Protein kinase C transiently activated heteromeric N-methyl-D-aspartate receptor channels independent of the phosphorylatable C-terminal splice domain and of consensus phosphorylation sites.
We have expressed dual subunit combinations of isoforms of the N-methyl-D-aspartate receptor, NR1A-NR2A and NR1C-NR2A, in Xenopus oocytes. We show that both forms of the receptor are stereospecifically activated by low concentrations (10 nM) of the phorbol ester 4-beta-phorbol 12-myristate 13-acetate, known to activate protein kinase C (PKC). The activation is transient, and, after reaching a maximum in about 10 min, it decreases rapidly in spite of the continuous presence of phorbol ester. The addition of 2 microM oleoylacetylglycerol had similar consequences. NR1C differs from NR1A by a deletion of 37 amino acids that include four consensus phosphorylation sites for PKC in the C-terminal region. The corresponding peptide has been shown to become phosphorylated upon activation of PKC in neurons (Tingley, W. G., Roche, K. W., Thompson, A. K., and Huganir, R. L. (1993) Nature 364, 70-73). However, the activity of NR1C-NR2A receptors was stimulated 7-fold, twice the potentiation observed for NR1A-NR2A. By site-specific mutagenesis of NR1C and NR2A, we removed additional consensus PKC phosphorylation sites located between TM3 and TM4. Coexpression of these mutant subunits showed a similar response to phorbol esters as wild type receptors. Our results indicate that neither the predicted consensus phosphorylation sites between transmembrane sequences TM3 and TM4 nor the phosphorylatable C-terminal splice domain is essential for the modulation of N-methyl-D-aspartate receptors by PKC.
Sigel
E
E
Department of Pharmacology, University of Bern, Switzerland.
Baur
R
R
Malherbe
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002540:Cerebral Cortex / Q000378:metabolism*; D016384:Consensus Sequence; D017931:DNA Primers; D005260:Female; D007473:Ion Channels / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016202:N-Methylaspartate / Q000494:pharmacology*; D009865:Oocytes / Q000378:metabolism / Q000502:physiology; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000096:biosynthesis / Q000378:metabolism*; D012326:RNA Splicing; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014981:Xenopus
D017931:DNA Primers; D007473:Ion Channels; D046911:Macromolecular Substances; D016194:Receptors, N-Methyl-D-Aspartate; D016202:N-Methylaspartate; D011494:Protein Kinases; D013755:Tetradecanoylphorbol Acetate