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Probing the Ligand-binding Domain of the mGluR4 Subtype of Metabotropic Glutamate Receptor

1999; Elsevier BV; Volume: 274; Issue: 47 Linguagem: Inglês

10.1074/jbc.274.47.33488

1083-351X

David R. Hampson, Xi‐Ping Huang, Roman Pekhletski, Vanya Peltekova, Geoffrey Hornby, C. Thomsen, Henning Thøgersen,

Photoreceptor and optogenetics research

Metabotropic glutamate receptors (mGluRs) are G-protein-coupled glutamate receptors that subserve a number of diverse functions in the central nervous system. The large extracellular amino-terminal domains (ATDs) of mGluRs are homologous to the periplasmic binding proteins in bacteria. In this study, a region in the ATD of the mGluR4 subtype of mGluR postulated to contain the ligand-binding pocket was explored by site-directed mutagenesis using a molecular model of the tertiary structure of the ATD as a guiding tool. Although the conversion of Arg78, Ser159, or Thr182 to Ala did not affect the level of protein expression or cell-surface expression, all three mutations severely impaired the ability of the receptor to bind the agonist l-[3H]amino-4-phosphonobutyric acid. Mutation of other residues within or in close proximity to the proposed binding pocket produced either no effect (Ser157 and Ser160) or a relatively modest effect (Ser181) on ligand affinity compared with the Arg78, Ser159, and Thr182 mutations. Based on these experimental findings, together with information obtained from the model in which the glutamate analog l-serineO-phosphate (l-SOP) was “docked” into the binding pocket, we suggest that the hydroxyl groups on the side chains of Ser159 and Thr182 of mGluR4 form hydrogen bonds with the α-carboxyl and α-amino groups on l-SOP, respectively, whereas Arg78 forms an electrostatic interaction with the acidic side chains of l-SOP or glutamate. The conservation of Arg78, Ser159, and Thr182 in all members of the mGluR family indicates that these amino acids may be fundamental recognition motifs for the binding of agonists to this class of receptors. Metabotropic glutamate receptors (mGluRs) are G-protein-coupled glutamate receptors that subserve a number of diverse functions in the central nervous system. The large extracellular amino-terminal domains (ATDs) of mGluRs are homologous to the periplasmic binding proteins in bacteria. In this study, a region in the ATD of the mGluR4 subtype of mGluR postulated to contain the ligand-binding pocket was explored by site-directed mutagenesis using a molecular model of the tertiary structure of the ATD as a guiding tool. Although the conversion of Arg78, Ser159, or Thr182 to Ala did not affect the level of protein expression or cell-surface expression, all three mutations severely impaired the ability of the receptor to bind the agonist l-[3H]amino-4-phosphonobutyric acid. Mutation of other residues within or in close proximity to the proposed binding pocket produced either no effect (Ser157 and Ser160) or a relatively modest effect (Ser181) on ligand affinity compared with the Arg78, Ser159, and Thr182 mutations. Based on these experimental findings, together with information obtained from the model in which the glutamate analog l-serineO-phosphate (l-SOP) was “docked” into the binding pocket, we suggest that the hydroxyl groups on the side chains of Ser159 and Thr182 of mGluR4 form hydrogen bonds with the α-carboxyl and α-amino groups on l-SOP, respectively, whereas Arg78 forms an electrostatic interaction with the acidic side chains of l-SOP or glutamate. The conservation of Arg78, Ser159, and Thr182 in all members of the mGluR family indicates that these amino acids may be fundamental recognition motifs for the binding of agonists to this class of receptors. metabotropic glutamate receptors l-amino-4-phosphonobutyric acid serineO-phosphate γ-aminobutyric acid, type B amino-terminal domain leucine/isoleucine/valine-binding protein human embryonic kidney polymerase chain reaction (2S,3S,4S)-CCG/(2S,1′S,2′S)-2-(carboxycyclopropyl)glycine (RS)-α-cyclopropyl-4-phosphonophenylglycine phosphate-buffered saline Metabotropic glutamate receptors (mGluRs)1 are a family of eight G-protein-coupled receptors that are expressed throughout the central nervous system and in sensory cells of the retina and tongue. The mGluR family has been divided into three subgroups based on sequence homology, pharmacology, and signal transduction properties; in cell lines, group I mGluRs couple to phosphoinositide turnover, whereas group II and III receptors couple to the inhibition of forskolin-stimulated cAMP via Gi/Go proteins (1Pin J.