RAPID PURIFICATION, SITE-DIRECTED MUTAGENESIS, AND INITIAL CHARACTERIZATION OF RECOMBINANT RC3 NEUROGRANIN

RC3/Neurogranin is a postnatal-onset, forebrain-specific, thyroid horm one-regulated, protein kinase C (PKC) substrate that binds calmodulin (CaM) and accumulates in dendritic spines. We bacterially expressed an d purified RC3 and, for comparison, GAP-43/neuromodulin to near homoge neity using relatively simple procedures. We then raised antisera agai nst recombinant RC3 that does not crossreact with GAP-43 and is suitab le for immunohistochemical analysis of brain slices. We also construct ed over 30 RC3 sequence variants by PCR-mediated, site-directed mutage nesis, and purified four of these to near homogeneity. The elution pro files displayed by RC3 and sequence variants during purification on Ca M-Sepharose columns suggest that two different affinity forms of the R C3 . CaM complex coexist when Ca2+ is absent and that GAP-43 . CaM int eractions are far more sensitive to salt than those that occur between recombinant RC3 and CaM. Variant proteins in which serine 36 was chan ged failed to serve as a substrate for PKC, implicating this as the ta rget residue.