Calcium Sensing Receptor Activation by a Calcimimetic Suggests a Link between Cooperativity and Intracellular Calcium Oscillations

• Published in: The Journal of biological chemistry Vol. 277; no. 51; pp. 49691 - 49699
• Main Authors: Miedlich, Susanne, Gama, Lucio, Breitwieser, Gerda E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.12.2002; American Society for Biochemistry and Molecular Biology
• Subjects: Allosteric Site; Aniline Compounds - pharmacology; Arginine - chemistry; Binding Sites; Calcium - agonists; Calcium - metabolism; Cell Line; Dimerization; Dose-Response Relationship, Drug; Genetic Linkage; Histidine - chemistry; Humans; Phenethylamines; Plasmids - metabolism; Point Mutation; Propylamines; Protein Binding; Protein Structure, Tertiary; Receptors, Calcium-Sensing; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - metabolism; Spectrometry, Fluorescence; Thapsigargin - pharmacology; Time Factors; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M205578200

Activation of the calcium sensing receptor (CaR) by small increments in extracellular calcium (Ca2+e) induces intracellular calcium (Ca2+i) oscillations that are dependent on thapsigargin-sensitive intracellular calcium stores. Phenylalkylamines such as NPS R-568 are allosteric modulators (calcimimetics) that activate CaR by increasing the apparent affinity of the receptor for calcium. We determined, by fluorescence imaging with fura-2, whether the calcimimetic NPS R-568 could activate Ca2+ioscillations in HEK-293 cells expressing human CaR. NPS R-568 was more potent than Ca2+e at eliciting Ca2+i oscillations, particularly at low [Ca2+]e (as low as 0.1 mm). The oscillation frequencies elicited by NPS R-568 varied over a 2-fold range from peak to peak intervals of 60–70 to 30–45 s, depending upon the concentrations of both Ca2+e and NPS R-568. Finally, NPS R-568 induced sustained (>15 min after drug removal) Ca2+i oscillations, suggesting slow release of the drug from its binding site. We exploited the potency of NPS R-568 for eliciting Ca2+i oscillations for structural studies. Truncation of the CaR carboxyl terminus from 1077 to 886 amino acids had no effect on the ability of Ca2+ or NPS R-568 to induce Ca2+i oscillations, but further truncation (to 868 amino acids) eliminated both highly cooperative Ca2+-dependent activation and regular Ca2+i oscillations. Alanine scanning within the amino acid sequence from Arg873 to His879 reveals a linkage between the cooperativity for Ca2+-dependent activation and establishment and maintenance of intracellular Ca2+ oscillations. The amino acid residues critical to both functions of CaR may contribute to interactions with either G proteins or between CaR monomers within the functional dimer.