Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.13 (protein kinase C)
49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ethanol has been demonstrated to potentiate GABAergic mechanisms in several different cell types and also appears to be active on GABAA receptors in specific brain regions. By expressing brain mRNA and cloned GABA receptor subunits in Xenopus oocytes, we have investigated the requirements for ethanol modulation of the GABAA receptor. Using hybrid arrest techniques, the GABA receptor subunits alpha 1, beta 1, gamma 1, gamma 2S+ gamma 2L, gamma 2L and gamma 3 were individually prevented from expressing. None of these treatments altered potentiation by pentobarbital, the gamma 2S+ gamma 2L and gamma 2L reduced diazepam potentiation, however only the gamma 2L hybridization reduced ethanol sensitivity. By expression of cloned GABA subunits the effects could be reproduced by comparing ethanol sensitivity of alpha 1 beta 1 gamma 2S and alpha 1 beta 1 gamma 2L. The gamma 2L is an alternatively spliced variant of gamma 2 containing an extra eight amino acids bearing a consensus site for phosphorylation by protein kinase C. By using in vitro mutagenesis techniques, alteration of key residues in the phosphorylation site prevented ethanol modulation of the receptors containing mutant gamma 2 subunits. These results suggest that phosphorylation of a site on the gamma 2L subunit can alter the modulatory effects of ethanol on GABAA receptors containing this subunit.
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PMID:GABAA receptor subunit expression and sensitivity to ethanol. 774 19

A partial cDNA sequence indicated that the T lymphocyte early-activation gene (Tea) encodes a protein related to the dual-function ecotropic retrovirus receptor/cationic amino acid transporter (ecoR/CAT1), and RNA blots suggested highest Tea expression in T lymphocytes and liver (MacLeod, C.L., Finley, K., Kakuda, D. Kozad, C.A., and Wilkinson, M.F. (1990) Mol. Cell. Biol. 7, 3663-3674). The sequence of full-length Tea cDNA from liver (3683 bases) predicts a 657-amino-acid protein (CAT2 alpha) with 12-14 transmembrane domains. A long (515 base) region with six initiation codons and termination codons precedes the translation start codon. The liver Tea cDNA is identical to Tea cDNA from T lymphocytes (encoding CAT2 beta) with the exception of an apparent alternatively spliced sequence encoding a hydrophilic loop of 43 amino acids. The liver-specific sequence contains unique consensus sites for phosphorylation by cyclic AMP-dependent protein kinase and by protein kinase C. Injection of Xenopus oocytes with CAT2 alpha or CAT2 beta messenger RNA resulted in expression of Na(+)-independent cationic amino acid transport that was detected by current measurements under voltage-clamp. Although the amino acid sequences of the isoforms differ in only 21 of 43 residues with the majority of substitutions being conservative, the apparent affinity of CAT2 beta for arginine uptake was 70-fold higher than the CAT2 alpha isoform (Km 38 microM versus 2.7 mM). Neither isoform functioned as a receptor for ecotropic or amphotropic murine retroviruses. However, CAT1-CAT2 chimeric proteins that contain the first three putative extracellular loops of ecoR/CAT1 functioned as ecotropic receptors despite a diminished capacity to bind the viral envelope glycoprotein. The chimeric proteins also functioned as basic amino acid transporters with substrate affinities corresponding to the CAT2 isoform constituting the carboxyl-terminal portion. These results demonstrate that domains of these transporters can function in chimeric combinations to control viral receptor and transport functions.
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PMID:Control of cationic amino acid transport and retroviral receptor functions in a membrane protein family. 819 86

