Review: neurotransmitter receptors. II. AMPA and kainate receptors.
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Animals↗
Biomedical subjects
B Bettler
Publications and source records attributed to B Bettler.
Agonist selectivity of glutamate receptors is specified by two domains structurally related to bacterial amino acid-binding proteins.
By exchanging portions of the AMPA receptor subunit GluR3 and the kainate receptor subunit GluR6, we have identified two discontinuous segments of approximately 150 amino acid residues each that control the agonist pharmacology of these glutamate receptors. The first segment (S1) is adjacent and N-terminal to the putative transmembrane domain 1 (TM1), whereas the second segment (S2) is located between the putative TM3 and TM4. Only the simultaneous exchange of S1 and S2 converts the pharmacological profile of the recipient to that of the donor subunit. The two segments identified in this study share sequence similarities with the ligand-binding site of several bacterial periplasmic amino acid-binding proteins. Based on the X-ray structure of these proteins, we propose a model for the glutamate-binding site of ionotropic glutamate receptors.
Amino Acid Sequence↗
The gene encoding the glutamate receptor subunit GluR5 is located on human chromosome 21q21.1-22.1 in the vicinity of the gene for familial amyotrophic lateral sclerosis.
Genomic clones of the human non-N-methyl-D-aspartate (non-NMDA) glutamate receptor subunit GluR5 were isolated by high-stringency screening of a cosmid library using the rat cDNA as a probe. The chromosomal localization of the human GluR5 gene has been established. Southern hybridization of DNA isolated from mapping panels of Chinese hamster-human hybrid cell lines and high-resolution in situ suppression hybridization localize the GluR5 gene to chromosome 21q21.1-22.1. This coincides with the localization of a mutant gene causing familial amyotrophic lateral sclerosis (ALS), as Siddique et al. established by linkage analyses [Siddique, T., Figlewicz, D. A., Pericak-Vance, M. A., Haines, J. L., Rouleau, G., Jeffers, A. J., Sapp, P., Hung, W. Y., Bebout, J., McKenna-Yasek, D., Deng, G., Horvitz, H. R., Gusella, J. F., Brown, R. H. & Roses, A. D. (1991) N. Engl. J. Med. 324, 1381-1384]. Convergent evidence from other investigators suggests that chronic pathologic activation of motor neurons via non-NMDA glutamate receptors might induce excitotoxic injury of motor neurons, culminating in ALS. Together with the demonstration that GluR5 transcripts are expressed in the ventral horn of the spinal cord, the region in which susceptible motor neurons reside, the chromosomal localization suggests that a mutated GluR5 gene may be responsible for the familial form of ALS.
Amyotrophic Lateral Sclerosis↗
Immunoglobulin E-binding site in Fc epsilon receptor (Fc epsilon RII/CD23) identified by homolog-scanning mutagenesis.
The IgE-binding site of the human low-affinity receptor for IgE (Fc epsilon RII/CD23) has previously been mapped to the extracellular domain between amino acid residues 160 and 287. We now have investigated which conformational epitope within this domain specifies the receptor-ligand interaction. The analysis of homolog-scanning mutants expressed in mammalian cells demonstrates that amino acid side chains that affect IgE binding are located in two discontinuous segments, between residues 165-190 and 224-256. The overall structure of the chimeric binding domains, as probed with 11 conformation-sensitive monoclonal antibodies, is generally not distorted, except by replacement of residues 165-183. In this region, disruption of binding function appears to be caused by global conformational constraints on the binding site. Substitution and deletion mutants demonstrate that six out of eight extracellular cysteines, Cys163, Cys174, Cys191, Cys259, Cys273, and Cys282, are necessary for IgE binding and are most likely involved in intramolecular disulfide bridges. We show that the Fc epsilon RII domain delineated by Cys163 and Cys282 encodes all the structural information required to form the IgE-binding site.
Amino Acid Sequence↗
Cytokine effects of CD23 are mediated by an epitope distinct from the IgE binding site.
Human CD23 and its soluble forms (sCD23) display various biological activities, in addition to their IgE binding function (IgE/BF). The IgE binding domain was recently mapped to residues between Cys163 and Cys282 but its involvement in IgE-independent, CD23 functions remains unknown. In order to clarify this point, a series of N-terminal, C-terminal and internal deletion mutants of CD23 or sCD23 were expressed in CHO cells and tested for their ability (i) to bind to IgE, (ii) to induce colony formation by human myeloid precursor cells, (iii) to promote mature T cell marker expression by early prothymocytes, and (iv) to regulate IgE synthesis. The present study indicates that cytokine activities require the presence of Cys288, while this amino acid is not necessary for IgE/BF. Blocking experiments using various conformation-sensitive monoclonal antibodies further suggest that active epitope(s) of CD23 in cytokine assays is(are) distinct from those involved in IgE/BF.
Animals↗
Cloning of a putative glutamate receptor: a low affinity kainate-binding subunit.
