except mGluR 4B and mGluR 6. Confirming our assignment of the CaM binding region, a previously described fusion protein lacking amino acids 864–876 within the CaM binding region of GST-7A (GST-7A-ΔCaM; see Ref. 9O'Connor V. El Far O. Bofill-Cardona E. Nanoff C. Freissmuth M. Karschin A. Airas J.M. Betz H. Boehm S. Science. 1999; 286: 1180-1184Crossref PubMed Scopus (135) Google Scholar) also failed to bind to CaM agarose (data not shown). Since this fusion protein was very sensitive to proteolysis, we also immobilized GST-7A-ΔCaM on glutathione-Sepharose. Again, the resulting matrix displayed no detectable CaM binding (Fig. 3 C). Our results contrast with previous observations showing that CaM binding is a unique feature of mGluR 7 (16Nakajima Y. Yamamoto T. Nakayama T. Nakanishi S. J. Biol. Chem. 1999; 274: 27573-27577Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). To corroborate that indeed native group III mGluRs other than mGluR 7 bind Ca2+/CaM, we also performed CaM affinity adsorption of mGluR 4A solubilized from cerebellar membranes. Native mGluR 4A receptors were specifically retained on CaM-agarose in the presence of Ca2+ but not EGTA. 2V. O'Connor, unpublished data. This result corroborates the pull-down results wit" @default.
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• W2159672893 title "Mapping of Calmodulin and Gβγ Binding Domains within the C-terminal Region of the Metabotropic Glutamate Receptor 7A" @default.
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