Multiple Ca2+-Binding Sites in the Extracellular Domain of the Ca2+-Sensing Receptor Corresponding to Cooperative Ca2+ Response
- 22 December 2008
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 48 (2), 388-398
- https://doi.org/10.1021/bi8014604
Abstract
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 noncontinuous Ca2+-binding sites in the ECD. We dissect the intact CaSR into three globular subdomains, each of which contains two to three 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 a location 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 the switching on and off the CaSR by the transition between its open inactive form and closed active form.Keywords
This publication has 40 references indexed in Scilit:
- Identification and Dissection of Ca2+-binding Sites in the Extracellular Domain of Ca2+-sensing ReceptorPublished by Elsevier BV ,2007
- Design of a Calcium-Binding Protein with Desired Structure in a Cell Adhesion MoleculeJournal of the American Chemical Society, 2005
- The Venus Fly Trap Domain of the Extracellular Ca2+-sensing Receptor Is Required for l-Amino Acid SensingPublished by Elsevier BV ,2004
- SWISS-MODEL: an automated protein homology-modeling serverNucleic Acids Research, 2003
- Autosomal Dominant Hypocalcemia Caused by a Novel Mutation in the Loop 2 Region of the Human Calcium Receptor Extracellular DomainJournal of Bone and Mineral Research, 2002
- Clinical disorders of extracellular calcium-sensing and the molecular biology of the calcium-sensing receptorAnnals of Medicine, 2002
- Comparative Protein Structure Modeling of Genes and GenomesAnnual Review of Biophysics and Biophysical Chemistry, 2000
- Quantitative endoproteinase GluC footprinting of cooperative Ca2+ binding to calmodulin: proteolytic susceptibility of e31 and e87 indicates interdomain interactionsBiochemistry, 1995
- Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroidNature, 1993
- Structure of calmodulin refined at 2.2 Å resolutionJournal of Molecular Biology, 1988