Molecular Determinants of High Affinity Binding to Group III Metabotropic Glutamate Receptors
2002; Elsevier BV; Volume: 277; Issue: 9 Linguagem: Inglês
10.1074/jbc.m110476200
ISSN1083-351X
AutoresErica Rosemond, Vanya Peltekova, Mark Naples, Henning Thøgersen, David R. Hampson,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoThe amino-terminal domain containing the ligand binding site of the G protein-coupled metabotropic glutamate receptors (mGluRs) consists of two lobes that close upon agonist binding. In this study, we explored the ligand binding pocket of the Group III mGluR4 receptor subtype using site-directed mutagenesis and radioligand binding. The selection of 16 mutations was guided by a molecular model of mGluR4, which was based on the crystal structure of the mGluR1 receptor. Lysines 74 and 405 are present on lobe I of mGluR4. The mutation of lysine 405 to alanine virtually eliminated the binding of the agonist [3H]l-amino-4-phosphonobutyrate ([3H]l-AP4). Thus lysine 405, which is conserved in all eight mGluRs, likely represents a fundamental recognition residue for ligand binding to the mGluRs. Single point mutations of lysines 74 or 317, which are not conserved in the mGluRs, to alanine had no effect on agonist affinity, whereas mutation of both residues together caused a loss of ligand binding. Mutation of lysine 74 in mGluR4, or the analogous lysine in mGluR8, to tyrosine (mimicking mGluR1 at this position) produced a large decrease in binding. The reduction in binding is likely due to steric hindrance of the phenolic side chain of tyrosine. The mutation of glutamate 287 to alanine, which is present on lobe II and is not conserved in the mGluR family, caused a loss of [3H]l-AP4 binding. We conclude that the determinants of high affinity ligand binding are dispersed across lobes I and II. Our results define a microenvironment within the binding pocket that encompasses several positively charged amino acids that recognize the negatively charged phosphonate group of l-AP4 or the endogenous compoundl-serine-O-phosphate. The amino-terminal domain containing the ligand binding site of the G protein-coupled metabotropic glutamate receptors (mGluRs) consists of two lobes that close upon agonist binding. In this study, we explored the ligand binding pocket of the Group III mGluR4 receptor subtype using site-directed mutagenesis and radioligand binding. The selection of 16 mutations was guided by a molecular model of mGluR4, which was based on the crystal structure of the mGluR1 receptor. Lysines 74 and 405 are present on lobe I of mGluR4. The mutation of lysine 405 to alanine virtually eliminated the binding of the agonist [3H]l-amino-4-phosphonobutyrate ([3H]l-AP4). Thus lysine 405, which is conserved in all eight mGluRs, likely represents a fundamental recognition residue for ligand binding to the mGluRs. Single point mutations of lysines 74 or 317, which are not conserved in the mGluRs, to alanine had no effect on agonist affinity, whereas mutation of both residues together caused a loss of ligand binding. Mutation of lysine 74 in mGluR4, or the analogous lysine in mGluR8, to tyrosine (mimicking mGluR1 at this position) produced a large decrease in binding. The reduction in binding is likely due to steric hindrance of the phenolic side chain of tyrosine. The mutation of glutamate 287 to alanine, which is present on lobe II and is not conserved in the mGluR family, caused a loss of [3H]l-AP4 binding. We conclude that the determinants of high affinity ligand binding are dispersed across lobes I and II. Our results define a microenvironment within the binding pocket that encompasses several positively charged amino acids that recognize the negatively charged phosphonate group of l-AP4 or the endogenous compoundl-serine-O-phosphate. The family of eight metabotropic glutamate receptors (mGluRs) 1mGluRmetabotropic glutamate receptorHEKhuman embryonic kidney 293 cellsl-AP4l-amino-4-phosphonobutyratel-SOPl-serine-O-phosphateCPPG(R,S)-α-cyclopropyl-4-phosphonophenylglycinePBSphosphate-buffered saline 1mGluRmetabotropic glutamate receptorHEKhuman embryonic kidney 293 cellsl-AP4l-amino-4-phosphonobutyratel-SOPl-serine-O-phosphateCPPG(R,S)-α-cyclopropyl-4-phosphonophenylglycinePBSphosphate-buffered saline has been subdivided into three groups based on sequence homology, signal transduction mechanisms, and pharmacological profiles (1Pin J.