Differential Activation of Intracellular Calcium Oscillations by Multiple Calcium and L-Phenylalanine Binding Sites Located at the Extracellular Domain of Calcium-Sensing-Receptor

Abstract

Calcium sensing receptor (CaSR), along other members of the family C G protein-coupled receptors (GPCRs), play very important roles in responding to changes in the extracellular calcium concentrations and in circulating levels of amino acids and integrating these extracellular signals into alterations in intracellular signaling pathways. We have reported several potential calcium-binding sites located within the CaSR's extracellular domain using our developed computational algorithms based on geometric factors and surface electrostatic potentials. In the present study, we first report the differential effects of several disease-related mutations located at the predicted calcium binding sites on the inhibition and activation of intracellular calcium responses using both a cell population assay and single cell imaging. We then identify a potential amino acid binding site using computational methods and site-directed mutagenesis. The effect of amino acid binding in altering intracellular calcium responses, especially calcium oscillations, and its synergistic interaction with the effects of extracellular calcium on these parameters are also investigated. A common activation mechanism for CaSR and other family C GPCRs, such as mGluR1 by extracellular calcium and amino acid is been proposed. These results could have important implications for our understanding of how the CaSR integrates information about these two completely different classes of agonists--one an inorganic divalent cation, the other a nutrient--and how the receptor senses these agonists in health and in disease states.