Multiple Ca(2+)-binding sites in the extracellular domain of the Ca(2+)-sensing receptor corresponding to cooperative Ca(2+) response.
A small change in the extracellular Ca(2+) concentration ([Ca(2+)](o)) integrates cell signaling responses in multiple cellular and tissue networks and functions via activation of Ca(2+)-sensing receptors (CaSR). Mainly through binding of Ca(2+) to the large extracellular domain (ECD) of the dimeric CaSR, intracellular Ca(2+) responses are highly cooperative with an apparent Hill coefficient ranging from 2 to 4. We have previously reported the identification of two continuous putative Ca(2+)-binding sites by grafting CaSR-derived, Ca(2+)-binding peptides to a scaffold protein, CD2, that does not bind Ca(2+). In this paper, we predict more potential noncontinuous Ca(2+)-binding sites in the ECD. We dissect the intact CaSR into three globular subdomains, each of which contains two to three predicted Ca(2+)-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. Tb(3+) luminescence energy transfer, ANS fluorescence, and NMR studies show biphasic metal-binding components and Ca(2+)-dependent conformational changes in these subdomains. Removing the predicted Ca(2+)-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.
Huang
Yun
Y
Department of Chemistry, Center of Drug Design and Advanced Biotechnology, Georgia State University, Atlanta, Georgia 30303, USA.
Zhou
Yubin
Y
Castiblanco
Adriana
A
Yang
Wei
W
Brown
Edward M
EM
Yang
Jenny J
JJ
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D007477:Ions / Q000378:metabolism*; D051379:Mice; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D044169:Receptors, Calcium-Sensing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013816:Thermodynamics
C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D007477:Ions; D044169:Receptors, Calcium-Sensing; D011994:Recombinant Proteins; D002118:Calcium