Date of Award

8-12-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Jenny Yang

Abstract

As a highly versatile signal, Ca2+ operates over a wide temporal range to regulate many different cellular processes, impacting nearly every aspect of cellular life including excitability, exocytosis, motility, apoptosis, and transcription. While it has been well recognized that Ca2+ acts as both a second messenger to regulate cell-cell communication upon external stimuli and as a first messenger to integrate extracellular with intracellular signaling in various cell types. Molecular bases for such regulation and related human diseases are largely hampered by the challenges related to key membrane proteins. In the present study, we first investigated the regulatory role of intracellular Ca2+ ([Ca2+]i) on Connexin45 (Cx45) gap junction through a ubiquitous Ca2+ sensor protein-Calmodulin (CaM). Using bioluminescence resonance energy transfer assay, this study provides the first evidence of direct association of Cx45 and CaM in a Ca2+-dependent manner in cells. Complementary approaches including bioinformatics analysis and various biophysical methods identified a putative CaM-binding site in the intracellular loop of Cx45 with high Ca2+/CaM-binding affinity and Ca2+-dependent binding mode that is different from alpha family of connexins. To understand the role of extracellular calcium in regulation of gap junction hemichannels, we would like to prove a possible Ca2+-binding site predicted by our computational algorithm MUGSR in Connexin 26 (Cx26) through mutagenesis study, metal binding affinity measurement, conformational changes examination of purified Cx26 protein from Sf9; however, we failed to achieve this goal due to either the limitation of available methods or lethal effect of mutating the predicted Ca2+-binding ligand. Additionally, in this study, we identified a putative Ca2+-binding site in metabotropic glutamate receptor 5 (mGluR5) and demonstrated the importance of this Ca2+-binding site in activation of mGluR5 and modulating the actions of other orthosteric ligands on mGluR5. In addition, we successfully solved the first crystal structure of the extracellular domain of Ca2+-sensing receptor (CaSR) bound with Mg2+ and an unexpected Trp derivative. The extensive study of mechanism of CaSR function specifically through Mg2+-binding site and the unexpected ligand-binding site was done using several cell-based assays in wild type CaSR and mutants. Studies in this dissertation provides more information on how Ca2+ regulates gap junction channels, modulates mGluR5 activities and structural basis for regulation of CaSR by Mg2+ and an unexpected Trp derivative co-agonist.

Available for download on Sunday, July 29, 2018

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