-P. Duvoisin R. Neuropharmacology. 1995; 34: 1-26Crossref PubMed Scopus (1233) Google Scholar, 2Conn P.J. Pin J.-P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2716) Google Scholar). The Group III mGluRs, which include mGluR4, -6, -7, and -8, are selectively activated by the agonist L(+)-2-amino-4-phosphonobutyric acid (l-AP4; see Fig. 1). With the exception of mGluR6, which is localized postsynaptically in cells of the retina, the other Group III mGluRs show primarily a presynaptic localization (3Shigemoto R. Kinoshita A. Wade E. Nimura 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, 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), although at some synapses these receptors may also be present postsynaptically. Presynaptic Group III mGluRs act as autoreceptors to inhibit the release of l-glutamate from glutamatergic nerve terminals (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, 5Pekhletski R. Gerlai R. Overstreet L. Huang X.-P. Agoypan N. Slater N.T. Abramow-Newerly W. Roder J.C. Hampson D.R. J. 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Based on these studies and the observation that mGluR4 and other Group III mGluRs display selectivity and high affinity for l-SOP, we have proposed that this phosphonated amino acid may be an endogenous neurotransmitter or neuromodulator at synapses expressing Group III mGluRs (17Hampson D.R. Huang X-P. Pekhletski R. Peltekova V. Hornby G. Thomsen C. Thøgersen H. J. Biol. Chem. 1999; 274: 33488-33495Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The analysis of truncated mGluRs that contain only the extracellular amino-terminal domain has demonstrated that the glutamate binding site is contained within this domain of the receptor protein (18Okamoto 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, 19Han G. Hampson D.R. J. Biol. Chem. 1999; 274: 10008-10013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 20Peltekova V. Han G. Soleymanlou N. Hampson D.R. Mol. Brain Res. 2000; 76: 180-190Crossref PubMed Scopus (21) Google Scholar). Earlier modeling studies based on structural similarities between mGluRs and a group of bacterial proteins known as periplasmic binding proteins, suggested that the amino-terminal domains of mGluRs possess a bilobed “Venus's flytrap” configuration whereby the ligand binds within a cleft between the two lobes causing a closing of the flytrap (21O'Hara P.J. Sheppard P.O. Thøgerson 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 recent elucidation of the crystal structure of the amino-terminal domain of the mGluR1 (Group I) receptor subtype has confirmed this basic arrangement and provides a much more detailed picture of the binding domain of mGluR1 (22Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1098) Google Scholar). Despite the availability of the information provided by the x-ray structure of mGluR1, the molecular determinants that confer receptor subtype selectivity among the mGluRs are not known. Previous mutagenesis and modeling studies have identified residues embedded in the binding pocket that function as fundamental recognition sites common to all eight receptor subtypes (17Hampson D.R. Huang X-P. Pekhletski R. Peltekova V. Hornby G. Thomsen C. Thøgersen H. J. Biol. Chem. 1999; 274: 33488-33495Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 21O'Hara P.J. Sheppard P.O. Thøgerson 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, 23Jensen A.A. Sheppard P.O. O'Hara P.J. Krogsgaard-Larsen P. Bräuner-Osborne H. Eur. J. Pharmacol. 2000; 397: 247-253Crossref PubMed Scopus (27) Google Scholar, 24Costantino G. Macchiarulo A. Pellicciari R. J. Med. Chem. 1999; 42: 5390-5401Crossref PubMed Scopus (32) Google Scholar, 25Bessis A.-S. Bertrand H-O. Galvez T., De Colle C. Pin J.