Ray K, Hauschild B C, Steinbach P J, Goldsmith P K, Hauache O, Spiegel A M
Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1999 Sep 24;274(39):27642-50. doi: 10.1074/jbc.274.39.27642.
We analyzed the effect of substituting serine for each of the 19 cysteine residues within the amino-terminal extracellular domain of the human Ca(2+) receptor on cell surface expression and receptor dimerization. C129S, C131S, C437S, C449S, and C482S were similar to wild type receptor; the other 14 cysteine to serine mutants were retained intracellularly. Four of these, C60S, C101S, C358S and C395S, were unable to dimerize. A C129S/C131S double mutant failed to dimerize but was unique in that the monomeric form expressed at the cell surface. Substitution of a cysteine for serine 132 within the C129S/C131S mutant restored receptor dimerization. Mutation of residues Cys-129, Cys-131, and Ser-132, singly and in various combinations caused a left shift in Ca(2+) response compared with wild type receptor. These results identify cysteines 129 and 131 as critical in formation of intermolecular disulfide bond(s) responsible for receptor dimerization. In a "venus flytrap" model of the receptor extracellular domain, Cys-129 and Cys-131 are located within a region protruding from one lobe of the flytrap. We suggest that this region represents a dimer interface for the receptor and that mutation of residues within the interface causes important changes in Ca(2+) response of the receptor.
我们分析了将人Ca(2+)受体氨基末端细胞外结构域内的19个半胱氨酸残基中的每一个替换为丝氨酸对细胞表面表达和受体二聚化的影响。C129S、C131S、C437S、C449S和C482S与野生型受体相似；其他14个半胱氨酸到丝氨酸的突变体保留在细胞内。其中四个，C60S、C101S、C358S和C395S，无法二聚化。C129S/C131S双突变体无法二聚化，但独特之处在于其单体形式在细胞表面表达。在C129S/C131S突变体内将半胱氨酸替换为丝氨酸132可恢复受体二聚化。与野生型受体相比，单独或多种组合突变Cys-129、Cys-131和Ser-132残基会导致Ca(2+)反应向左偏移。这些结果表明半胱氨酸129和131对于形成负责受体二聚化的分子间二硫键至关重要。在受体细胞外结构域的“捕蝇草”模型中，Cys-129和Cys-131位于捕蝇草一个叶瓣突出的区域内。我们认为该区域代表受体的二聚化界面，并且该界面内残基的突变会导致受体Ca(2+)反应发生重要变化。
