Identification and dissection of Ca2+-binding sites in the extracellular domain of Ca2+-sensing receptor

Abstract

Ca2+-sensing receptors (CaSRs) represent a class of receptors that respond to changes in the extracellular Ca2+ concentration ([Ca2+]o) and activate multiple signaling pathways. A major barrier to advancing our understanding of the role of Ca2+ in regulating CaSRs is the lack of adequate information about their Ca 2+-binding locations, which is largely hindered by the lack of a solved three-dimensional structure and rapid off rates due to low Ca 2+-binding affinities. In this paper, we have reported the identification of three potential Ca2+-binding sites in a modeled CaSR structure using computational algorithms based on the geometric description and surface electrostatic potentials. Mutation of the predicted ligand residues in the full-length CaSR caused abnormal responses to [Ca2+] o, similar to those observed with naturally occurring activating or inactivating mutations of the CaR, supporting the essential role of these predicted Ca2+-binding sites in the sensing capability of the CaSR. In addition, to probe the intrinsic Ca2+-binding properties of the predicted sequences, we engineered two predicted continuous Ca 2+-binding sequences individually into a scaffold protein provided by a non-Ca2+-binding protein, CD2. We report herein the estimation of the metal-binding affinities of these predicted sites in the CaSR by monitoring aromatic-sensitized Tb3+ fluorescence energy transfer. Removing the predicted Ca2+-binding ligands resulted in the loss of or significantly weakened cation binding. The potential Ca2+-binding residues were shown to be involved in Ca2+/Ln3+ binding by high resolution NMR and site-directed mutagenesis, further validating our prediction of Ca2+-binding sites within the extracellular domain of the CaSR. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.