Kainate, a glutamate receptor agonist, is a potent neuroexcitatory agent that produces epileptiform activity and selective neuronal degeneration. Binding studies using neuronal membrane homogenates or brain sections have identified sites having either high or low affinity for [3H]kainate. Here we report the cloning of a gene, GluR7, with approximately 75% sequence identity with the previously cloned GluR5 and GluR6 subunit genes. Transcripts of the GluR7 gene are evident in brain areas that bind [3H]kainate and are susceptible to kainate-induced neurotoxicity. We have performed ligand binding studies with membranes of transfected HeLa cells expressing GluR6 or GluR7 subunits. Our data show that the GluR6 and GluR7 subunits have a rank order of agonist affinity (domoate greater than kainate much greater than L-glutamate, quisqualate much greater than AMPA, NMDA) and a dissociation constant for kainate (95 and 77 nM, respectively) characteristic of the low affinity kainate-binding sites described in the brain.
Amino Acid Sequence↗
Molecular biology of the glutamate receptors.
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Amino Acids↗
Native and recombinant soluble CD23 fragments with IgE suppressive activity.
CD23-bearing cells are known to release 37,000 33,000 and 25,000 MW soluble CD23 (sCD23) fragments that were reported to display multiple biological activities, including the potentiation of IgE synthesis. We previously reported that tunicamycin treatment of RPMI-8866 cells switched the biological activity of the sCD23 released by these cells from IgE potentiation to IgE suppression. In this study we show that tunicamycin-treated cells release small CD23 fragments with a MW of 16,000. These fragments are formed by truncation of the N-terminal 160 amino acids and truncation of the carboxy-terminal end of CD23. Two observations indicate that the cleavage of surface CD23 into 16,000 MW fragments is not caused by tunicamycin-mediated inhibition of the N-glycosylation of CD23 but rather by the deletion of the carboxy terminal end of the molecule: (1) Chinese hamster ovary (CHO) transfectants expressing a CD23 mutant lacking the N-glycosylation site release 37,000-33,000 MW sCD23 unless they are treated with tunicamycin; (2) transfectants expressing a CD23 deletion mutant lacking the last 33 carboxy-terminal amino acids release 16,000 MW sCD23. Highly purified native and recombinant 16,000 MW sCD23 bind to IgE and down-regulate the ongoing and the interleukin-4 (IL-4)-stimulated synthesis of IgE.
Animals↗
Cloning of a cDNA for a glutamate receptor subunit activated by kainate but not AMPA.
Fast excitatory transmission in the vertebrate central nervous system is mediated mainly by L-glutamate. On the basis of pharmacological, physiological and agonist binding properties, the ionotropic glutamate receptors are classified into NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate) and kainate subtypes. Sequence homology between complementary DNA clones encoding non-NMDA glutamate receptor subunits reveals at least two subunit classes: the GluR1 to GluR4 class and the GluR5 class. Here we report the cloning and expression of a functional rat glutamate receptor subunit cDNA, GluR6, which has a very different pharmacology from that of the GluR1-GluR4 class. Receptors generated from the GluR1-GluR4 class have a higher apparent affinity for AMPA than for kainate. When expressed in Xenopus oocytes the homomeric GluR6 receptor is activated by kainate, quisqualate and L-glutamate but not by AMPA, and the apparent affinity for kainate is higher than for receptors from the GluR1-GluR4 class. Desensitization of the receptor was observed with continuous application of agonist. The homomeric GluR6 glutamate receptor exhibits an outwardly rectifying current-voltage relationship. In situ hybridizations reveal a pattern of GluR6 gene expression reminiscent of the binding pattern obtained with [3H]kainate.
Amino Acid Sequence↗
Expression, structure, and function of the CD23 antigen.
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Animals↗
Cloning of a novel glutamate receptor subunit, GluR5: expression in the nervous system during development.
We have isolated cDNAs encoding a glutamate receptor subunit, designated GluR5, displaying 40%-41% amino acid identity with the kainate/AMPA receptor subunits GluR1, GluR2, GluR3, and GluR4. This level of sequence similarity is significantly below the approximately 70% intersubunit identity characteristic of kainate/AMPA receptors. The GluR5 protein forms homomeric ion channels in Xenopus oocytes that are weakly responsive to L-glutamate. The GluR5 gene is expressed in subsets of neurons throughout the developing and adult central and peripheral nervous systems. During embryogenesis, GluR5 transcripts are detected in areas of neuronal differentiation and synapse formation.
Aging↗
Binding site for IgE of the human lymphocyte low-affinity Fc epsilon receptor (Fc epsilon RII/CD23) is confined to the domain homologous with animal lectins.
