The venus's-flytrap and cysteine-rich domains of the human Ca2+ receptor are not linked by disulfide bonds

The extracellular N-terminal domain of the human Ca2+ receptor (hCaR) consi sts 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 tra nsmission from the VFT domain to the seven-transmembrane domain. The VFT do main contains 10 cysteines: two of them (Cys(129) and Cys(131)) were identi fied as involved in intermolecular disulfide bonds necessary for homodimeri zation, and six others (Cys(60)- Cys(101), Cys(358)-Cys(395), and Cys(437)- Cys(449)) are predicted to form three intramolecular disulfide bonds. The C ys-rich domain contains nine cysteines, the involvement of which in disulfi de bond formation has not been defined. In this work, we asked whether the remaining cysteines in the hCaR VFT, namely Cys(236) and Cys(482), form dis ulfide bond(s) with cysteines in the Cys-rich domain. We constructed mutant hCaRs with a unique tobacco etch virus (TEV) protease recognition site ins erted between the VFT domain and the Cys-rich domain. These mutant hCaRs re main 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 d isulfide bond between the VFT and the Cys-rich domains in the hCaR.