We propose to elucidate the physiological function of RC3 (also known as neurogranin). RC3 is postnatal onset, forebrain-enriched, Calmodulin (CaM)-binding, Protein Kinase C (PKC) substrate that is abundant in dendritic spines and loosely associated with postsynaptic densities. Based on several line of evidence, we hypothesize that RC3 plays a critical role in dendritic spine development and the regulation of intracellular Ca2+ ([Ca2+]i). We also hypothesize that RC3 and Ca2+/CaM dependent kinase II (CaMKII), and the degree to which they are phosphorylated, are both major, although opposing, determinants of the sliding LTD/LTP threshold theorized by Bienenstock, Cooper and Munro (BCM). Thus, the phosphorylation of RC3 and CaMKII may be principle mechanisms by which postsynaptic machinery keeps track of prior activity. We will inactivate RC3 by gene targeting in embryonic stem cells and will also create transgenic mice that over-express RC3 or one of two sequence variants of RC3. In one variant, serine 36, the target of PKC phosphorylation has been changed to an alanine (S36A), thus preventing phosphorylation but not CaM binding. The other variant contains an aspartate residue at position 36 (S36D) so that it no longer binds CaM. We will investigate the compensatory and pathological effects of these genetic perturbation by measuring expression levels and phosphorylation states of other proteins involved in pre- and post-synaptic signal transduction. We will also test the hypothesis that RC3 plays a critical role in dendritic spine development and the regulation of intracellular Ca2+ ([Ca2+]i) by studying primary hippocampal cell cultures derived from our knockout and transgenic mouse lines and we will test the hypothesis that the state of RC3 phosphorylation regulates the sliding LTD/LTP threshold by examining LTP in hippocampal slice preparations derived from the mouse line generated in aim 1.
| van Dalen, Jacqueline J W; Gerendasy, Dan D; de Graan, Pierre N E et al. (2003) Calcium dynamics are altered in cortical neurons lacking the calmodulin-binding protein RC3. Eur J Neurosci 18:13-22 |
| Krucker, Thomas; Siggins, George R; McNamara, Robert K et al. (2002) Targeted disruption of RC3 reveals a calmodulin-based mechanism for regulating metaplasticity in the hippocampus. J Neurosci 22:5525-35 |
| Gerendasy, D (1999) Homeostatic tuning of Ca2+ signal transduction by members of the calpacitin protein family. J Neurosci Res 58:107-19 |
| Ramakers, G M; Gerendasy, D D; de Graan, P N (1999) Substrate phosphorylation in the protein kinase Cgamma knockout mouse. J Biol Chem 274:1873-4 |
