Identification and Dissection of Ca2+-binding Sites in the Extracellular Domain of Ca2+-sensing Receptor

Abstract

[Ca 2ϩ ] o ) the receptor and that cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism (in the former) and autosomal dominant hypoparathyroidism (in the latter case) (20, 21).The CaSR and other members of family C of the G proteincoupled receptor consist of a large extracellular domain (ECD), a transmembrane domain with seven transmembrane segments, and an intracellular C-tail segment (22,23). Based on the functional responses at the cellular level, the ECD regions have been proposed to contain the major Ca 2ϩ -binding sites and to respond to [Ca 2ϩ ] o for both the mGluRs and CaSRs. The Hill coefficient suggests that 3-5 Ca 2ϩ ions bind cooperatively to the CaSR (22,24

Identification of an l-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium

“…3 and 4). Such population studies were also reported in our previous studies (25,26). Results from Western blot and immunofluorescence staining using an anti-CaSR antibody indicated essentially equivalent expression of WT CaSR as well as its variants on the cell surface (Fig.…”

“…By using our own computational algorithms, we previously identified five putative Ca 2ϩ -binding sites in the modeled CaSR ECD (Fig. 1) (25,26,45). Among these, site 1 is located in the hinge region between the two lobes in the Venus flytrap motif.…”

“…The cells were then alternately illuminated with 340-or 380-nm light, and the fluorescence at an emission wavelength 510 nm was recorded in real time as the concentration of extracellular Ca 2ϩ was increased in a stepwise manner in the presence or absence of 5 mM L-Phe. The ratio of the emitted fluorescence intensities resulting from excitation at both wavelengths was utilized as a surrogate for changes in [ (25). Briefly, CaSR-transfected HEK293 cells were grown on 13.5 ϫ 20-mm coverslips.…”

“…2ϩ -Binding Sites from a Model Structure-The structure of the extracellular domain of CaSR (residues 25-530) was modeled based on the crystal structure of metabotropic glutamate receptor 1 (mGluR1) (Protein Data Bank codes 1EWT, 1EWK, and 1ISR), and the potential Ca 2ϩ -binding sites in the CaSR ECD were predicted using MetalFinder (25,27). Prediction of the L-Phe-binding site was performed by AutoDock-Vina (28).…”

“…Nevertheless, we still lack a thorough understanding of the molecular mechanisms by which CaSR is activated by [Ca 2ϩ ] o and amino acids, which, in turn, regulate CaSR functional positive cooperativity. In addition, in a clinical setting, the molecular basis for the alterations in this cooperativity caused by disease-associated mutations is largely unknown because of the lack of knowledge of the structure of this receptor and its weak binding affinities for [Ca 2ϩ ] o and amino acids (13,15,25,26).…”