-P. Acher F. Protein Sci. 2000; 9: 2200-2209Crossref PubMed Scopus (61) Google Scholar). Examples of this class of residues in mGluR4 include arginine 78, serine 159, and threonine 182. However, in addition to the complement of binding residues that are conserved in all mGluRs, an additional subset of amino acids in the binding pocket must be responsible for the unique pharmacological profiles of each of the three subgroups of mGluRs. The goal of the present study was to identify residues within the binding pocket of Group III receptors that mediate high affinityl-AP4 binding. To accomplish this task, we used a combination of molecular modeling, site-directed mutagenesis, and radioligand binding. l-AP4, l-SOP, (R,S)-α-cyclopropyl-4-phosphonophenylglycine (CPPG),l-glutamate, [3H]l-AP4 (specific activity 49 Ci/mol), and [3H]CPPG (specific activity 15.5 Ci/mmol) were purchased from Tocris Cookson Inc. (Bristol, UK). The three-dimensional structure of the proposed ligand binding domain of the rat mGluR4 receptor was formulated by homology modeling using the experimentally determined structure of the closed form of mGluR1 (22Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1098) Google Scholar). The atomic coordinates were obtained from the Protein Data Base (PDB code, 1ewk). The QUANTA program (version 2000, Molecular Simulations Inc.) and the SYBYL program (version 6.7, TRIPOS Corp.) were used to view the model that encompassed the region from methionine 40 to tryptophan 508 of the rat mGluR4 sequence. The sequence alignment used to build the mGluR4 model is shown in Fig. 2. Backbone atom coordinates were assigned the corresponding residue coordinates from the crystal structure of the mGluR1, and side chain atom coordinates were based on maximal side chain atom fitting to the mGluR1 structure using the modeler software from within Quanta. Regions with insertions or deletions were modeled using known substructures identified by loop searching techniques; regions 1–39 and 125–147, which are absent in the mGluR1 structure, were not included in the model. Thel-SOP molecule was docked into the binding site of mGluR4 in an orientation corresponding to that observed for glutamate binding to mGluR1. 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 the mGluR1 using the CHARMm force field. A steepest descent followed by a conjugate gradient method was used for energy minimization until the energy change per cycle was less than 0.0001 kcal/mol. The wild-type rat mGluR4a with a c-Myc epitope tag inserted between the amino acids histidine 38 and proline 39 (mGluR4a-cmyc-N) was subcloned into pcDNA3 (Invitrogen) as described previously (19Han G. Hampson D.R. J. Biol. Chem. 1999; 274: 10008-10013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The wild-type rat mGluR8a containing a c-Myc-polyhistidine epitope fused to the 3′ region was subcloned into pcDNA3.1 (Invitrogen) as described previously (20Peltekova V. Han G. Soleymanlou N. Hampson D.R. Mol. Brain Res. 2000; 76: 180-190Crossref PubMed Scopus (21) Google Scholar). An alternative version of the rat mGluR4a with a c-Myc-His epitope fused to the 3′ end of the mGluR4a cDNA was also produced (termed mGluR4a-cmyc-C). To delete the 3′ untranslated region of mGluR4a and fuse the c-Myc-His epitope, a PCR was performed on a 3838-bp HindIII-XbaI fragment using the forward primer (5′-CCG GGA GCT GAG CTA-3′) and the reverse primer (5′-TCC CCG CGG GAT GGC ATG GTT GTT-3′). The reverse primer deleted the stop codon to allow fusion of the tag and contained aSacII restriction enzyme site. The PCR product was purified and digested with AvrII and SacII, and the 766-bp product was gel-purified. For subcloning into the pcDNA3.1 vector (Invitrogen), full-length mGluR4a cDNA was digested withHindIII and AvrII yielding a 2.1-kb fragment that was gel-purified. The 766-bp AvrII/SacII PCR product and the 2.1-kb HindIII/AvrII mGluR4a fragment were ligated into the HindIII/SacII sites of pcDNA3.1- c-Myc-His vector. The resultant mGluR4a-cymc-C was sequenced from the 3′ end to confirm in-frame fusion of the c-Myc and His tags. The mGluR8a mutants K71A, K71Y, R75A, and several of the mGluR4a mutants including K74A, K74Y, K74N, K74Q, and H77Q were made using a cassette mutagenesis method. Briefly, the 1.8-kb KpnI fragment of mGluR8a and the 1.8-kb KpnI fragment of mGluR4a were both subcloned into pBluescript SK− (Stratagene) and transformed into the CJ236 strain of Escherichia coli. Single-stranded DNA was isolated using the Muta-Gene phagemid in vitro mutagenesis kit from Bio-Rad. Mutagenic oligonucleotides were annealed to the single-stranded template and used to make double-stranded mutant DNA. Double-stranded DNA was transformed into DH5α E. coli (Invitrogen)-competent cells. Mutated cassettes were then excised from pBluescript SK− and ligated back into their corresponding vectors. All remaining mutations were made in mGluR4a-cmyc-C and produced by the QuikChange site-directed mutagenesis strategy (Stratagene) according to the manufacturer's instructions