Fan G F, Ray K, Zhao X M, Goldsmith P K, Spiegel A M
Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
FEBS Lett. 1998 Oct 9;436(3):353-6. doi: 10.1016/s0014-5793(98)01165-x.
Mammalian calcium receptors (CaRs) share with the metabotropic glutamate receptors (mGluRs) the relative positions of 16 cysteine residues in the amino-terminal extracellular domain. To investigate the role of these cysteines, a series of mutants in the extracellular domain of the human CaR was prepared in which each of these 16 cysteine residues and three others not conserved in the mGluRs were replaced by serines. Wild-type and mutant CaR cDNAs were expressed in HEK-293 cells, and evaluated for expression and response to extracellular calcium. Mutation of three non-conserved cysteines and of two conserved cysteines produced proteins with near wild-type phenotype. In contrast, mutation of the other conserved cysteines gave proteins that showed drastic reduction in cell surface expression and/or failed to respond to calcium. We identified 14 cysteines essential for proper trafficking and function of the receptor, two of which may be involved in formation of a disulfide-linked dimer.
哺乳动物钙受体（CaRs）与代谢型谷氨酸受体（mGluRs）在氨基末端细胞外结构域中16个半胱氨酸残基的相对位置相同。为了研究这些半胱氨酸的作用，制备了一系列人CaR细胞外结构域的突变体，其中这16个半胱氨酸残基中的每一个以及mGluRs中不保守的另外三个半胱氨酸残基都被丝氨酸取代。野生型和突变型CaR cDNA在HEK-293细胞中表达，并评估其表达情况以及对细胞外钙的反应。三个非保守半胱氨酸和两个保守半胱氨酸的突变产生了具有接近野生型表型的蛋白质。相比之下，其他保守半胱氨酸的突变产生的蛋白质在细胞表面表达上大幅降低和/或对钙无反应。我们确定了14个对于受体正常转运和功能至关重要的半胱氨酸，其中两个可能参与二硫键连接的二聚体的形成。
