Subtractive cDNA cloning of RC3, a rodent cortex‐enriched mRNA encoding a novel 78 residue protein

Abstract

A rodent cortex-enriched mRNA, RC3, was identified by screening a rat brain cDNA library with a cortex-minus-cerebellum subtracted cDNA probe. Conceptual translation of RC3 cDNA sequences indicates that the rat and mouse mRNAs encode identical, novel 78 amino acid proteins. The RC3 protein amino terminus contains a cysteine-rich domain similar to those found in snake venom neurotoxins; the carboxyl terminus contains a collagen-like motif that may function in the assembly of RC3 subunits into a multimeric protein. Western blot experiments with an antiserum to a synthetic peptide corresponding to 27 residues of the 78 residue sequence identify an immunoreactive polypeptide with 18 kDa gel mobility that is likely to correspond to the RC3 protein. Northern blot analysis and in situ hybridization experiments show that RC3 mRNA is highly enriched in rat brain, with restricted expression in neuronal subsets primarily in the cortex, striatum, and hippocampus as well as certain nuclei within the thalamus, hypothalamus, the olfactory bulb.

Golgi Cell Dendrites Are Restricted by Purkinje Cell Stripe Boundaries in the Adult Mouse Cerebellar Cortex

“…Antizebrin II is a mouse monoclonal antibody produced by immunization with a crude cerebellar homogenate from the weakly electric fish Apteronotus (Brochu et al, 1990) and subsequently shown to bind the respiratory isoenzyme aldolase C (Ahn et al, 1994;Hawkes and Herrup, 1996); it was used directly from spent hybridoma culture medium. Rabbit anti-neurogranin was raised against full-length recombinant rat neurogranin protein (Millipore, Billerica, MA; catalog #AB5620; used diluted 1:1000); it recognizes Purkinje cells in the neonatal cerebellum and Golgi cells in the adult cerebellum, as previously reported (Singec et al, 2003;Larouche et al, 2006), and on Western blots of newborn or postnatal day 20 cerebellar homogenates reveals a single polypeptide band of apparent molecular weight 10 kDa (Larouche et al, 2006), consistent with that of neurogranin/RC3 (Watson et al, 1990). Mouse hybridoma cell line TIB200 secreting monoclonal anti-HNK-1 (Abo and Balch, 1981) was obtained from the American Type Culture Collection (Rockville, MD).…”

Involvement of neurogranin in the modulation of calcium/calmodulin-dependent protein kinase II, synaptic plasticity, and spatial learning: A study with knockout mice

“…PKC is the only known Ng kinase in vitro (5), and deletion of PKC ␥ gene in mice negates both the glutamate-and depolarization-mediated phosphorylation of Ng (9). The close functional relationship between Ng and PKC ␥ is further illustrated by their similar developmental expression patterns in the cerebral cortex (10)(11)(12). Previous studies also suggest that Ng plays a critical role in synaptic plasticity.…”

Mechanism of protein kinase C activation during the induction and maintenance of long-term potentiation probed using a selective peptide substrate.

“…Identification of specific protein kinases activated in vivo has in general been limited by the lack of potent and selective protein kinase substrates. We have developed a peptide substrate selective for protein kinase C (PKC) that corresponds to the phosphorylation site (aa 28-43) of neurogranin/ RC3 [NG- (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)], a neuronal protein that is an endogenous selective PKC substrate (11)(12)(13). In the present studies, we have characterized the properties of NG-(28-43) phosphorylation in hippocampal homogenates and used the peptide to test the hypothesis that PKC is activated in the induction and maintenance phases of LTP.…”