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Journal of Neuroscience, Vol 12, 2144-2155, Copyright © 1992 by Society for Neuroscience
Characterization of a novel synapse-specific protein. II. cDNA cloning and sequence analysis of the F1-20 protein
S Zhou, R Sousa, NH Tannery and EM Lafer
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
The F1-20 protein is a novel neuronal-specific, synapse-associated protein
that is expressed nonuniformly in mouse brain. Expression of the F1-20
protein is developmentally regulated in a pattern coincident with active
synaptogenesis and synaptic maturation. Here we report the cloning of the
cDNA sequence for the F1-20 protein. We found two distinct isoforms of
F1-20 cDNA that differed by the presence of 15 additional nucleotides,
which does not interrupt the open reading frame. RNase protection analysis
and PCR amplification of mouse brain RNA revealed that both isoforms are
present in cellular RNA. It is likely that the two F1-20 mRNA isoforms are
derived from RNA splicing events utilizing alternative 3' acceptor sites.
Analysis of the deduced amino acid sequence for the complete open reading
frame revealed that the predominant F1-20 mRNA encodes an 896 amino acid
polypeptide with a molecular weight of 91,319 Da. The deduced amino acid
sequence does not contain a signal sequence, or any extensive hydrophobic
regions. The deduced amino acid sequence does contain a number of consensus
sequences for protein kinases. Searches of the protein and nucleic acid
sequence data bases revealed that the F1-20 protein has not been previously
characterized at the primary structure level, although a weak similarity
was found between rabbit calpastatin and the C-terminal portion of the
F1-20 protein. We then determined biochemically that the F1-20 protein is a
substrate for Ca(2+)-dependent proteolysis, which is specifically inhibited
by calpain inhibitors in vitro. This indicates that the F1-20 protein is a
substrate for neuronal calpain. We observed that treatment of a
synaptosomal lysate with alkaline phosphatase led to an increase in the
electrophoretic mobility of the F1-20 protein, as well as to an increase in
the sharpness of the electrophoretic band. This indicates that the F1-20
protein is phosphorylated in vivo.
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