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J. Biol. Chem., Vol. 277, Issue 51, 49691-49699, December 20, 2002
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From the Department of Biology, Syracuse University,
Syracuse, New York 13244
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+i
oscillations 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.
Calcium Sensing Receptor Activation by a Calcimimetic Suggests a
Link between Cooperativity and Intracellular Calcium Oscillations*
,
*
This work was supported in part by National Institutes of
Health Grant GM 58578 and funds from Novartis Pharma AG (to G. E. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported in part by a fellowship from the Bundesministerium fuer
Bildung und Forschung and Interdisciplinary Centre for Clinical Research (IZKF) at the University of Leipzig (IZKF Leipzig Stipendium 01KS9504).
§
Current address: Division of Comparative Medicine, Johns Hopkins
University School of Medicine, Baltimore, MD 21205.
¶
To whom correspondence should be addressed: 122 Lyman Hall,
108 College Place, Syracuse, NY 13244. Tel.: 315-443-2964; Fax: 315-443-2510; E-mail: gebreitw@syr.edu.
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