MUTATIONAL AND BIOPHYSICAL STUDIES SUGGEST RC3 NEUROGRANIN REGULATES CALMODULIN AVAILABILITY/

RC3/neurogranin is a forebrain-enriched, postnatal-onset, thyroid horm one-dependent, protein kinase C substrate of dendritic spines that int eracts with calmodulin. These characteristics suggest a prominent role within the Ca2+-mediated second messenger cascades associated with ne onatal synaptogenesis and adult neural plasticity. To understand the m olecular interactions between RC3 and calmodulin, we characterized rec ombinant RC3 and four sequence variants: Ser-36 --> Ala, Ser-36 --> As p, Ser-36 --> Lys, and Phe-37 --> Trp. Interactions between CaM and va riant Phe-37 --> Trp can be monitored by fluorescence spectroscopy, al lowing 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 dis section of hydrophobic, ionic, and structural components of the RC3-Ca M 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 detect able RC3-CaM interactions. We propose a model wherein a Ca2+-''sensiti ve,'' bimodal interaction between RC3 and CaM regulates the transducti on of postsynaptic Ca2+ fluxes into physiological responses through th e modulation of Ca2+/ CaM availability.