STUDIES ON A CALCIUM SENSING RECEPTOR

Brown和他的同事(《自然》1993)克隆了一种 新的钙敏感受体(CAR)是G 蛋白质偶联受体超家族，结构类似于 代谢性谷氨酸受体。这辆车用一个 有限的细胞类型，包括肾、脑、甲状腺C细胞， 最突出的是甲状旁腺细胞。CAR基因预测了一种 7跨膜核心典型的G蛋白偶联受体，但 具有大的(约600个残基)N-末端胞外 域(ECD)。我们正在研究受体的几个方面 为了了解钙离子结合的结构和功能 结合受体导致G蛋白激活。我们已经筹集了 几种合成多肽的多克隆抗血清 在受体的大胞外域中的序列。这些 识别蛋白质印迹上的受体。单克隆抗体 是针对免疫原性最强的两个ECD多肽而提出的。这些 单克隆体已经被广泛地描述并证明 在免疫印迹、免疫细胞化学和Flow中非常有用 受体的细胞学研究。例如，我们已经能够 以证明ECD的糖基化对CAR是必不可少的 在细胞表面表达。我们也通过突变来定义， 受体关键的200个残基C-末端区域 表达和G蛋白偶联。详细的诱变研究 关键残基(例如保守的半胱氨酸、假定的糖基化位点) 在ECD中也进行了，并定义了半胱氨酸 和糖基化位点对细胞中受体的表达至关重要 薄膜。我们已经成功地表达和纯化了 ECD。含有N-末端的ECD的生化特性 用于确定信号肽切割位置的测序，定义 碳水化合物含量，Cd的二级结构，以及 胰酶卵裂。我们发现ECD是一种分子间分子。 二硫键连接的二聚体，解释了二聚体的性质 完整的受体。我们还成功地产生了一组 抗纯化的ECD的单抗具有 汽车上有趣的功能效果，以及正在被 对它们的表位进行评估，以进一步确定受体 结构/功能。最后，我们描述了泛函 常染色体错义突变对受试者的影响 显性低钙血症。大多数这样的突变会导致 受体对钙的敏感性，但在第7位 跨膜结构域导致真正的结构性激活 受体，即使在一个被截断的受体缺乏 ECD。

Brown and colleagues (Nature 1993) have cloned a novel calcium-sensing receptor (CaR) which is a member of the G protein-coupled receptor superfamily similar in structure to the metabotropic glutamate receptors. The CaR is expressed in a limited range of cell types including kidney, brain, thyroid C cells, and most prominently parathyroid cells. The CaR cDNA predicts a 7 transmembrane core typical of G protein-coupled receptors but with a large (approximately 600 residue) N-terminal extracellular domain (ECD). We are studying several aspects of the receptor's structure and function in order to understand how calcium binding to the receptor leads to G protein activation. We have raised polyclonal antisera to several synthetic peptides corresponding to sequences in the large extracellular domain of the receptor. These recognize the receptor on western blots. Monoclonal antibodies were raised against the 2 most immunogenic ECD peptides. These monoclonals have been extensively characterized and have proved very useful in immunoblot, immunocytochemistry, and flow cytometry studies of the receptor. For example we have been able to show that glycosylation of the ECD is essential for CaR expression at the cell surface. We have also defined by mutagenesis, regions of the 200 residue C-terminus critical for receptor expression and G protein coupling. Detailed mutagenesis studies of key residues (e.g. conserved cysteines, putative glycosylation sites) in the ECD have also been performed, and have defined cysteines and glycosylation sites critical for receptor expression at the cell membrane. We have succeeded in expressing and purifying the ECD. Biochemical characterization of the ECD included N-terminal sequencing to define site of signal peptide cleavage, definition of carbohydrate content, secondary structure by CD, and sites of tryptic cleavage. We found that the ECD is an intermolecular disulfide-linked dimer that accounts for the dimeric nature of the intact receptor. We have also succeeded in generating a battery of monoclonal antibodies against the purified ECD which have interesting functional effects on the CaR, and which are being evaluated for their epitopes in a further effort to define receptor structure/function. Finally, we have characterized the functional effects of missense mutations identified in subjects with autosomal dominant hypocalcemia. Most such mutations cause increased sensitivity of the receptor to calcium, but one in the 7th transmembrane domain causes true constitutive activation of the receptor, even in the context of a truncated receptor lacking the ECD.