Solubilization and Purification of an α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding Protein from Bovine Brain

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) is a selective ligand for an excitatory amino acid receptor subtype in mammalian brain. We have solubilized an AMPA binding protein from bovine brain membranes with 1% Triton X-100 in 0.5 M phosphate buffer and 20% glycerol at 37 degrees C and purified the stable binding sites using a series of chromatographic steps. Scatchard analysis of the purified preparation showed a curvilinear plot with dissociation constants of 10.6 and 323 nM and Bmax values of 670 and 1,073 pmol/mg of protein for the high- and low-affinity sites, respectively. Inhibition constants for several excitatory amino acid analogues were similar to those obtained for other membrane and solubilized preparations. Gel filtration of the soluble AMPA binding protein showed a single peak of [3H]AMPA binding activity at Mr approximately 500,000. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified AMPA binding protein showed a single major band at Mr = 110,000. Previously, we have shown that a monoclonal antibody (KAR-B1) against a frog brain kainate binding protein selectively recognizes an unknown protein in mammalian brain migrating at Mr approximately 100,000. We now show that this antibody recognizes the major component of the purified AMPA binding protein, supporting a structural similarity between the frog brain kainate binding protein and the mammalian AMPA binding protein.     

Interaction of the frog brain kainate receptor expressed in Chinese hamster ovary cells with a GTP-binding protein.

A protein that binds kainate with high affinity has been purified and cloned from frog brain (Rana pipiens) and has approximately 35% sequence homology with mammalian non-N-methyl-D-aspartate glutamate receptors, some of which have been shown to be ligand-gated ion channels. Frog brain membranes and membranes from Chinese hamster ovary (CHO) cells transfected with the cDNA coding for the frog kainate-binding protein (CHO-4 cells) bound kainate with essentially identical affinity (KD values of 1.9 and 2.1 nM, respectively). In both tissues, the affinity for kainate decreased 9-fold in the presence of 100 microM GTP gamma S (guanosine 5'-O-(3-thio)triphosphate). No specific kainate binding to nontransfected CHO cell membranes was observed. GTP gamma S and GDP were effective inhibitors of kainate binding, while cGMP and adenosine 5'-O-(3-thio)triphosphate had no effect in either frog brain membranes or CHO-4 membranes. Pretreatment of CHO-4 cell membranes with pertussis toxin led to a 34% decrease in kainate binding. Kainate increased the binding of [3H]5'-guanylyl imidodiphosphate by 61%, and the rate of GTP hydrolysis by up to 5-fold. These results indicate that the kainate receptor cloned from frog brain can interact functionally with a G protein present in CHO-4 cell membranes.     

Preparation of Antibodies to β Subunits of γ-Aminobutyric AcidA Receptors

Antisera were produced in rabbits against synthetic peptides based on two regions of the cDNA sequence of the beta 1 subunit of bovine gamma-aminobutyric acidA (GABAA) receptors. The deduced amino acid sequences were similar in other beta subunits of bovine, rat, and chick receptors, predicting cross-reactability with all beta subunits. One antiserum (anti-beta e) was raised against an extracellular moiety near the invariant disulfide loop thought to be located near the neurotransmitter binding domain; the other (anti-beta c) was raised against an intracellular moiety containing a consensus sequence for cyclic AMP-dependent protein kinase phosphorylation of a serine residue. Predicted secondary structures suggested high potential immunogenicity for the chosen antigen peptides. Both antisera at high dilutions recognized the same polypeptide bands on western blots of GABAA receptors purified from three regions of bovine brain (four bands at 57, 54, 53, and 52 kDa in cerebral cortex) but fewer bands (57, 54, and 52 kDa) in hippocampus and cerebellum (one major band at 54 kDa, traces at 57 and 53 kDa). This is consistent with the presence of multiple beta subunits whose expression varies with brain region, as shown by molecular cloning. The anti-beta c antibody was able to immunoprecipitate purified GABAA receptor [3H]-muscimol binding, 87% in bovine cortex and 75% in total rat brain; the anti-beta e was unable to immunoprecipitate any antigen. These antibodies indicate a region-dependent heterogeneity of beta subunits and should be useful for analyzing structure, function, and localization of GABAA receptor subtypes in brain.     

