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Originally published In Press as doi:10.1074/jbc.C000865200 on January 19, 2001

J. Biol. Chem., Vol. 276, Issue 10, 6901-6904, March 9, 2001
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ACCELERATED PUBLICATION
The Venus's-flytrap and Cysteine-rich Domains of the Human Ca2+ Receptor Are Not Linked by Disulfide Bonds*

Jianxin HuDagger , Guadalupe Reyes-Cruz, Paul K. Goldsmith, and Allen M. Spiegel

From the Molecular Pathophysiology Section, NIDCD, National Institutes of Health, Bethesda, Maryland 20892

The extracellular N-terminal domain of the human Ca2+ receptor (hCaR) consists of a Venus's-flytrap (VFT) domain and a cysteine-rich (Cys-rich) domain. We have shown earlier that the Cys-rich domain is critical for signal transmission from the VFT domain to the seven-transmembrane domain. The VFT domain contains 10 cysteines: two of them (Cys129 and Cys131) were identified as involved in intermolecular disulfide bonds necessary for homodimerization, and six others (Cys60-Cys101, Cys358-Cys395, and Cys437-Cys449) are predicted to form three intramolecular disulfide bonds. The Cys-rich domain contains nine cysteines, the involvement of which in disulfide bond formation has not been defined. In this work, we asked whether the remaining cysteines in the hCaR VFT, namely Cys236 and Cys482, form disulfide bond(s) with cysteines in the Cys-rich domain. We constructed mutant hCaRs with a unique tobacco etch virus (TEV) protease recognition site inserted between the VFT domain and the Cys-rich domain. These mutant hCaRs remain fully functional compared with the wild type hCaR. After TEV protease digestion of the mutant hCaR proteins, dimers of the VFT were identified on Western blot under nonreducing conditions. We concluded that there is no disulfide bond between the VFT and the Cys-rich domains in the hCaR.

* 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.

Dagger To whom correspondence should be addressed: NIDCD, National Institutes of Health, Bldg. 10, Rm. 8C-101, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-9212; Fax: 301-402-0374; E-mail: jianxinh@intra.niddk.nih.gov.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.




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