The extracellular calcium concentration ([Ca2+]0) directly regulates parathyroid (PT) and certain renal cells [e.g., the proximal tubular cells synthesizing 1,25(OH)2D and those of the medullary thick ascending limb (MTAL)], which, in turn, contribute to maintaining Ca2+ homeostasis. Although indirect evidence suggests that [Ca2+]0 regulates these cells via a cell surface Ca2+-ensing receptor, the latter has been neither isolated in pure form nor characterized in detail. In our preliminary studies, we have cloned a PI-coupled, Ca2+-sensing receptor from bovine PT gland (BoPCaR) with properties very similar to those of the native receptor. Receptor transcripts are present in tissues that respond directly to [Ca2+]0, including kidney cortex (which has the proximal tubular segment(s) synthesizing 1,25(OH)2D) and outer medulla (which includes the MTAL). Moreover, we have subsequently obtained a cross-hybridizing clone from a rat kidney cDNA library derived from outer medulla, which appears to encode a renal Ca2+-sensing receptor. The overall goal of this proposal is to characterize the structure, function, regulation and detailed cellular localization of PT and kidney extracellular Ca2+-sensing receptor(s) by addressing the following specific issues. (1) We will study the molecular diversity of the Ca2+-sensing receptors by (a) comparing the nucleotide and deduced amino acid sequences and structural featrues of the rat renal Ca2+- receptor cDNA, RaKCaR, with those of BoPCaR and other receptors and proteins in the databases and (b) searching for additional PT and kidney forms of the Ca2+-sensing receptors using homology-based strategies. (2). We will characterize the pharmacology and signal transduction pathways of the extracellular Ca2+-sensing receptors by (a) comparing their pharmacological profiles for interacting with polycations and (b) determining whether a given form of PT or kidney Ca2+-receptor can couple to a single or to multiple second messenger pathways. (3) We will raise polyclonal antisera to the Ca2+-sensing receptors for subsequent studies on receptor regulation and localization. (4) We will study the factors involved in Ca2+-sensing receptor regulation by investigating (a) whether protein kinase C (PKC)-mediated phosphorylation of BoPCaR controls its coupling to phospholipase C (PLC), (b) if key physiological regulators of PT and kidney function (e.g., [Ca2+]o, 1,25(OH)2D, etc.) regulate Ca2+- sensing receptor mRNA levels or receptor protein levels and (vc) whether there are differences in receptor mRNA or protein levels or degree of phosphorylation that could account for the marked increase in set-point of dispersed and particularly cultured calf PT cells relative to dispersed cow PT cells. (5) Finally, we will determine the tissue distribution of Ca2+- sensing receptor mRNA using (a) in situ hybridization and (b) reverse transcriptase-dependent PCR of individually dissected nephron segments and glomeruli and assess the membrane localization of Ca2+-sensing receptors by immunohistochemistry. These studies should provide fundamental new insights into the manner in which extracellular Ca2+ directly regulates the function of these two critical Ca2+-regulating tissues.
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