Isolation of a human cDNA encoding a 25 kDa FK-506 and rapamycin binding protein.

Recently, the nearly complete peptide sequence of a 25 kDa rapamycin and FK-506 binding protein that had been isolated from calf thymus, brain, and spleen was reported (1). Based upon the amino acid sequence of this bovine protein, bFKBP25, we have isolated from a JURKAT cDNA library the cDNA encoding the human homolog, hFKBP25. Translation of the open reading frame contained within this cDNA clone yields a sequence that, in its C-terminal half, is 41% identical to the major human FK-506 binding protein, hFKBP12, and 43% identical to hFKBP13. The N-terminal half of hFKBP25 is unrelated to any known protein.     

6,7-Dinitroquinoxaline-2,3-Dione Blocks the Cytotoxicity of N-Methyl-D-Aspartate and Kainate, but Not Quisqualate, in Cortical Cultures

Based on radioligand binding and electrophysiological studies, quinoxalinediones such as 6,7-dinitroquinoxaline-2,3-dione (DNQX) have been shown to be potent competitive antagonists at the quisqualate and kainate subtypes of the glutamate receptor. In this report we have examined the effects of DNQX on excitatory amino acid neurotoxicity and evoked neurotransmitter release. DNQX was found to be a potent neuroprotective agent against glutamate and N-methyl-D-aspartate (NMDA) neurotoxicity. The data suggest that this neuroprotective activity of DNQX is due to its antagonism of the coagonist activity of glycine at the NMDA receptor-channel complex. The specificity of DNQX for the glycine site associated with the NMDA receptor-channel complex was confirmed in radioligand binding and neurotransmitter release studies. DNQX also prevented kainate neurotoxicity and kainate-evoked neurotransmitter release, presumably by direct competition for the kainate receptor. DNQX, however, did not prevent quisqualate neurotoxicity, suggesting that a novel quisqualate-preferring receptor insensitive to DNQX may mediate quisqualate toxicity.     

Purification and characterization of a high-molecular-weight endogenous glutamate-binding inhibitor in porcine brain

A high-molecular-weight glutamate-binding inhibitor (HGBI) from porcine brain extract was purified to homogeneity. The results of this purification process show that glutamate receptor activity can be regulated by a high-molecular-weight protein, which inhibits [³H]L-glutamate binding to excitatory amino acid (EAA) receptors. The purified HGBI appears to be a protein with a molecular weight of approximately 70 kD. The purified HGBI is negatively charged, suggesting that it may contain acidic amino acids, and most likely,L-glutamate- andL-aspartate-enriched regions, responsible for its surface charge as well as for its binding to glutamate receptors. Inhibition of [³H]L-glutamate binding by the purified HGBI is reversible, and appears to change the binding kinetics. This endogenous ligand for glutamate receptors has unique characteristics separating it from all the other ligands found so far in the EAA receptor system. This HGBI represents a new class of modulator for the EAA receptor, thus further investigation of the function and structure of the HGBI should provide new understanding of the mechanisms of EAA-mediated neurotransmission.     

Purification and characterization of the human brain insulin receptor

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.     

Identification and characterization of the ligand binding subunit of a kainic acid receptor using monoclonal antibodies and peptide mapping

