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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Smith, Ward
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Georgia State University
Schools of Arts and Sciences
United States
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