Multiple Ca2+ Binding Sites in the Extracellular Domain of Ca2+-Sensing Receptor Corresponding to Cooperative Ca2+ Response

摘要

A small change in the extracellular Ca²⁺ concentration ([Ca²⁺]o) integrates cell signaling responses in multiple cellular and tissue networks and functions via activation of Ca²⁺-sensing receptors (CaSR). Mainly through binding of Ca²⁺ to the large extracellular domain (ECD) of the dimeric CaSR, intracellular Ca²⁺ responses are highly cooperative with an apparent Hill coefficient ranging from 2 to 4. We have previously reported the identification of two continuous putative Ca²⁺-binding sites by grafting CaSR-derived, Ca²⁺-binding peptides to a scaffold protein, CD2, that does not bind Ca²⁺. In this paper, we predict more potential non-continuous Ca²⁺-binding sites in the ECD. We dissect the intact CaSR into three globular subdomains, each of which contains 2 to 3 predicted Ca²⁺-binding sites. This approach enables us to further understand the mechanisms underlying the binding of multiple metal ions to extended polypeptides derived from within the ECD of the CaSR, which would be anticipated to more closely mimic the structure of the native CaSR ECD. Tb³⁺-luminescence energy transfer, ANS fluorescence, and NMR studies show biphasic metal-binding components and Ca²⁺-dependent conformational changes in these subdomains. Removing the predicted Ca²⁺-binding ligands in site 1 and site 3 abolishes the first binding step and second binding step, respectively. Studies on these subdomains suggest the existence of multiple metal-binding sites and metal-induced conformational changes that might be responsible for switching on/off the CaSR by transition between its open inactive form and closed active form.