The orphan glutamate-like receptor GluR␦2 is predominantly expressed in Purkinje cells of the central nervous system. The classification of GluR␦2 to the ionotropic glutamate receptor family is based on sequence similarities, because GluR␦2 does not form functional homomeric glutamate-gated ion channels in transfected cells. Studies in GluR␦2 ؊/؊ knockout mice as well as in mice with naturally occurring mutations in the GluR␦2 gene have demonstrated an essential role of GluR␦2 in cerebellar long-term depression, motor learning, motor coordination, and synaptogenesis. However, the lack of a known agonist has hampered investigations on the function of GluR␦2. In this study, the ligand-binding core of GluR␦2 (GluR␦2-S1S2) was found to bind neutral amino acids such as D-serine and glycine, as demonstrated by isothermal titration calorimetry. Direct evidence for binding of D-serine and structural rearrangements in the binding cleft of GluR␦2-S1S2 is provided by x-ray structures of GluR␦2-S1S2 in its apo form and in complex with D-serine. Functionally, D-serine and glycine were shown to inactivate spontaneous ion-channel conductance in GluR␦2 containing the lurcher mutation (EC 50 values, 182 and 507 M, respectively). These data demonstrate that the GluR␦2 ligand-binding core is capable of binding ligands and that cleft closure of the ligandbinding core can induce conformational changes that alter ion permeation.crystal structure ͉ electrophysiology ͉ isothermal titration calorimetry ͉ ligand-binding core

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A comparison of the pharmacological and physiological properties of the metabotropic glutamate 1 alpha and 1 beta receptors (mGluR1 alpha and mGluR1 beta) expressed in baby hamster kidney (BHK 570) cells was performed. The mGluR1 beta receptor is an alternatively spliced form of mGluR1 alpha with a modified carboxy terminus. Immunoblots of membranes from the two cell lines probed with receptor-specific antipeptide antibodies showed that mGluR1 alpha migrated with an M(r) = 154,000, whereas mGluR1 beta migrated with an M(r) = 96,000. Immunofluorescence imaging of receptors expressed in BHK 570 cells revealed that the mGluR1 alpha receptor was localized to patches along the plasmalemma and on intracellular membranes surrounding the nucleus, whereas mGluR1 beta was distributed diffusely throughout the cell. Agonist activation of the mGluR1 alpha and the mGluR1 beta receptors stimulated phosphoinositide hydrolysis. At both receptors, glutamate, quisqualate, and ibotenate were full agonists, whereas trans-(+)-1-aminocyclopentane-1,3-dicarboxylate appeared to act as a partial agonist. The stimulation of phosphoinositide hydrolysis by mGluR1 alpha showed pertussis toxin-sensitive and insensitive components, whereas the mGluR1 beta response displayed only the toxin-insensitive component. The mGluR1 alpha and mGluR1 beta receptors also increased intracellular calcium levels by inducing release from intracellular stores. These results indicate that the different carboxy terminal sequences of the two receptors directly influences G protein coupling and subcellular deposition of the receptor polypeptides and suggest that the two receptors may subserve different roles in the nervous system.

Replacement of the methyl group of the AMPA receptor agonist 2-amino-3-[3-hydroxy-5-(2-methyl-2H-5-tetrazolyl)-4-isoxazolyl]propionic acid (2-Me-Tet-AMPA) with a benzyl group provided the first AMPA receptor agonist, compound 7, capable of discriminating GluR2-4 from GluR1 by its more than 10-fold preference for the former receptor subtypes. An X-ray crystallographic analysis of this new analogue in complex with the GluR2-S1S2J construct shows that accommodation of the benzyl group creates a previously unobserved pocket in the receptor, which may explain the remarkable pharmacological profile of compound 7.