-P. Duvoisin R. Neuropharmacology. 1995; 34: 1-26Crossref PubMed Scopus (1233) Google Scholar, 2Gomeza J. Mary S. Brabet I. Parmentier M.-L. Restituito S. Bockaert J. Pin J.-P. Mol. Pharmacol. 1996; 50: 923-930PubMed Google Scholar). mGluR4 together with mGluR6, mGluR7, and mGluR8 constitute the group III subclass of mGluRs that are selectively sensitive to the phosphono derivative of l-glutamate,l-amino-4-phosphonobutyric acid (l-AP4), and the endogenous amino acid l-serine O-phosphate (l-SOP). The group III mGluRs are important regulators of synaptic transmission in the central nervous system. Electrophysiological experiments have shown that activation of l-AP4-sensitive receptors causes a suppression of synaptic transmission by inhibiting neurotransmitter release from nerve terminals (3Macek T.A. Winder D.G. Gereau IV, R.W. Ladd L.O. Conn J.P. J. Neurosci. 1996; 76: 3798-3806Google Scholar), and immunocytochemical studies have confirmed that group III mGluRs are localized presynaptically (4Risso Bradley S. Standaert D.G. Rhodes K.J. Rees H.D. Testa C.M. Levey A.I. Conn P.J. J. Comp. Neurol. 1999; 407: 33-46Crossref PubMed Scopus (133) Google Scholar, 5Shigemoto 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, 6Stowell J.N. Craig A.M. Neuron. 1999; 22: 525-536Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The characterization of mutant mice lacking the mGluR4 subtype of mGluR has provided additional insight into the function of this receptor in the nervous system. For example, observations from electrophysiological analyses demonstrating impaired presynaptic functions in the mutant mice led to the suggestion that this receptor may be required for sustaining synaptic transmission during periods of high-frequency neurotransmission (7Pekhletski R. Gerlai R. Overstreet L. Huang X.-P. Agoypan N. Slater N.T. Roder J.C. Hampson D.R. J. Neurosci. 1996; 16: 6364-6373Crossref PubMed Google Scholar). Behavioral studies on mGluR4 mutant mice have shown that this receptor plays a role in motor and spatial learning (7Pekhletski R. Gerlai R. Overstreet L. Huang X.-P. Agoypan N. Slater N.T. Roder J.C. Hampson D.R. J. Neurosci. 1996; 16: 6364-6373Crossref PubMed Google Scholar,8Gerlai R. Roder J.C. Hampson D.R. Behav. Neurosci. 1998; 112: 1-8Crossref Scopus (54) Google Scholar). The potential use of group III mGluR ligands as therapeutic agents in epilepsy and neurodegenerative disorders has provided a persuasive argument for conducting more detailed structural analyses of this class of neurotransmitter receptors (9Nicoletti F. Bruno V. Copani A. Casabona G. Knopfel T. Trends Neurosci. 1996; 19: 267-272Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 10Thomsen C. Dalby N.O. Neuropharmacology. 1998; 37: 1465-1473Crossref PubMed Scopus (65) Google Scholar). The amino acid sequences of the mGluRs are homologous to the periplasmic amino acid-binding proteins in bacteria (11O'Hara P.J. Sheppard P. Thøgersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (617) Google Scholar), the calcium-sensing receptor of the parathyroid gland (12Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M. Lytton J. Hebert S.C. 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Cell. 1997; 90: 775-788Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar), and a class of taste receptors expressed in lingual tissue (18Moon M.A. Alder E. Lindemeier J. Battey J.F. Ryba N.J.P. Zucker C.S. Cell. 1999; 96: 541-551Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar). The basic structural domains of mGluRs include a large extracellular amino-terminal domain (ATD), seven putative transmembrane domains, and an intracellular carboxyl terminus. The homology of the ATDs of mGluRs to the leucine/isoleucine/valine-binding protein (LIVBP) and other bacterial periplasmic binding proteins that mediate the transport of amino acids in prokaryotes is fortuitous because the mGluRs appear to possess a similar three-dimensional fold and the crystal structures of the bacterial proteins are known (11O'Hara P.J. Sheppard P. Thøgersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (617) Google Scholar). Data obtained from experiments on chimeric constructs of the ATD of human mGluR4 with the transmembrane domains and carboxyl-terminal regions of mGluR1b (19Tones M.A. Bendali H. Flor P.J. Knopfel T. Kuhn R. Neuroreport. 