using the wild-type mGluR4a 1.8-kbKpnI cassette in pBluescript SK− as a template. Briefly, two complementary oligonucleotides (31–34 bases) contained the desired base pair changes in the center of the oligos. The mGluR4a cassette was amplified using Pfu Turbo DNA polymerase (Stratagene) for 12–16 cycles in a programmable thermal controller (MJ Research Inc.). Digestion of template (nonmutated) DNA was accomplished with DpnI, and the resultant muted DNA was transformed into DH5α E. coli-competent cells. The amplified DNA was digested with transformed into E. coli DH5α-competent cells. All resultant mutated KpnI cassettes were sequenced via automatic sequencing on a LiCor LongReadIR sequencer prior to subcloning back into the full-length expression vector. Transient transfections of human embryonic kidney (HEK) 293-TSA-201 cells were conducted using a calcium phosphate precipitation protocol as described previously (19Han G. Hampson D.R. J. Biol. Chem. 1999; 274: 10008-10013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The cells were cultured in modified Eagle's medium with 6% fetal bovine serum and antibiotics. All experiments were conducted on cells or membranes collected 48 h after transfection. The membrane preparation and the [3H]l-AP4 and [3H]CPPG radioligand binding assays were carried out as described previously (26Eriksen L. Thomsen C. Br. J. Pharmacol. 1995; 116: 3279-3287Crossref PubMed Scopus (46) Google Scholar, 27Naples M.A. Hampson D.R. Neuropharmacology. 2001; 40: 170-177Crossref PubMed Scopus (41) Google Scholar). Briefly, HEK-293 cells transiently transfected were harvested 48 h post-transfection and pelleted by centrifugation (3,840 × g, 20 min, 4 °C). Pelleted cells were suspended in 20 ml of ice-cold lysis buffer (30 mm HEPES, 5 mmMgCl2·6H2O, 1 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, pH 7.4) and homogenized with a Polytron prior to centrifugation (48,400 × g, 20 min, 4 °C). The cell pellets were resuspended in 15 ml of lysis buffer supplemented with 0.08% Triton X-100 and incubated for 10 min at 37 °C. Following incubation, an additional 15 ml of lysis buffer was added, and the membranes were recentrifuged at 48,400 ×g for 15 min. Cell membranes were washed by resuspension in 15 ml of assay buffer (30 mm HEPES, 110 mmNaCl, 1.2 mm MgCl2·6 H2O, 5 mm KCl, 2.5 mm CaCl2, 0.1 mm phenylmethylsulfonyl fluoride, pH 8.0), centrifuged (48,400 × g, 15 min), and homogenized in 1–5 ml of assay buffer. Protein concentrations were determined using a kit from Bio-Rad. All membranes were diluted in assay buffer to a final concentration of 0.625 mg/ml and stored at −70 °C. For radioligand binding, frozen membranes were thawed and homogenized using a Polytron. 125 μg of membrane protein was used for all binding assays in a final volume of 250 μl. All assays were performed on ice using either 20 nm [3H]CPPG or 30 nm[3H]l-AP4; nonspecific binding of these ligands to cell membranes was defined as binding in the presence of 300 μml-SOP. Following a 30-min incubation, bound and free radioligand were separated by centrifugation (14,000 × g, 4 min). The membranes were washed with cold assay buffer and solubilized overnight in 1 m NaOH. The solubilized pellets were counted using standard liquid scintillation counting techniques, and all data were analyzed using GraphPad Prism 3.0. The procedures for immunoblotting were carried out as described previously (28Pickering D.S. Taverna F.A. Salter M.W. Hampson D.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12090-12094Crossref PubMed Scopus (71) Google Scholar). Electrophoresis samples containing 100 mm dithiothreitol were incubated at 37 °C for 15 min prior to gel electrophoresis. For immunocytochemical analyses, HEK cells were washed with phosphate-buffered saline (PBS) twice at 48 h post-transfection and fixed with PBS containing 4% paraformaldehyde and 4% sucrose for 10 min at 25 °C. The cells were air-dried for 15 min and then incubated in 10% bovine serum albumin in PBS for 30 min at 25 °C. The cells were subsequently incubated for 1 h at 25 °C with the anti-mGluR4a antibody (Upstate Biotechnology, Inc.) diluted to a final concentration of 0.15 μg/ml in 3% bovine serum albumin in PBS. The primary antibody was then removed, and the cells were washed five times for 5 min each with PBS. After washing, the cells were incubated for 60 min at 25 °C with biotin-conjugated anti-mouse