Mutational and biophysical studies suggest RC3/neurogranin regulates calmodulin availability

RC3/neurogranin is a forebrain-enriched, postnatal-onset, thyroid hormone-dependent, protein kinase C substrate of dendritic spines that interacts with calmodulin. These characteristics suggest a prominent role within the Ca(2+)-mediated second messenger cascades associated with neonatal synaptogenesis and adult neural plasticity. To understand the molecular interactions between RC3 and calmodulin, we characterized recombinant RC3 and four sequence variants: Ser-36-->Ala, Ser-36-->Asp, Ser-36-->Lys, and Phe-37-->Trp. Interactions between CaM and variant Phe-37-->Trp can be monitored by fluorescence spectroscopy, allowing us to determine, by competitive assays, the relative affinities of the wild-type and variant proteins for calmodulin. The effects of salt and Ca2+ on the rank order of these affinities permit partial dissection of hydrophobic, ionic, and structural components of the RC3-CaM interaction and suggest that it is bimodal. We demonstrate that RC3 binds preferentially to CaM when Ca2+ is absent and that the addition of a negative charge to residue 36 is sufficient to disrupt all detectable RC3-CaM interactions. We propose a model wherein a Ca(2+)-"sensitive," bimodal interaction between RC3 and CaM regulates the transduction of postsynaptic Ca2+ fluxes into physiological responses through the modulation of Ca2+/CaM availability.