Ionotropic glutamate receptors mediate most rapid excitatory synaptic transmission in the mammalian central nervous system, and their involvement in neurological diseases has stimulated widespread interest in their structure and function. Despite a large number of agonists developed so far, few display selectivity among (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA)-receptor subtypes. The present study provides X-ray structures of the glutamate receptor 2 (GluR2)-selective partial agonist (S)-2-amino-3-(1,3,5,6,7-pentahydro-2,4-dioxocyclopenta[e] pyrimidin-1-yl) propanoic acid [(S)-CPW399] in complex with the ligand-binding core of GluR2 (GluR2-S1S2J) and with a (Y702F)GluR2-S1S2J mutant. In addition, the structure of the nonselective partial agonist kainate in complex with (Y702F)GluR2-S1S2J was determined. The results show that the selectivity of (S)-CPW399 toward full-length GluR2 relative to GluR3 is reflected in the binding data on the two soluble constructs, allowing the use of (Y702F)GluR2-S1S2J as a model system for studying GluR2/GluR3 selectivity. Structural comparisons suggest that selectivity arises from disruption of a watermediated network between ligand and receptor. A D1-D2 domain closure occurs upon agonist binding. (S)-CPW399 and kainate induce greater domain closure in the Y702F mutant, indicating that these partial agonists here act in a manner more reminiscent of full agonists. Both kainate and (S)-CPW399 exhibited higher efficacy at (Y702F)GluR2(Q) i than at wild-type GluR2(Q) i . Whereas an excellent correlation exists between domain closure and efficacy of a range of agonists at full-length GluR2 determined by electrophysiology in Xenopus laevis oocytes, a direct correlation between agonist induced domain closure of (Y702F)GluR2-S1S2J and efficacy at the GluR3 receptor is not observed. Although it clearly controls selectivity, mutation of this residue alone is insufficient to explain agonist-induced conformational rearrangements occurring in this variant.Binding of (S)-glutamate to ionotropic glutamate receptors (iGluRs) is a key step in the predominant mechanism of rapid excitatory synaptic transmission among nerve cells within the mammalian central nervous system. iGluRs are important in the development and function of the central nervous system and are implicated in learning and memory formation. Furthermore, iGluRs also seem to be associated with certain neurological and psychiatric diseases (e.g., Alzheimer-type diseases, Parkinson's disease, epilepsy, stroke, amyotrophic lateral sclerosis, and schizophrenia). Therefore, the iGluRs are considered potential drug targets (Dingledine et al., 1999;Brä uner-Osborne et al., 2000). The iGluRs have been divided into three different classes: the (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA), The structures reported in this article have been deposited within the Protein Databank with accession numbers 1SYH, 1SYI, and 1XHY.Article, publication date, and citation information can be found at ...

Dimeric positive allosteric modulators of ionotropic glutamate receptors were designed, synthesized, and characterized pharmacologically in electrophysiological experiments. The designed compounds are dimers of arylpropylsulfonamides and have been constructed without a linker. The monomeric arylpropylsulfonamides were derived from known modulators and target the cyclothiazide-binding site at the AMPA receptors. The three stereoisomers--R,R, meso, and S,S--of the two constructed dimers were prepared, and in vitro testing showed the R,R forms to be the most potent stereoisomers. The biarylpropylsulfonamides have dramatically increased potencies, more than three orders of magnitude higher than the corresponding monomers. Dimer (R,R)-2a was cocrystallized with the GluR2-S1S2J construct, and an X-ray crystallographic analysis showed (R,R)-2a to bridge two identical binding pockets on two neighboring GluR2 subunits. Thus, this is biostructural evidence of a homomeric dimer bridging two identical receptor-binding sites.

The G-protein-coupled metabotropic glutamate receptor mGluRla and the ionotropic glutamate receptor GluR6 were examined for posttranslational palmitoylation. Recombinant receptors were expressed in baculovirusinfected insect cells or in human embryonic kidney cells and were metabolically labeled with [3HI palmitic acid. The metabotropic mGluRla receptor was not labeled whereas the GluR6 kainate receptor was labeled after incubation with Receptors for the excitatory neurotransmitter L-glutamate can be grouped into two major families, ionotropic receptors and metabotropic receptors. The metabotropic glutamate receptors are G-protein-linked receptors coupled to effectors such as phospholipase C and adenylyl cyclase, whereas the ionotropic receptors form homomeric and heteromeric ligandgated ion channels permeable to mono-and divalent cations. A further subdivision of the ionotropic family can be made on the basis of agonist selectivity, giving the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) (GluR1-4, also called GluRA-D), kainate (GluR5-7, KA-1, KA-2), and Nmethyl-D-aspartate (NMDA) classes of receptors (1).Several studies have examined various aspects of the structure and function of the GluR6 kainate receptor (2-10). The activity of GluR6 channels can be modulated by protein kinase A (6, 7) and protein kinase C (PKC) (8). Site-directed mutagenesis of asparagine-linked glycosylation sites (9-12) and epitope tagging studies (13) have been carried out to derive transmembrane topological models of kainate and AMPA receptors. In these models, a large extracellular aminoterminal domain is followed by either three or five transmembrane domains (TMDs), resulting in either case in a carboxyl terminus that is intracellular.Palmitoylation is a posttranslational modification whereby a C16 fatty acyl chain is covalently bound to a nascent protein in a post-endoplasmic reticulum or pre-Golgi compartment. The fatty acid can be attached to a serine, threonine, or cysteineThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. residue via an ester or thioester bond. In contrast to myristoylation, which occurs cotranslationally and without metabolic turnover (14), a rapid, dynamic turnover of palmitate has been demonstrated in several systems (15)(16)(17).Several G-protein-linked receptors, including rhodopsin (18) and 132-adrenergic (19)