STUDIES ON A CALCIUM SENSING RECEPTOR

钙敏感受体的研究

• 批准号: 6105446
• 负责人: ALLEN M. SPIEGEL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6105446
• 关键词: G protein; calcium; calcium indicator; glutamate receptor; hormone regulation /control mechanism; human tissue; membrane proteins; monoclonal antibody; parathyroid gland; polymerase chain reaction; protein purification; protein structure function; receptor; receptor coupling; receptor expression; tissue /cell culture; transfection; western blottings

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.

布朗和他的同事们（Nature 1993）克隆了一个 钙敏感受体（CaR）是G 蛋白偶联受体超家族在结构上类似于 代谢型谷氨酸受体CaR以一种 有限的细胞类型，包括肾脏、脑、甲状腺C细胞， 和最显著的甲状旁腺细胞。CaR cDNA预测， 7个典型的G蛋白偶联受体的跨膜核心， 具有大的（约600个残基）N末端细胞外 结构域（ECD）。我们正在研究受体的几个方面， 结构和功能，以了解钙结合 导致G蛋白激活。我们已经筹集 多克隆抗血清的几个合成肽对应 在受体的大胞外结构域中的序列。这些 在蛋白质印迹上识别受体。单克隆抗体 针对2种最具免疫原性的ECD肽产生。这些 单克隆抗体已被广泛表征，并已证明 在免疫印迹、免疫细胞化学和流式细胞术中非常有用 受体的细胞计数研究。例如，我们已经能够 表明ECD的糖基化对于CaR是必需的， 在细胞表面表达。我们还定义了诱变， 200个残基的C-末端区域对受体的关键作用 表达和G蛋白偶联。详细的诱变研究 关键残基（如保守的半胱氨酸、推定的糖基化位点） 在ECD中，也已经进行了，并定义了半胱氨酸 和细胞中受体表达的关键糖基化位点 膜的我们已经成功地表达和净化了 幼儿发展。ECD的生化特征包括N末端 测序以确定信号肽切割位点， 碳水化合物含量，CD二级结构，以及 胰蛋白酶裂解。我们发现ECD是一种分子间的 二硫键连接的二聚体，其解释了 完整受体我们还成功地制造了一组 针对纯化的ECD的单克隆抗体， 对CaR的有趣的功能影响， 评估其表位，以进一步确定受体 结构/功能。最后，我们描述了功能性 在常染色体显性遗传受试者中发现的错义突变的影响 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，但在第七 跨膜结构域引起真正的组成性激活的 受体，即使在缺乏受体的截短受体的情况下， 幼儿发展。