1995; 7: 117-120Crossref PubMed Google Scholar) and constructs containing various segments of the ATD of rat mGluR2 and the transmembrane domain and carboxyl terminus of mGluR1a (20Takahashi K. Tsuchida K. Tanabe Y. Masu M. Nakanishi S. J. Biol. Chem. 1993; 268: 19341-19345Abstract Full Text PDF PubMed Google Scholar) indicated that pharmacological selectivity is conferred by residues located in the ATDs of mGluRs. More recent studies demonstrating that the ATDs of mGluR1 (21Okamoto T. Sekiyama N. Otsu M. Shimada Y. Sato A. Nakanishi S. Jingami H. J. Biol. Chem. 1998; 273: 13089-13096Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and mGluR4 (22Han G. Hampson D.R. J. Biol. Chem. 1999; 274: 10008-10013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) can be expressed as soluble proteins that are secreted from transfected cells and that retain ligand-binding capabilities have corroborated the concept that the primary determinants of ligand binding to mGluRs are contained within the ATDs. In this study, we have employed molecular modeling in conjunction with site-directed mutagenesis to probe the ligand-binding pocket of mGluR4. Our results indicate that three conserved amino acids present in the ATDs may be key determinants of ligand binding to all members of the mGluR family. The three-dimensional structure of the proposed ligand-binding domain of rat mGluR4 was formulated by homology modeling using the experimentally determined structure of the closed form of LIVBP from Escherichia coli and the strategy outlined by Blundell et al. (23Blundell T.L. Sibanda B.L. Sternberg M.J.E. Thornton J.M. Nature. 1987; 326: 347-352Crossref PubMed Scopus (589) Google Scholar). The atomic coordinates for the closed form of LIVBP with leucine in the binding pocket were kindly provided by Dr. F. A. Quiocho (Baylor College of Medicine). The QUANTA program (Version 97, MSI Corp.) and the SYBYL program (Version 6.4, Tripos Associates) were used to view the model that encompassed the region from Gly47 to Lys490 in the ATD of mGluR4. The sequence alignment used in the mGluR4 model has been described previously (11O'Hara P.J. Sheppard P. Thøgersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (617) Google Scholar). Backbone atom coordinates were assigned the corresponding residue coordinates from the crystal structure of LIVBP, and side chain atom coordinates were based on maximal side chain atom fitting to the LIVBP structure. Regions with insertions or deletions were modeled using known substructures identified by loop-searching techniques; regions 1–46, 125–149, 353–401, and 426–439, which are absent in LIVBP, were not included in the model. The l-SOP molecule was docked into the binding site of mGluR4 in an orientation that corresponds to that observed for leucine binding to LIVBP. The model was energy-optimized using a restrained energy minimization with additional constraints applied to the backbone regions based on the x-ray structure of LIVBP using the CHARMm force field. A steepest descent followed by a conjugate gradient method were used for energy minimization until the energy change per cycle was 0.05, one-way analysis of variance and Dunnett's multiple comparison test) from that of the untagged receptor, indicating that the insertion of the epitope at this site did not affect ligand affinity or the level of expression of mGluR4a.Table IAffinity constants and maximal binding capacities from l-[ 3 H]AP4 saturation binding analyses conducted on wild-type mGluR4a, c-Myc-tagged mGluR4a, and mutant receptorsmGluR4aK DB maxnmpmol/mgWT1-aWild-type.504 ± 998.6 ± 2.9c-Myc-WT404 ± 648.7 ± 1.3S157A683 ± 526.3 ± 1.0S160A470 ± 725.0 ± 1.6S181A570 ± 524.2 ± 1.2Each value is the mean ± S.E. of three to four experiments conducted in triplicate.1-a Wild-type. Open table in a new tab Each value is the mean ± S.E. of three to four experiments conducted in triplicate. The molecular model of the ATD of mGluR4 suggests that Arg78, Ser159, and Thr182 interact directly with the glutamate ligand. When mutated to alanine, all three residues produced receptors that were nearly devoid of the ability to bind l-[3H]AP4 (Fig.4). The R78A, S159A, and T182A mutants displayed 2 ± 0.8, 5 ± 1, and 4 ± 2% (mean ± S.E. of three experiments) of control (wild-type mGluR4a) binding, respectively. Due to the very low level of binding of the radioligand, it was not possible to obtain estimates of affinities for these two mutants in saturation or competition experiments. To further probe the ligand-binding domain of mGluR4, several additional mutations were made at amino acid residues that were predicted to be in or very near the binding pocket, but not directly involved in ligand binding. Saturation experiments showed that neither the dissociation constants nor the maximum numbers of binding sites of the S157A, S160A, and S181A mutants were significantly different from those of the wild-type receptor (p > 0.05, one-way analysis of variance and Dunnett's multiple comparison test) (Table I). To assess the pharmacological profile of these mutants, competition experiments were conducted using the agonists l-glutamate,l-SOP, and l-CCG-1 and the group III antagonist CPPG (31Toms N.J. Jane D.E. Kemp M.C. Bedingfield J.S. Roberts P.J. Br. J. Pharmacol. 1996; 119: 851-854Crossref PubMed Scopus (77) Google Scholar). The rank order of potency in the S157A, S160A, and S181A mutants was similar to that observed in the wild-type receptor (l-SOP > l-CCG-1 >l-glutamate > CPPG) (Fig.5). The inhibition constants for these drugs with the S157A and S160A mutants were also similar to those seen with the wild-type receptor (Table II). However, the inhibition constants for the S181A mutant were ∼3–5 times higher than those for the wild-type receptor, indicating that this mutation produced a moderate decrease in affinity for the series of compounds tested.Table IIInhibition constants of various drugs forl-[ 3 H]AP4 binding to wild-type mGluR4a and the S157A, S160A, and S181A mutantsmGluR4aIC50l-SOPl-CCG-1l-GlutamateCPPG μmWild-type2.7 ± 0.54.0 ± 1.55.7 ± 0.524 ± 2.7S157A2.2 ± 0.52.3 ± 0.611 ± 3.610.3 ± 4.3S160A3.2 ± 0.53.8 ± 0.34.0 ± 2.621.3 ± 1.1S181A10 ± 216 ± 4.129 ± 5.369 ± 13The concentration of l-[3H]AP4 was 30 nm. Data are the means ± S.E. of three experiments. Open table in a new tab The concentration of l-[3H]AP4 was 30 nm. Data are the means ± S.E. of three experiments. Although the results from the immunoblot experiments indicated that the R78A, S159A, and T182 mutant polypeptides were translated and expressed at levels comparable to those of the wild-type receptor, it is possible that the very low level of ligand binding of the mutants was caused by misfolding and/or lack of cell-surface expression. To investigate this possibility, an immunocytochemical analysis was carried out on the c-Myc-tagged wild-type receptor, the R78A and T182A mutant receptors, and the untagged S159A receptor. Cell-surface expression was assessed by labeling lightly fixed HEK cells (4% paraformaldehyde for 10 min) with the anti-mGluR4a or anti-c-Myc antibody, followed by a biotinylated anti-rabbit or anti-mouse secondary antibody and a fluorescein isothiocyanate-avidin conjugate. Cells expressing c-Myc-tagged wild-type mGluR4a labeled with the anti-mGluR4a antibody and treated with Triton X-100 to permeabilize the cells showed intense labeling in and particularly around the periphery of the cells, whereas similarly transfected cells not treated with Triton X-100 displayed only background labeling (Fig.6, A and B). The absence of specific immunostaining in unpermeabilized transfected cells indicates that the fixation protocol used (without Triton X-100 treatment) did not cause permeabilization of the cells. The immunolabeling pattern observed wit