Monoclonal antibodies (mAb) and a polyclonal antiserum were produced against a kainic acid receptor (KAR) purified from frog brain. Several of the mAb and the antiserum immunoprecipitated [3H]kainic acid binding activity from solubilized preparations of frog brain and labeled a group of proteins on immunoblots that migrated at Mr = 48,000. These results confirm that the ligand binding subunit of the frog brain KAR is contained in the Mr = 48,000 proteins. Immunoblots from different frog tissues demonstrated that the antibody reactivity was highly concentrated in the frog nervous system with no detectable immunoreactivity observed in non-neuronal tissues. The purified KAR was radioiodinated and subjected to two-dimensional gel electrophoresis and autoradiography. A series of proteins was detected at Mr = 48,000 with isoelectric points from 5.5 to 6.3. The anti-KAR mAb and the antiserum reacted with the same group of proteins from frog whole brain after separation by two-dimensional gel electrophoresis. Peptide maps of the 125I-labeled KAR separated by two-dimensional gel electrophoresis demonstrated that the group of proteins clustered at Mr = 48,000 is homologous. mAb KAR-B1 reacted on immunoblots with a protein in rat brain with a Mr = 99,000. This protein comigrated with an unreduced form of the KAR in frog brain. It was present in rat cerebral cortex, hippocampus, and cerebellum but was not detected in thalamus, globus pallidus, or brain stem, nor was it detected in rat non-neuronal tissues. The presence of the Mr = 99,000 immunoreactive polypeptide in discrete areas of rat brain suggests that this protein may be part of a mammalian KAR or a related receptor.     

Domoic acid, the alleged "mussel toxin," might produce its neurotoxic effect through kainate receptor activation: an electrophysiological study in the dorsal hippocampus

Domoic acid, an excitatory amino acid structurally related to kainate, was recently identified as being presumably responsible for the recent severe intoxication presented by more than 100 people having eaten mussels grown in Prince Edward Island (Canada). The amino acid kainate has been shown to be highly neurotoxic to the hippocampus, which is the most sensitive structure in the central nervous system. The present in vivo electrophysiological studies were undertaken to determine if domoic acid exerts its neurotoxic effect via kainate receptor activation. Unitary extracellular recordings were obtained from pyramidal neurons of the CA1 and the CA3 regions of the rat dorsal hippocampus. The excitatory effect of domoic acid applied by microiontophoresis was compared with that of agonists of the three subtypes of glutamatergic receptors: kainate, quisqualate, and N-methyl-D-aspartate. In CA1, the activation induced by domoic acid was about threefold greater than that induced by kainate; identical concentrations and similar currents were used. In CA3, domoic acid was also three times more potent than kainate. However, the most striking finding was that domoic acid, similar to kainate, was more than 20-fold more potent in the CA3 than in the CA1 region, whereas no such regional difference could be detected with quisqualate and N-methyl-D-aspartate. As the differential regional response of CA1 and CA3 pyramidal neurons to kainate is attributable to the extremely high density of kainate receptors in the CA3 region, these results provide the first electrophysiological evidence that domoic acid may produce its neurotoxic effects through kainate receptor activation.     

Receptors for L-glutamate and GABA in the nervous system of an insect (Periplaneta americana)

The nervous system of the cockroach Periplaneta americana is well suited to studies of invertebrate amino acid receptors. Using a combination of radioligand binding and electrophysiological techniques, several distinct receptors have now been identified. These include an l-glutamate-gated chloride channel which has no known counterpart in the vertebrate nervous system, and a putative kainate/quisqualate receptor with pharmacological properties different from those of the existing categories of vertebrate excitatory amino acid receptors. GABA receptors have also been characterized in the cockroach nervous system. Bicuculline, benzodiazepines and steroids have revealed important differences between certain insect GABA-gated chloride channels and vertebrate GABA receptors. Identifiable neurones may facilitate the allocation of specific functions to amino acid receptor subtypes. In view of the existence of subtypes of amino acid receptors in insects, it is of interest to examine how this is reflected at the molecular level in terms of receptor subunit composition and amino acid sequence. Preliminary molecular cloning studies on insect GABA receptors are described.     

Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisqualate.