440-kD ankyrinB is an alternatively spliced variant of 220-kD ankyrinB, with a predicted 220-kD sequence inserted between the membrane/spectrin binding domains and COOH-terminal domain (Kunimoto, M., E. Otto, and V. Bennett. 1991. J. Cell Biol. 236:1372-1379). This paper presents the sequence of 2085 amino acids comprising the alternatively spliced portion of 440-kD ankyrinB, and provides evidence that much of the inserted sequence has the configuration of an extended random coil. Notable features of the inserted sequence include a hydrophilicity profile that contains few hydrophobic regions, and 220 predicted sites for phosphorylation by protein kinases (casein kinase 2, protein kinase C, and proline-directed protein kinase). Secondary structure and folding of the inserted amino acid residues were deduced from properties of recombinant polypeptides. Frictional ratios of 1.9-2.4 were calculated from Stokes radii and sedimentation coefficients, for polypeptides comprising 70% of the inserted sequence, indicating a highly asymmetric shape. Circular dichroism spectra of these polypeptides indicate a nonglobular structure with negligible alpha-helix or beta sheet folding. These results suggest a ball-and-chain model for 440-kD ankyrinB with a membrane-associated globular head domain and an extended filamentous tail domain encoded by the inserted sequence. Immunofluorescence and immunoblot studies of developing neonatal rat optic nerve indicate that 440-kD ankyrinB is selectively targeted to premyelinated axons, and that 440-kD ankyrinB disappears from these axons coincident with myelination. Hypomyelinated nerve tracts of the myelin-deficient Shiverer mice exhibit elevated levels of 440-kD ankyrinB. 440-kD ankyrinB thus is a specific component of unmyelinated axons and expression of 440-kD ankyrinB may be downregulated as a consequence of myelination.
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PMID:440-kD ankyrinB: structure of the major developmentally regulated domain and selective localization in unmyelinated axons. 825 44

Dynamin is a GTP-, microtubule-, and phospholipid-binding protein that is expressed primarily in brain. In Drosophila, the shibire gene encodes a homologue of dynamin; mutations in this gene result in a defect in endocytosis, suggesting a function for dynamin in endocytic membrane traffic. In the present study we show that there are at least two distinct dynamin genes in mammals whose products are referred to as dynamins I and II. The two dynamins are similar to each other (79% identity) and are both equally homologous to the Drosophila shibire gene product (66% identity). The highest degree of identity between dynamins is observed in their N-terminal halves, whereas their C termini exhibit little homology. Transcripts of both dynamin genes are subject to at least two alternative splicing events, the first of which is identically found in both dynamins, whereas the second site of alternative splicing is different between the two types of dynamins. The first alternatively spliced sequence of the dynamins consists of an interior region that is present in two distinct but homologous forms in both dynamins, suggesting alternative use of exons in both genes at identical positions. The second site of alternative splicing results in the generation of different C termini in dynamin I and in the inclusion or exclusion of an interior four-amino acid sequence in dynamin II. The two dynamins exhibit remarkable differences in their tissue distribution and regulation. Dynamin I is almost exclusively expressed in the central nervous system. Conversely, dynamin II is expressed ubiquitously in all tissues tested. Previous studies revealed that the GTPase activity of dynamin I is regulated by phosphorylation by protein kinase C in nerve terminals. Expression of dynamins I and II by transfection in COS cells demonstrates that only dynamin I but not dynamin II is a substrate for protein kinase C. Our data suggest a specialization in the endocytic functions and the regulation of dynamins between neural and non-neural tissues in mammals.
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PMID:Differential expression and regulation of multiple dynamins. 830 25

The NMDA (N-methyl D-aspartate) receptors in the brain play a critical role in synaptic plasticity, synaptogenesis and excitotoxicity. Molecular cloning has demonstrated that NMDA receptors consist of several homologous subunits (NMDAR1, 2A-2D). A variety of studies have suggested that protein phosphorylation of NMDA receptors may regulate their function and play a role in many forms of synaptic plasticity such as long-term potentiation. We have examined the phosphorylation of the NMDA receptor subunit NMDAR1 (NR1) by protein kinase C (PKC) in cells transiently expressing recombinant NR1 and in primary cultures of cortical neurons. PKC phosphorylation occurs on several distinct sites on the NR1 subunit. Most of these sites are contained within a single alternatively spliced exon in the C-terminal domain, which has previously been proposed to be on the extracellular side of the membrane. These results demonstrate that alternative splicing of the NR1 messenger RNA regulates its phosphorylation by PKC, and that mRNA splicing is a novel mechanism for regulating the sensitivity of glutamate receptors to protein phosphorylation. These results also provide evidence that the C-terminal domain of the NR1 protein is located intracellularly, suggesting that the proposed transmembrane topology model for glutamate receptors may be incorrect.
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PMID:Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain. 831 1

