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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK048330-02
Application #
2148546
Study Section
General Medicine B Study Section (GMB)
Project Start
1994-07-11
Project End
1999-06-30
Budget Start
1995-07-01
Budget End
1996-06-30
Support Year
2
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
Bandyopadhyay, Sanghamitra; Jeong, Kyeong-Hoon; Hansen, Jacob Tfelt et al. (2007) Calcium-sensing receptor stimulates secretion of an interferon-gamma-induced monokine (CXCL10) and monocyte chemoattractant protein-3 in immortalized GnRH neurons. J Neurosci Res 85:882-95
Chattopadhyay, Naibedya; Jeong, Kyeong-Hoon; Yano, Shozo et al. (2007) Calcium receptor stimulates chemotaxis and secretion of MCP-1 in GnRH neurons in vitro: potential impact on reduced GnRH neuron population in CaR-null mice. Am J Physiol Endocrinol Metab 292:E523-32
Yano, Shozo; Mentaverri, Romuald; Kanuparthi, Deepthi et al. (2005) Functional expression of beta-chemokine receptors in osteoblasts: role of regulated upon activation, normal T cell expressed and secreted (RANTES) in osteoblasts and regulation of its secretion by osteoblasts and osteoclasts. Endocrinology 146:2324-35
Tfelt-Hansen, Jacob; Ferreira, Ana; Yano, Shozo et al. (2005) Calcium-sensing receptor activation induces nitric oxide production in H-500 Leydig cancer cells. Am J Physiol Endocrinol Metab 288:E1206-13
Mallya, Sanjay M; Gallagher, James J; Wild, Yvette K et al. (2005) Abnormal parathyroid cell proliferation precedes biochemical abnormalities in a mouse model of primary hyperparathyroidism. Mol Endocrinol 19:2603-9
Tfelt-Hansen, Jacob; Yano, Shozo; John Macleod, R et al. (2005) High calcium activates the EGF receptor potentially through the calcium-sensing receptor in Leydig cancer cells. Growth Factors 23:117-23
Tfelt-Hansen, J; Chattopadhyay, N; Yano, S et al. (2004) Calcium-sensing receptor induces proliferation through p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase but not extracellularly regulated kinase in a model of humoral hypercalcemia of malignancy. Endocrinology 145:1211-7
Ye, Chian Ping; Yano, Shozo; Tfelt-Hansen, Jacob et al. (2004) Regulation of a Ca2+-activated K+ channel by calcium-sensing receptor involves p38 MAP kinase. J Neurosci Res 75:491-8
Kifor, Olga; McElduff, Aidan; LeBoff, Meryl S et al. (2004) Activating antibodies to the calcium-sensing receptor in two patients with autoimmune hypoparathyroidism. J Clin Endocrinol Metab 89:548-56
Chattopadhyay, Naibedya; T-Felt Hansen, Jacob; Godbole, Madan M et al. (2004) Transforming growth factor beta receptor family ligands inhibit hepatocyte growth factor synthesis and secretion from astrocytoma cells. Brain Res Mol Brain Res 121:146-50

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