The long-term goal of this research project is to understand the mechanisms underlying Ca2+-mediated signaling and the molecular basis for diseases associated with alterations in Ca2+ homeostasis. Extracellular Ca2+ ([Ca2+]o) has been proposed to function as a first messenger to trigger diverse cellular processes. Ca2+-sensing receptors (CaRs) represent a class of receptors that respond to changes in [Ca2+]o and activate multiple signaling pathways. By serving as the body's """"""""thermostats"""""""" for [Ca2+]o, CaRs play a central role in the regulation of [Ca2+]o homeostasis and represent important therapeutic targets. A major barrier to advancing our understanding of the role of Ca2+ in regulating CaRs is the lack of adequate information about the location of their Ca2+-binding sites and the structural information of this class of membrane proteins. Obtaining site-specific Ca2+-binding affinities of naturally-occurring proteins is hampered by the complexities encountered in cooperative, multi-site systems. The delineation of the Ca2+-binding sites in the CaR and related proteins is further hindered by limitations of crystallization conditions, rapid off-rates owing to low Ca2+-binding affinities and the existence of multiple conformations that are in equilibrium with one another.The immediate goals of this proposal are to 1) probe Ca2+-binding sites in the Ca2+-sensing receptors and 2) verify our prediction of specific Ca2+-binding sites by correlating the site-specific and domain-specific Ca2+-binding information with the biological activity of the w.t. receptor in mammalian cells as well as receptors with mutations in these Ca2+-binding sites. Results from our proposed work will have a major impact on the understanding of the mechanisms underlying the biological activities carried out by Ca2+-modulated receptors. These proposed investigations will provide novel methods for identifying Ca2+- binding sites in the CaR and related proteins, thus overcoming the major obstacles encountered in visualizing Ca2+-binding sites with weak binding affinities. Success in identifying Ca2+-binding sites and clarifying how Ca2+ regulates the CaR will not only promote an understanding of how Ca2+ functions as an extracellular messenger, but will also provide insights into the molecular basis of the clinical disorders associated with this receptor. Our success in designing and engineering metal-binding sites into arbitrary proteins could also lead to new ways of developing valuable reagents for diagnostic tests and chemotherapy. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM081749-01
Application #
7303386
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Basavappa, Ravi
Project Start
2007-09-01
Project End
2011-08-31
Budget Start
2007-09-01
Budget End
2008-08-31
Support Year
1
Fiscal Year
2007
Total Cost
$282,261
Indirect Cost
Name
Georgia State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
837322494
City
Atlanta
State
GA
Country
United States
Zip Code
30302
Zou, Juan; Salarian, Mani; Chen, Yanyi et al. (2017) Direct visualization of interaction between calmodulin and connexin45. Biochem J 474:4035-4051
Gorkhali, Rakshya; Huang, Kenneth; Kirberger, Michael et al. (2016) Defining potential roles of Pb(2+) in neurotoxicity from a calciomics approach. Metallomics 8:563-78
Zhang, Chen; Zhang, Tuo; Zou, Juan et al. (2016) Structural basis for regulation of human calcium-sensing receptor by magnesium ions and an unexpected tryptophan derivative co-agonist. Sci Adv 2:e1600241
Tang, Shen; Reddish, Florence; Zhuo, You et al. (2015) Fast kinetics of calcium signaling and sensor design. Curr Opin Chem Biol 27:90-7
Zhuo, You; Solntsev, Kyril M; Reddish, Florence et al. (2015) Effect of Ca²? on the steady-state and time-resolved emission properties of the genetically encoded fluorescent sensor CatchER. J Phys Chem B 119:2103-11
Zhang, Chen; Miller, Cassandra Lynn; Brown, Edward M et al. (2015) The calcium sensing receptor: from calcium sensing to signaling. Sci China Life Sci 58:14-27
Hsieh, Ying-Hsin; Zou, Juan; Jin, Jin-Shan et al. (2015) Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes. Anal Biochem 480:58-66
Zhang, Chen; Huang, Yun; Jiang, Yusheng et al. (2014) Identification of an L-phenylalanine binding site enhancing the cooperative responses of the calcium-sensing receptor to calcium. J Biol Chem 289:5296-309
Jiang, Jason Y; Nagaraju, Mulpuri; Meyer, Rebecca C et al. (2014) Extracellular calcium modulates actions of orthosteric and allosteric ligands on metabotropic glutamate receptor 1?. J Biol Chem 289:1649-61
Zou, Juan; Salarian, Mani; Chen, Yanyi et al. (2014) Gap junction regulation by calmodulin. FEBS Lett 588:1430-8

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