The N-methyl-D-aspartate (NMDA) receptor NR1 gene encodes RNA that is alternatively spliced to generate at least seven variants. The variants arise from splicing in or out of three exons; one encodes a 21-amino acid insert in the N-terminal domain, and two encode adjacent sequences of 37 and 38 amino acids in the C-terminal domain. Splicing out of the second C-terminal exon deletes a stop codon and results in an additional open reading frame encoding an unrelated sequence of 22 amino acids before arriving at a second stop codon. We denote the NR1 variants by the presence or absence of the three alternatively spliced exons (from 5' to 3'); thus, NR1(111) has all three exons, NR1(000) has none, and NR1(100) has only the N-terminal exon. We report here electrophysiological characterization of six splice variants of the NR1 receptor expressed in Xenopus oocytes. NR1 receptors that lacked the N-terminal exon (NR1(000), NR1(010), and NR1(011)) exhibited a relatively high affinity for NMDA (EC50 approximately 13 microM) and marked potentiation by spermine. In contrast, those receptor variants with the N-terminal insert (NR1(100), NR1(101), and NR1(111)) showed a lower agonist affinity and little or no spermine potentiation at saturating glycine. All six variants showed spermine potentiation at low glycine and inhibition by spermine at more negative potentials. Variants differing only in the C-terminal domain differed little in agonist affinity and spermine potentiation. These findings indicate that the N-terminal insert either participates in agonist and polyamine binding domains or indirectly modifies their conformations. The splice variants differed in the extent to which they could be potentiated by activators of protein kinase C (PKC) from 3- to 20-fold. Presence of the N-terminal insert and absence of the C-terminal sequences increased potentiation by PKC. These findings identify the contributions of the separate polypeptide domains to modulation by polyamines and PKC and provide further support for the concept that subunit composition determines functional properties of NMDA receptors.
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PMID:Splice variants of the N-methyl-D-aspartate receptor NR1 identify domains involved in regulation by polyamines and protein kinase C. 834 92

The gamma 2 subunit of the GABA receptor (GABAA-R) is alternatively spliced. The long variant (gamma 2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca2+/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336-351 of the intracellular loop of the gamma 2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The Km values for PKC- and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 microM, respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the gamma 2L subunit has been shown to be necessary for ethanol potentiation of the GABAA-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.
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PMID:Ca2+/calmodulin-dependent protein kinase II and protein kinase C phosphorylate a synthetic peptide corresponding to a sequence that is specific for the gamma 2L subunit of the GABAA receptor. 839 May 66

