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Title page for ETD etd-02202019-113322


Type of Document Dissertation
Author Marks, Christian Randal
URN etd-02202019-113322
Title Role of Ca2+/Calmodulin-Dependent Protein Kinase II in Regulating the Metabotropic Glutamate Receptor 5
Degree PhD
Department Molecular Physiology and Biophysics
Advisory Committee
Advisor Name Title
Danny Winder, Ph.D. Committee Chair
Brian Wadzinski, Ph.D. Committee Member
Colleen Niswender, Ph.D. Committee Member
David Jacobson, Ph.D. Committee Member
Eric Delpire, Ph.D. Committee Member
Keywords
  • kinase activity
  • calicum imaging
  • neuronal activity
  • camkii
  • protein interactions
  • metabotropic glutamate receptor
  • phosphorylation
  • protein binding
Date of Defense 2019-01-29
Availability unrestricted
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.

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