Footprint of Calcium on Regulation of Extracellular Calcium-Sensing Receptor and Connexin26

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

Calcium Dynamics Mediated by the Endoplasmic/Sarcoplasmic Reticulum and Related Diseases

The flow of intracellular calcium (Ca2+) is critical for the activation and regulation of important biological events that are required in living organisms. As the major Ca2+ repositories inside the cell, the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of muscle cells are central in maintaining and amplifying the intracellular Ca2+ signal. The morphology of these organelles, along with the distribution of key calcium-binding proteins (CaBPs), regulatory proteins, pumps, and receptors fundamentally impact the local and global differences in Ca2+ release kinetics. In this review, we will discuss the structural and morphological differences between the ER and SR and how they influence localized Ca2+ release, related diseases, and the need for targeted genetically encoded calcium indicators (GECIs) to study these events

Molecular Basis of the Extracellular Ligands Mediated Signaling by the Calcium Sensing Receptor

Ca2+-sensing receptors (CaSRs) play a central role in regulating extracellular calcium concentration ([Ca2+]o) homeostasis and many (patho)physiological processes in multiple organs. This regulation is orchestrated by a cooperative response to extracellular stimuli such as small changes in Ca2+, Mg2+, amino acids and other ligands. In addition, CaSR is a pleiotropic receptor regulating several intracellular signaling pathways, including calcium mobilization and intracellular calcium oscillation. Nearly 200 mutations and polymorphisms have been found in CaSR in relation to a variety of human disorders associated with abnormal Ca2+ homeostasis. In this review, we summarize efforts directed at identifying binding sites for calcium and amino acids. Both homotropic cooperativity among multiple calcium binding sites and heterotropic cooperativity between calcium and amino acid were revealed using computational modeling, predictions, and site-directed mutagenesis coupled with functional assays. The hinge region of the bilobed Venus flytrap (VFT) domain of CaSR plays a pivotal role in coordinating multiple extracellular stimuli, leading to cooperative responses from the receptor. We further highlight the extensive number of disease-associated mutations that have also been shown to affect CaSR’s cooperative action via several types of mechanisms. These results provide insights into the molecular bases of the structure and functional cooperativity of this receptor and other members of family C of the G protein-coupled receptors (cGPCRs) in health and disease states, and may assist in the prospective development of novel receptor-based therapeutics

Structural basis for regulation of human calcium-sensing receptor by magnesium ions and an unexpected tryptophan derivative co-agonist

Ca2+-sensing receptors (CaSRs) modulate calcium and magnesium homeostasis and many (patho)physiological processes by responding to extracellular stimuli, including divalent cations and amino acids. We report the first crystal structure of the extracellular domain (ECD) of human CaSR bound with Mg2+ and a tryptophan derivative ligand at 2.1 Å. The structure reveals key determinants for cooperative activation by metal ions and aromatic amino acids. The unexpected tryptophan derivative was bound in the hinge region between two globular ECD subdomains, and represents a novel high-affinity co-agonist of CaSR. The dissection of structure-function relations by mutagenesis, biochemical, and functional studies provides insights into the molecular basis of human diseases arising from CaSR mutations. The data also provide a novel paradigm for understanding the mechanism of CaSR-mediated signaling that is likely shared by the other family C GPCR [G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor] members and can facilitate the development of novel CaSR-based therapeutics