EXPRESSION AND STRUCTURE OF THE CALCIUM SENSING RECEPTOR

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DESCRIPTION (provided by applicant): The objective of this R21 grant application, submitted in response to program announcement, PA-01-127, "Pilot and Feasibility Program Related to the Kidney", is to develop methods for the expression and crystallization of the extracellular domain of the calcium-sensing receptor (CaSR), an important seven- transmembrane domain, G-protein coupled receptor, involved in the control of parathyroid hormone secretion and renal calcium reabsorption. The hypothesis of his grant application is that the extracellular domain of the GaSR forms a homodimer in which Ca -binding sites are present along the outer surface of the dimer pair. The binding of calcium (Ca2+) to the homodimer results in a more compact structure of the homodimer. This Ca2+-induced structural change in the extracellular domain of the CaSR is the signal that alters the activity of the receptor, and allows the modulation of intra-cellular events that alter the release of parathyroid hormone from parathyroid cells. Our specific aims are: Specific Aim 1: To develop methods for the expression of the following domains of the human CaSR - the extracellular domain (amino acids Tyr20-Glu610), an abbreviated form of the extracellular domain (ammo acids Tyr20- Glu535) and the cysteine-rich domain (amino acids Glu536-Glu610) of the human CaSR. Specific Aim 2: To test the Ca2+-binding properties of the expressed extracellular domain of the CaSR (the extracellular domain construct, amino acids Tyr20-Glu610, and the abbreviated form of the extracellular domain construct amino acids Tyr20-Glu535) and determine whether changes in secondary structure occur upon the binding of Ca2+. Specific Aim 3: To determine appropriate conditions for crystallization of the extracellular domain of the human CaSR in the presence and absence of calcium. Significance: The CaSR is an exceptionally important receptor that plays a role in many physiologically relevant processes such as the secretion of parathyroid hormone and the renal reabsorption of Ca2+. The structure of the receptor is unknown. If successful, our experiments will provide required, new methods that will allow us to determine the structure of the extracellular domain of this important receptor. It will allow us to determine how various agonists, antagonists and allosteric activators or inhibitors bind to, and alter the activity of the receptor. Comparison between structures of wild-type and mutant receptors might aid understanding of the clinical data collected on individuals with either activating or inactivating mutations of the CaSR. Furthermore, this information will serve as the basis for more extensive experiments involving the structure of the full-length receptor.