The complete amino acid sequences of two mouse glutamate receptor subunits (GluR1 and GluR2) have been deduced by cloning and sequencing the cDNAs. Xenopus oocytes injected with mRNA derived from the GluR1 cDNA exhibit current responses both to kainate and to quisqualate as well as to glutamate, whereas oocytes injected with mRNA derived from the GluR2 cDNA show little response. Injection of oocytes with both the mRNAs produces current responses larger than those induced by the GluR1-specific mRNA and the dose-response relations indicate a positively cooperative interaction between the two subunits. These results suggest that kainate and quisqualate can activate a common glutamate receptor subtype and that glutamate-gated ionic channels are hetero-oligomers of different subunits.     

Inositol hexakisphosphate receptor identified as the clathrin assembly protein AP-2.

To clarify the function of the receptor binding protein for inositol hexakisphosphate (IP6), we obtained a partial amino acid sequence from the purified protein and a partial nucleotide sequence from a cDNA clone of the gene. The sequences are essentially identical to those of the alpha-subunit of the clathrin assembly protein AP-2. The IP6 receptor protein analyzed by SDS-PAGE contains a series of subunits which are the same as those of AP-2. Antibodies to AP-2 react with the IP6 receptor protein in immunoblot analysis.     

Isolation and Characterization of a Rat Brain Triakontatetraneuropeptide, a Posttranslational Product of Diazepam Binding Inhibitor: Specific Action at the Ro 5-4864 Recognition Site

This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17-50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octadecaneuropeptide, DBI 33-50) antibodies coupled to CNBr-Sepharose 4B followed by two reverse-phase HPLC steps. The amino acid sequence of the purified peptide is: Thr-Gln-Pro-Thr-Asp-Glu-Glu-Met-Leu-Phe-Ile-Tyr-Ser-His-Phe-Lys-Gln-Ala-Thr-Val - Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. Synthetic TTN injected intracerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific "peripheral" benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the "central" BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5-4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 microM. TTN also enhances picrotoxinin inhibition of gamma-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at "Ro 5-4864 preferring" BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor-mediated effects.     

Yotiao protein, a ligand for the NMDA receptor, binds and targets cAMP-dependent protein kinase II(1)

A yeast two-hybrid screen revealed that regulatory subunits (RII) of PKAII bind the Yotiao protein. Yotiao interacts with the NR1 subunit of the NMDA receptor. A purified C-terminal fragment of Yotiao binds PKAII, via an RII binding site constituted by amino acid residues 1452-1469, with a dissociation constant (K(d)) between 50 and 90 nM in vitro. A stable complex composed of Yotiao, RII and NR1 was immunoprecipitated from whole rat brain extracts. Immunostaining analysis disclosed that Yotiao, RIIbeta and NR1 colocalize in striatal and cerebellar neurons. Co-assembly of Yotiao/PKAII complexes with NR1 subunits may promote cAMP-dependent modulation of NMDA receptor activity at synapses, thereby influencing brain development and synaptic plasticity.     

Molecular Cloning, Expression, and Pharmacological Characterization of humEAA1, a Human Kainate Receptor Subunit

Abstract: Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high-affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [³H] kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L-glutamate > ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid > dihydro-kainate > 6, 7-dinitroquinoxaline-2, 3-dione > 6-cyano-7-nitroquinoxaline-2, 3-dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand-gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high-affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1–16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.     

Characterization and partial purification of a chloride- and calcium-dependent glutamate-binding protein from rat brain

A glutamate-binding protein was solubilized from rat brain synaptic plasma membranes using sodium cholate. Its properties were characterized after addition of exogenous phospholipids and formation of proteoliposomes. Glutamate binding was dependent on calcium and chloride ions with maximal binding at concentrations of 10(-5) M calcium and 10 mM chloride ions. The effects of the two ions were synergistic rather than additive. In addition, glutamate binding was not affected by inhibitors specific for N-methyl-D-aspartate and kainate receptor subtypes, but was inhibited by quisqualate (Ki = 50 microM) and DL-2-amino-4-phosphonobutyrate (Ki = 1.3 mM). Furthermore, binding was abolished by 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1 mM dithiothreitol. These properties resemble those of the chloride- and calcium-dependent binding site. Starting from the detergent extract, the glutamate-binding protein was purified 123-fold using fractionated ammonium sulfate precipitation, chromatography on hydroxyapatite and on DEAE-Sephacel as sequential purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein fraction showed two major bands migrating with Mr values of 51,000 and 105,000. The properties of the partially purified binding protein were similar to those of the detergent extract. Glutamate binding to the partially purified protein is not due to a sequestration process or product binding to N-acetylated alpha-linked dipeptidase. Thus, the functional role of the binding protein remains to be established.     

