Role of Ca2+/Calmodulin-Dependent Protein Kinase II in Regulating the Metabotropic Glutamate Receptor 5

Abstract

Multi-protein complexes formed through protein-protein interactions in the dendrites of neurons are highly regulated to facilitate proper synaptic function. The work presented in this dissertation highlights the importance of a novel interaction between two synaptic regulators: Calcium/Calmodulin Dependent Protein Kinase II (CaMKII) and the metabotropic glutamate receptor 5 (mGlu5). Here, I show that CaMKII can bind to and phosphorylate the mGlu5 C-terminal domain (CTD). In vitro characterization showed that the mGlu5-CTD directly interacts with Thr286-autophosphorylated CaMKII. I identified three basic residues on the mGlu5-CTD necessary for the mGlu5-CaMKII interaction. Mutagenesis of these residues allowed for the construction of a full-length mGlu5 construct with reduced CaMKII binding. I hypothesized that the mGlu5-CaMKII interaction could modulate mGlu5 signaling. Activation of mGlu5 results in downstream signals that increase intracellular Ca2+ release and activate ERK to regulate many cellular processes. The co-expression of active CaMKII increased basal mGlu5 surface expression and ERK activation in heterologous cells. In addition, CaMKII modulated mGlu5-mediated Ca2+ release in heterologous cells, decreasing the initial Ca2+ amplitude, but prolonging the relative Ca2+ signal. Therefore, I hypothesized that knockdown of CaMKII in neurons would result in increased mGlu5-mediated Ca2+ signals. However, knockdown of CaMKII reduced mGlu5-specific Ca2+ signals in neuronal cultures. This effect was specific to mGlu5 because CaMKII knockdown had no effect on L-type voltage dependent Ca2+ channel (LTCC) signals. However, knockdown of an important synaptic scaffolding protein, Shank-3, reduced mGlu5 and LTCC Ca2+ responses.