Date of Award

8-13-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Jenny Yang

Second Advisor

Donald Hamelberg

Third Advisor

Zhi-Ren Liu

Fourth Advisor

Ming Luo

Abstract

Calcium (Ca2+) functions as a primary and secondary messenger regulating crucial cellular processes. We study two central membrane proteins, extracellular calcium-sensing receptor (CaSR) and connexin26 (Cx26) gap junction (GJ) channel, that are regulated by Ca2+ and play critical roles in regulating Ca2+ homeostasis and communication between the intra- and extra- cellular milieu. Mutations in CaSR are associated with abnormal Ca2+ homeostasis, hypoparathyroidism, myocardial infarction and cancers. Similarly, mutations in Cx26 are implicated in many hereditary deafness and dermatological disorders. The role of Ca2+ in CaSR biosynthesis, CaSR mediated intracellular Ca2+ signaling, Cx26 regulation and tuning of their regulators in biological and pathological is reported, however, the knowledge of exact molecular mechanism is obscure due to challenges associated with membrane proteins.

In this dissertation, we first report the discovery of 98 novel putative CaSR interactors using co-immunoprecipitation, mass-spectrometry and confocal imaging. Our findings suggest that extracellular Ca2+ dependent CaSR mediated intracellular signaling facilitates ER quality control and trafficking by upregulating the interaction with proteins affiliated with ubiquitination, chaperoning and glycosylation. Next, the cooperative activation of CaSR by Ca2+, Mg2+ and aromatic amino acids is validated in wild type CaSR in HEK293 cells. CaSR mutations at conserved metal binding sites reduce Ca2+/Mg2+ evoked intracellular Ca2+ mobilization and Ca2+ oscillation. This work further uses single cell imaging, immunoassay and sequencing to report tissue specific expression and differential capabilities of cations and drugs to tune CaSR mediated signaling in prostate (PCa) and thyroid cancer cells. We report a presence of wild type CaSR in PCa cell using RT-PCR. Additionally, proteomics and gene ontology show differential proteostasis between prostate cancer and HEK293 cells. Finally, this study endeavored at expressing and purifying a challenging protein, Cx26, and established binding affinity for Tb3+ and Ca2+ as 1.8 µM and 37 mM, respectively. The N-terminal lobe of CaM was found to bind Ca2+ tighter by 2.5-folds greater than C-lobe in the presence of Cx26p1-21. Our study on role of Ca2+ on regulation of CaSR and Cx26 allows for greater understanding of their function and provides avenue for potential therapeutic targets.

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