Several lines of evidence suggest that N-methyl-D-aspartate (NMDA) receptors significantly contribute to the development of kindling. In addition, a lasting enhancement of the NMDA receptor function has been suggested to play a significant role in the chronic hyperexcitability occurring in the hippocampus after kindling epileptogenesis. We have investigated whether hippocampal kindling induces changes in the NMDA receptor at the molecular level by assessing the expression of mRNAs of the different spliced variants at the N-terminal (exon 5) and C-terminal (exon 21) position of the NMDA receptor 1 (NR1) gene by means of the reverse transcription-polymerase chain reaction. Alternative splicing at exon 5 confers different sensitivity of the NMDA receptor to polyamines while exon 21 encodes a 37-amino acid insert containing the major phosphorylation sites for protein kinase C. One week after the acquisition of stage 5 of kindling in rats (generalized tonic-clonic seizures), the relative abundance of the two alternatively spliced forms at the C-terminal domain, respectively containing (+) or lacking (-) exon 21, was reversed compared to controls (implanted with electrodes but not stimulated) in the dorsal hippocampus ipsilateral and contralateral to the electrical stimulation. The exon 21+/exon 21- mRNA ratio for controls was 1.3 +/- 0.04 (mean +/- SE); for ipsilaterally kindled rats it was 0.64 +/- 0.05 (P < 0.05), and for contralaterally kindled rats it was 0.48 +/- 0.07 (P < 0.01). Similar bilateral effects were observed in the ventral hippocampus (temporal pole). No changes were found 4 weeks after stage 5 seizures and 1 week after the induction of a single afterdischarge. No significant alterations were induced by kindling in the relative abundance of the spliced variants containing or lacking exon 5. Our findings show selective changes in alternative splicing of the NR1 gene after repeated application of an epileptogenic stimulus. This may generate receptors with different functional properties, which may contribute to the increased sensitivity for the induction of generalized seizures during kindling.
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PMID:Alternative splicing at the C-terminal but not at the N-terminal domain of the NMDA receptor NR1 is altered in the kindled hippocampus. 884 57

Two subtypes of the thromboxane A2 (TxA2) receptor (TxA2R-E and TxA2R-P), which differ in their alternatively spliced cytoplasmic tails, have been identified. The initial concentration of the TxA2 mimetic IBOP required to reduce peak intracellular Ca2+ concentration ([Ca2+]i) induced by a second addition of IBOP (100 nmol/L) was similar (IC50 for TxA2R-E and TxA2R-P, 0.46 +/- 0.16 and 0.40 +/- 0.07 nmol/L) in fibroblasts overexpressing either the TxA2R-E or -P subtype. Although the number of TxA2 binding sites decreased in TxA2R-P cells after prolonged stimulation with a TxA2 mimetic, those in the TxA2R-E cells increased markedly. To determine whether the mechanism for desensitization differs between subtypes, the effect of activation of protein kinase C (PKC) or cAMP-dependent kinase on TxA2-induced [Ca2+]i mobilization was measured. Forskolin reduced the IBOP-induced peak [Ca2+]i in neither TxA2R-E nor TxA2R-P cells; however, treatment with phorbol esters (IC50, 0.57 +/- 0.70 nmol/L) strongly prevented IBOP-mediated [Ca2+]i rise in TxA2R-E but not in TxA2R-P cells. Desensitization of TxA2R-E by phorbol esters was prevented by the PKC inhibitor calphostin C or by downregulation of PKC-alpha. Thus, the response of TxA2R-E to prolonged stimulation differs from that of TxA2R-P in both the regulation of the number of binding sites and the mechanism for desensitization; agonists that activate PKC-alpha might interfere with TxA2R-E-mediated signaling.
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PMID:Differential desensitization of thromboxane A2 receptor subtypes. 911 86

Metabotropic glutamate receptors, which are members of a G protein-coupled receptor family, mediate the glutamate responses by coupling to the intracellular signal transduction pathway. We herein report that calmodulin (CaM) interacts with the metabotropic glutamate receptor subtype 5 (mGluR5) in a Ca2+-dependent manner in vitro. CaM is capable of binding on two distinct sites in the COOH-terminal intracellular region of the receptor with different affinities. The CaM binding domains are separated by an alternatively spliced exon cassette present in one of the splicing isoforms of mGluR5. By using fusion proteins and synthetic peptides we showed that protein kinase C phosphorylates both CaM binding regions. This phosphorylation is inhibited by the binding of CaM to the receptor, and conversely the binding is inhibited by the phosphorylation. These antagonisms of the CaM binding and phosphorylation thus suggest the possibility that they regulate the receptor responses in vivo.
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PMID:Phosphorylation and calmodulin binding of the metabotropic glutamate receptor subtype 5 (mGluR5) are antagonistic in vitro. 924 10


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