The calcium sensing receptor (CaR) is a G protein-coupled receptor transducer alterations in extracellular Ca2+ and polyvalent cation concentrations into activation of cellular signaling pathways involved in hormone secretion, growth and differentiation, and ion transport. While the cell and tissue distributions of CaR expression have been established, and the basic features of CaR signaling in several cell types have been described, there is little information about the molecular details of CaR function as a G protein-coupled receptor. The long term objective of this study is to define the molecular mechanism(s) of CaR activation and regulation. Human CaR will be studied in either transiently or stably transfected HEK-293 cells.
Aim 1 addresses the kinetics of agonist-mediated CaR activation. Fluorescence single cell imaging of intracellular Ca2+ will be used to characterize the basic features and regulation of CaR activation, including agonist dose/responses and the origins of CaR cooperativity.
Aim 2 will determine the contributions of disulfide bonds to CaR function/regulation. We will determine the contributions of disulfide bond-mediated dimerization to CaR function as well as the role(s) of disulfide bonds in transducing agonist binding into receptor activation.
Aim 3 will characterize the molecular mechanism(s) involved in acute CaR desensitization. The kinetics of desensitization/recovery will be quantified, and modulation by protein kinases/phosphatases will be assesesed both functionally and in protein kinase site mutants of CaR. These studies will establish the molecular mechanism(s) of CaR regulation by agonist, an important first step in establishing pharmacological interventions which can modulate the disparate functions of CaR in the wide range of cell types in which it is endogenously expressed. As a member of the superfamily encompassing metabotropic glutamate, GABAB, and pheromone receptors, these studies of CaR structure/function will likely be generalizable to a broad class of receptors important to human physiology and pathophysiology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058578-04
Application #
6525492
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Program Officer
Cole, Alison E
Project Start
1999-09-01
Project End
2005-08-31
Budget Start
2002-09-01
Budget End
2005-08-31
Support Year
4
Fiscal Year
2002
Total Cost
$316,190
Indirect Cost
Name
Syracuse University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
002257350
City
Syracuse
State
NY
Country
United States
Zip Code
13244
Cavanaugh, Alice; Huang, Ying; Breitwieser, Gerda E (2012) Behind the curtain: cellular mechanisms for allosteric modulation of calcium-sensing receptors. Br J Pharmacol 165:1670-1677
Huang, Ying; Cavanaugh, Alice; Breitwieser, Gerda E (2011) Regulation of stability and trafficking of calcium-sensing receptors by pharmacologic chaperones. Adv Pharmacol 62:143-73
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Breitwieser, Gerda E (2006) Calcium sensing receptors and calcium oscillations: calcium as a first messenger. Curr Top Dev Biol 73:85-114
Huang, Ying; Niwa, Jun-ichi; Sobue, Gen et al. (2006) Calcium-sensing receptor ubiquitination and degradation mediated by the E3 ubiquitin ligase dorfin. J Biol Chem 281:11610-7
Zhang, Mingliang; Breitwieser, Gerda E (2005) High affinity interaction with filamin A protects against calcium-sensing receptor degradation. J Biol Chem 280:11140-6
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Breitwieser, Gerda E; Miedlich, Susanne U; Zhang, Mingliang (2004) Calcium sensing receptors as integrators of multiple metabolic signals. Cell Calcium 35:209-16
Breitwieser, Gerda E (2004) G protein-coupled receptor oligomerization: implications for G protein activation and cell signaling. Circ Res 94:17-27
Maldonado-Perez, David; Breitwieser, Gerda E; Gama, Lucio et al. (2003) Human calcium-sensing receptor can be suppressed by antisense sequences. Biochem Biophys Res Commun 311:610-7

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