Excitatory amino acid receptors in normal and abnormal vestibular function

Although excitatory amino acid (EAA) receptors have been investigated extensively in the limbic system and neocortex, less is known of the function of EAA receptors in the brainstem. A number of biochemical and electrophysiological studies suggest that the synapse between the ipsilateral vestibular (VIIIth) nerve and the brainstem vestibular nucleus (VN) is mediated by an EAA acting predominantly on kainate or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors. In addition, there is electrophysiological evidence that input from the contralateral vestibular nerve via the contralateral VN is partially mediated by N-methyl-D-aspartate (NMDA) receptors. Input to the VN from the spinal cord may also be partially mediated by NMDA receptors. All of the electrophysiological studies conducted so far have used in vitro preparations, and it is possible that denervation of the VN during the preparation of an explant or slice causes changes in EAA receptor function. Nonetheless, these results suggest that EAA receptors may be important in many different parts of the vestibular reflex pathways. Studies of the peripheral vestibular system have also shown that EAAs are involved in transmission between the receptor hair cells and the vestibular nerve fibers. A number of recent studies in the area of vestibular plasticity have reported that antagonists for the NMDA receptor subtype disrupt the behavioral recovery that occurs following unilateral deafferentation of the vestibular nerve fibers (vestibular compensation). It has been suggested that vestibular compensation may be owing to an upregulation or increased affinity of NMDA receptors in the VN ipsilateral to the peripheral deafferentation; however; at present, there is no clear evidence to support this hypothesis.     

Monospecific antibodies against a synthetic peptide predicted from the alpha-3 nicotinic receptor cDNA inhibit binding of [3H]nicotine to rat brain nicotinic cholinergic receptor

Polyclonal antibodies were raised against a synthetic decapeptide (designated S3) predicted from a segment of the alpha-3 subunit cDNA (amino acid residues 130-139) encoding the rat brain nicotinic cholinergic receptor. This segment was selected because it may be proximate to the nicotine/acetylcholine-binding site of the receptor (1). By radioligand binding assays and sucrose density gradient centrifugation, these monospecific antibodies were shown to inhibit the binding of [3H]nicotine to both the large molecular weight rat brain receptor (240 kDa) and to an SDS-disaggregated nicotine-binding subunit species (80 kDa), in a dose-dependent manner. The neutralizing effect of the anti-S3 antibodies supports the view that this region of the protein is closely related to the agonist binding site.     

Role of Guanine Nucleotides as Endogenous Ligands of a Kainic Acid Binding Site Population in the Mammalian Cerebellum

Abstract: An endogenous inhibitor of the membrane binding of kainic acid was extracted from pig brain tissue and purified. The substance was identified as GMP by structural analysis: Most likely it corresponds to an inhibitor previously extracted from the rat brain. The nucleotide is active as an inhibitor for kainate binding on goldfish brain synaptosomes, probably owing to direct displacement on receptor sites; it is also active on a low-affinity kainate site population in membranes from rat cerebellum. The interaction of GMP with the latter sites leads to a concentration-dependent kainate binding increase or inhibition, thus demonstrating that these sites can bind the nucleotide and cooperatively increase their affinity. Other guanine nucleotides show interaction with these sites, by either an increase (GTP) or inhibition (cyclic GMP or GDP) of kainate binding. These findings support the view that a guanine nucleotide is the endogenous ligand of a receptor in the mammalian cerebellum similar to the kainate binding protein present with high density in the cerebellum of lower vertebrates, whose function is probably connected to the role of the glial cells in this zone.