STUDIES ON A CALCIUM SENSING RECEPTOR

Brown及其同事（Nature 1993）克隆了一种新的钙敏感受体（CaR），它是G蛋白偶联受体（GPCR）超家族的成员，特别是亚家族3，包括代谢型谷氨酸、GABA-B、味觉和推定的信息素受体。CaR在多种细胞类型中表达，包括肾、脑、甲状腺C细胞和最显著的甲状旁腺细胞，并且参与细胞外钙稳态。CaR cDNA预测GPCR典型的7个跨膜核心，但具有大的（约600个残基）N-末端胞外结构域（ECD）。我们正在研究受体的结构和功能，以了解钙与受体的结合如何导致G蛋白激活。我们已经提出了多克隆和单克隆抗体的合成肽对应于在ECD的受体的序列。这些抗体在受体的免疫印迹、免疫细胞化学和流式细胞术研究中已被证明是非常有用的。我们还成功地表达和纯化了ECD，并制备了抗纯化ECD的单克隆抗体。这些对CaR具有有趣的功能作用，并且正在评估其表位以帮助定义受体结构/功能。ECD的生化表征包括N-末端测序以确定信号肽切割位点、碳水化合物含量的定义、CD二级结构和胰蛋白酶切割位点。我们发现，ECD是一个分子间二硫键连接的二聚体，占完整的受体的二聚体的性质。ECD半胱氨酸的突变已经确定了哪些是受体表达所必需的，并且已经鉴定了负责受体二聚化的半胱氨酸。ECD的二硫键定位正在进行中，我们也已经确定了细胞表面对受体表达至关重要的糖基化位点。我们已经确定了常染色体显性遗传性低钙血症患者中错义突变的功能效应。大多数这样的突变会导致增加的受体对钙的敏感性，但在第7跨膜结构域的一个真正的组成型激活的受体，即使在一个截短的受体缺乏的ECD.We的背景下已经建模的ECD结构的基础上，其同源性的细菌周质结合蛋白，已知有一个双叶，捕蝇草结构，并正在测试这个模型使用诱变和生物化学方法。- G蛋白偶联受体;高钙血症;低钙血症;甲状旁腺激素;钙稳态

Brown and colleagues (Nature 1993) have cloned a novel calcium-sensing receptor (CaR) which is a member of the G protein-coupled receptor (GPCR) superfamily, specifically subfamily 3 which includes metabotropic glutamate, GABA-B, taste, and putative pheromone receptors. The CaR is expressed in a variety of cell types including kidney, brain, thyroid C cells, and most prominently parathyroid cells, and is involved in extracellular calcium homeostasis. The CaR cDNA predicts a 7 transmembrane core typical of GPCR but with a large (approximately 600 residue) N-terminal extracellular domain (ECD). We are studying the receptors structure and function in order to understand how calcium binding to the receptor leads to G protein activation. We have raised polyclonal and monoclonal antibodies to synthetic peptides corresponding to sequences in the ECD of the receptor. These antibodies have proved very useful in immunoblot, immunocytochemistry, and flow cytometry studies of the receptor. We have alsosucceeded in expressing and purifying the ECD,and in generating monoclonal antibodies against the purified ECD. These have interesting functional effects on the CaR, and are being evaluated for their epitopes to help define receptor structure/function. 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. Mutagenesis of ECD cysteines has defined which are essential for receptor expression and has identified the cysteine responsible for receptor dimerization. Disulfide mapping of the ECD is in progress.We have also identified the glycosylation sites critical for receptor expression at the cell surface. 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.We have modeled the ECD structure based on its homology to bacterial periplasmic binding proteins known to have a bilobed, venus flytrap structure, and are testing this model using mutagenesis and biochemical approaches. - G protein-coupled receptor; hypercalcemia; hypocalcemia; parathyroid hormone; calcium homeostasis