IgG (Sigma, B0529) diluted to a final concentration of 2.75 μg/ml in 3% bovine serum albumin in PBS. After incubation, the cells were washed five times for 5 min each with PBS and treated with fluorescein isothiocyanate-conjugated ExtrAvidin (Sigma, E-2761) diluted to a final concentration of 5 μg/ml in 3% bovine serum albumin in PBS for 60 min at 25 °C in the dark; the cells were washed four times for 5 min with phosphate-buffered saline, mounted with 50% glycerol solution in PBS, and photographed with a Zeiss Axiovert 135 TV microscope equipped with a 485-nm excitation and 530-nm emission filter at a magnification of 400× using Kodak Tmax 400 film. An amino acid sequence alignment of a portion of the amino-terminal domain of the mGluRs is shown in Fig. 2. The amino acids targeted for mutagenesis are shown in bold; a total of 14 single and 2 double mutants were generated and characterized (TableI). The rationale for the choice of mutations was based on the homology model of mGluR4 (see below), which was in turn based on the crystal structure of mGluR1. All of the mutants analyzed were made in residues located within the region encompassing the putative glutamate binding pocket. The results of immunoblotting and radioligand binding experiments conducted at a single concentration of [3H]l-AP4 indicated that several of the mutant receptors were expressed at levels similar to that of wild-type rat mGluR4a (Fig.3). Further analysis based on radioligand binding competition experiments defined a class of mutants that were expressed at levels similar to the wild-type mGluR4a and showed no major changes in the affinity for l-AP4. This set of “no substantial effect” mutants included K74A, K74N, K74Q, H77Q, G158A, K317A, and K74A/R258A (Fig. 3 and Table I). “No substantial effect” was defined as mutants showing less than a 2-fold change in affinity compared with the wild-type receptor.Table ImGluR4 mutants generated and characterizedmGluR4 mutationIC50Equivalent residue in mGluR1LobemGluR4a0.420 ± 0.20 K74A0.488 ± 0.29Tyr74I K74YTyr74I K74N0.422 ± 0.25Tyr74I K74Q0.644 ± 0.09Tyr74I H77Q0.643 ± 0.33Gln77I G158A0.508 ± 0.24Ser164ImGluR4a-cmycC0.351 ± 0.13 D202A0.476 ± 0.15Asp208II Y230A0.328 ± 0.07Tyr236II R258A0.231 ± 0.03Ser263II N286A0.285 ± 0.16Glu292II E287AGly293II D312A0.445 ± 0.15Asp318II K317A0.330 ± 0.14Arg323II K405ALys409IIK74A/K317ATyr74/Arg323I and IIK74A/R258A0.266 ± 0.04Tyr74/Arg258I and IIFor mutagenesis, the wild-type amino acid was converted to the neutral amino acid alanine, or mutations were made to mimic another subtype of mGluR. These included K74Y, K74Q, and K74N to mimic mGluR1, mGluR6, and mGluR7, respectively. For the H77Q mutant, glutamine is present at this position at Group I and II mGluRs, whereas histidine is present in all members of the Group III mGluRs. The IC50 values are the means ± S.E. of three determinations using 30 nm[3H]L-AP4 and various concentrations of unlabeledl-AP4. Hyphens in the IC50 column indicates that the level of [3H]L-AP4 binding was too low to conduct competition analyses. mGluR4a-cmycC denotes the construct in which the carboxy terminus of mGluR4a was fused to the c-Myc and polyhistidine epitopes in the pcDNA3.1 vector. All mutants listed below mGluR4a-cmycC in the table were made in pcDNA3.1, whereas the mutants above mGluR4a-cmycC were constructed in pcDNA3, which lacks the c-Myc and polyhistidine tags. Open table in a new tab For mutagenesis, the wild-type amino acid was converted to the neutral amino acid alanine, or mutations were made to mimic another subtype of mGluR. These included K74Y, K74Q, and K74N to mimic mGluR1, mGluR6, and mGluR7, respectively. For the H77Q mutant, glutamine is present at this position at Group I and II mGluRs, whereas histidine is present in all members of the Group III mGluRs. The IC50 values are the means ± S.E. of three determinations using 30 nm[3H]L-AP4 and various concentrations of unlabeledl-AP4. Hyphens in the IC50 column indicates that the level of [3H]L-AP4 binding was too low to conduct competition analyses. mGluR4a-cmycC denotes the construct in which the carboxy terminus of mGluR4a was fused to the c-Myc and polyhistidine epitopes in the pcDNA3.1 vector. All mutants listed below mGluR4a-cmycC in the table were made in pcDNA3.1, whereas the mutants above mGluR4a-cmycC were constructed in pcDNA3, which lacks the c-Myc and polyhistidine tags. Another