The lymphocyte low-affinity receptor for IgE (Fc epsilon RII) is involved in two seemingly unrelated processes: (i) promotion of general B-cell growth and (ii) isotype-specific IgE synthesis. To characterize domains of Fc epsilon RII important for effector function, we have expressed Fc epsilon RII mutants in mammalian cells. The results show that the IgE-binding region of Fc epsilon RII corresponds almost exactly to a domain of 123 amino acid residues homologous with the carbohydrate-binding domain of C-type animal lectins. With the recent demonstration that Fc epsilon RII binds to IgE independently of any lectin-like activity [Vercelli, D., Helm, B., Marsh, P., Padlan, E., Geha, R.S. & Gould, H. (1989) Nature (London) 338, 649-651], it is now clear that, in this case, the lectin module has evolved to interact with a protein rather than a carbohydrate moiety. The epitopes of several independent monoclonal antibodies that inhibit the binding of IgE to Fc epsilon RII are clustered within the lectin-like domain. Some of these antibodies are also known to suppress, isotype-specifically, the interleukin 4-promoted IgE synthesis from peripheral blood mononuclear cells or the spontaneous synthesis of IgE by B cells isolated from atopic donors. The epitope of MHM6, an anti-F epsilon RII monoclonal antibody delivering an epitope-restricted growth-promoting effect on B cells, is also located within the lectin-like domain. Thus, the lectin module of Fc epsilon RII not only acts as a carbohydrate-independent, isotype-specific Fc receptor but may also participate in the general regulation of B-cell growth.
Animals↗
Molecular structure and expression of the murine lymphocyte low-affinity receptor for IgE (Fc epsilon RII).
The cDNA encoding the murine low-affinity receptor for IgE (Fc epsilon RII) has been isolated from a cDNA library prepared from B cells activated with lipopolysaccharide and interleukin 4. It encodes a 37-kDa protein of 331 amino acids with two potential N-linked glycosylation sites. Analogous to its human counterpart, there is no signal sequence and the putative transmembrane region is close to the amino terminus, indicating an inverse membrane orientation with the carboxyl terminus at the cell exterior. The predicted murine Fc epsilon RII amino acid sequence demonstrates a 57% identity with its human counterpart. The murine sequence has an additional internal repeat motif of 21 amino acids giving four repeats as compared to three in the human sequence. Furthermore, the murine Fc epsilon RII is truncated at the carboxyl terminus and the Arg-Gly-Asp sequence, a common recognition site of integrin receptors, which is found in the reverse configuration in human Fc epsilon RII, is missing. B cells activated with interleukin 4 and lipopolysaccharide have an increased amount of Fc epsilon RII mRNA as compared with resting or lipopolysaccharide-stimulated B cells. Con A-activated normal T cells, the TH-2 cell line D10, as well as the macrophage cell line J774 have no detectable Fc epsilon RII mRNA. Expression analysis using transiently transfected COS cells revealed that recombinant murine Fc epsilon RII binds anti-Fc epsilon RII as well as mouse and rat IgE but does not bind human IgE or mouse IgG. Fc epsilon RII expressed in COS cells has a molecular mass of 45 kDa whereas the Fc epsilon RII from B-cell lines is a 49-kDa protein.
Amino Acid Sequence↗
The upstream limit of nuclease-sensitive chromatin in Dictyostelium rRNA genes neighbors a topoisomerase I-like cluster.
In the chromatin of Dictyostelium ribosomal RNA (rRNA) genes, the coding and upstream flanking regions are sensitive to endonucleases. This sensitivity stops about 2.3 x 10(3) bases upstream from the transcription start, at a point we call the structural boundary. Upstream from the boundary an 850 base-pair region is strongly protected against micrococcal nuclease cleavage, particularly in rapidly transcribing vegetative cells, and upstream from this the pattern of nuclease protection suggests that positioned nucleosomes are present. On the gene side of the structural boundary nucleosomes are known to be absent in vegetative cells but present in differentiating slug cells where the rRNA synthesis rate is lower. We show that in slugs these nucleosomes are randomly distributed, in contrast to those upstream from the boundary. Close to the gene side of the boundary is a duplication of the putative promoter located 29 base-pairs distant from four clustered topoisomerase I recognition sequences, which are cleaved by endogenous topoisomerase I-like activity. An additional topoisomerase I recognition sequence found upstream from the structural boundary is not cleaved in chromatin. The possible significance of these sequences and structures in transcription is discussed.
Binding Sites↗
Ligands for expression cloning and isolation of GABA(B) receptors.
Outlined is the rationale behind the syntheses of radioligands [125I]CGP64213 and [125I]CGP71872, which led to the identification of cloned GABA(B) receptors 1a and 1b 17 years after the first pharmacological characterisation of native GABA(B) receptors by Bowery et al. [Nature 283 (1980) 92-94]. More recently it was shown that the N-terminal extracellular domains of GABA(B) receptors 1a and 1b contain the binding sites for agonists and antagonists [B. Malitschek et al., Mol. Pharmacol. 56 (1999) 448-454]. In order to isolate the extracellular domain(s) of GABA(B) receptors 1a (or 1b) and to purify and crystallise these proteins a third ligand [125I]CGP84963 was designed, which combines, in one molecule, a GABA(B) receptor binding part, an azidosalicylic acid as photoaffinity moiety and 2-iminobiotin, which binds to avidin in a reversible, pH-dependent fashion [W. Froestl et al., Neuropharmacology 38 (1999) 1641-1646].
Animals↗