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 Ca(2+) 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 (Cys(129) and Cys(131)) were identified as involved in intermolecular disulfide bonds necessary for homodimerization, 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 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 Cys(236) and Cys(482), 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.
Hu
J
J
Molecular Pathophysiology Section, NIDCD, National Institutes of Health, Bethesda, Maryland 20892, USA. jianxinh@intra.niddk.nih.gov
Reyes-Cruz
G
G
Goldsmith
P K
PK
Spiegel
A M
AM
D000595:Amino Acid Sequence; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry*; D019281:Dimerization; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D014162:Transfection; D014633:Valine / Q000737:chemistry
D002135:Calcium-Binding Proteins; D004220:Disulfides; D021382:Protein Sorting Signals; D018698:Glutamic Acid; D010450:Endopeptidases; C099488:TEV protease; D014633:Valine; D003545:Cysteine