subset of mutants that included D202A, Y230A, R258A, N286A, and D312A displayed expression levels that were slightly to moderately lower than that of the wild-type receptor. The lower level of protein expression seen on immunoblots was reflected in the level of [3H]l-AP4 binding (Fig. 3). However, this set of mutants also did not show major changes in the IC50values compared with the wild-type receptor in autocompetition experiments using radiolabeled and unlabeled l-AP4 (TableI). The absence of effect of the tyrosine 230 to alanine mutation indicates that a possible cation-pi interaction between the phenyl group of the tyrosine and the amino group of the bound ligand, as suggested for mGluR1 (22Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1098) Google Scholar), is not critical for the binding of ligands to Group III mGluRs. The most interesting class of mutants comprised those that showed levels of protein expression on immunoblots similar to or slightly lower than that of the wild-type receptor but with drastic reductions in the level of [3H]l-AP4 binding. These included K74Y, E287A, K405A, and the K74A/K317A double mutant, which showed 7.6, 4.5, 1.9, and 3.8%, respectively, of wild-type binding (Fig. 3). Lysine 405, located on lobe I, is conserved in all eight mGluRs. Thus, this amino acid is likely essential for ligand binding to all members of the mGluR family. In contrast, lysine 74 on lobe I and glutamate 287 and lysine 317 on lobe II are not conserved in the mGluR family and therefore are likely not fundamental ligand recognition motifs; these amino acids may contribute to subgroup selective ligand binding. Lysine 74 was mutated to several different amino acids; mutation to the neutral alanine or to glutamine, which mimics mGluR6, or to asparagine, which mimics mGluR7 at this position, did not affect the pharmacological profile (Table I). In contrast, mutation of lysine 74 to tyrosine, which mimics mGluR1 at this position, resulted in a great loss of binding (Fig. 3). The observations on the series of mutations of lysine 74 in mGluR4 prompted us to confirm these results in the mGluR8 receptor, which is 71% identical to mGluR4 in the amino-terminal domain. Consistent with the results in mGluR4, mutation of lysine 71 to tyrosine in mGluR8 (lysine 71 in mGluR8 is analogous to lysine 74 in mGluR4) induced a sharp decline in the level of [3H]l-AP4 binding (Fig. 4, A andB). In contrast, mutation of this residue to alanine increased the amount of binding to mGluR8; this was reflected as a nonsignificant increase in the affinity for [3H]l-AP4 (Fig. 4 C). In addition to lysine 71, arginine 75 in mGluR8 was also mutated to alanine; the analogous residues in mGluR1 and mGluR4 (arginine 78, see Table I) have been shown to be essential for ligand binding to these receptors (17Hampson D.R. Huang X-P. Pekhletski R. Peltekova V. Hornby G. Thomsen C. Thøgersen H. J. Biol. Chem. 1999; 274: 33488-33495Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar,23Jensen A.A. Sheppard P.O. O'Hara P.J. Krogsgaard-Larsen P. Bräuner-Osborne H. Eur. J. Pharmacol. 2000; 397: 247-253Crossref PubMed Scopus (27) Google Scholar). As seen with mGluR4, mutation of this critical arginine in mGluR8 virtually eliminated [3H]l-AP4 binding. The effects of these mutations on the binding of the phosphonophenylglycine antagonist [3H]CPPG to mGluR8 were also investigated. At low nanomolar concentrations, [3H]CPPG can be used as a selective probe for mGluR8 (27Naples M.A. Hampson D.R. Neuropharmacology. 2001; 40: 170-177Crossref PubMed Scopus (41) Google Scholar). As observed with [3H]l-AP4 binding, the R75A mGluR8 mutant also showed a large reduction in [3H]CPPG binding (Fig. 4 B). However, in contrast to the absence of any significant change in affinity in [3H]l-AP4 binding to the K71A mGluR8 mutant, the affinity of [3H]CPPG for K71A was ∼4-fold lower than for the wild-type receptor (Fig. 4 C). The lack of effect on [3H]l-AP4 binding combined with the 4-fold reduction in affinity of the mGluR8 K71A for [3H]CPPG, suggests the possibility that the longer length of the CPPG molecule compared with L-AP4 may place the terminal phosphonate group of CPPG in a better position for making an interaction with the side chain of lysine 71 in mGluR8 as compared with the analogous lysine 74 in mGluR4. The only single point mutation on
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