Li TianFollow

Author ORCID Identifier

https://orcid.org/0000-0001-9999-5308

Date of Award

5-6-2024

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Jenny J. Yang

Second Advisor

Angela Mabb

Third Advisor

Donald Hamelberg

Fourth Advisor

Jun Yin

Fifth Advisor

Ming Luo

Abstract

Calcium (Ca2+) serves as both a primary and secondary messenger, regulating various biological and pathological functions through calcium dynamics and interacting with crucial calcium-binding proteins like the calcium-sensing receptor (CaSR). Although there have been reports regarding the role of Ca2+ in CaSR biosynthesis and intracellular Ca2+ signaling, the precise molecular mechanisms remain elusive, primarily due to the complexities associated with membrane proteins. In addition, CaSR expression is associated with cancer, particularly in patients with primary renal cell tumors and bone metastasis, where it is highly expressed. Mutation of CaSR also causes diseases like autosomal dominant hypocalcemia (ADH), familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT). So far, only a few medications have received FDA approval for the treatment of various hyperparathyroidism forms. However, these medications often come with a multitude of side effects. Surprisingly, there are no CaSR antagonists, known as "calcilytics," in clinical use for conditions like osteoporosis or certain types of hypoparathyroidism. Thus, there is an urgent need for the development of thera(g)nostic compounds for early cancer detection and treatment of diseases associated with CaSR.

In this dissertation, we first presented the initial CaSR protein-protein interactome (PPI) and unveiled 98 novel potential CaSR interactors. Our findings suggest that CaSR-mediated intracellular signaling facilitates ER quality control and trafficking. Next, we focused on a key CaSR interactor, VAMP-associated protein A (VAPA). We demonstrated the direct interaction between VAPA and CaSR, as well as VAPA's crucial role in modulating CaSR sensitivity, CaSR-mediated calcium signaling, and Nuclear factor of an activated T cell (NFAT) translocation mediated by CaSR. Moreover, VAPA was found to be involved in the endocytosis of membrane CaSR. Third, we investigated the mechanism of cooperative binding and functional cooperativity of CaSR, wherein our data suggested the pivotal role of a hinge region calcium binding site. Additionally, we established a working model indicating that several Ca2+-binding sites within the CaSR-ECD, alongside endogenous ambient amino acids, cooperatively activate the receptor. Finally, we discuss efforts to develop novel CaSR-targeted thera(g)nostic PET imaging agents and genetically-encoded mitochondrial calcium indicators.

DOI

https://doi.org/10.57709/36898262

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