STUDIES ON A CALCIUM SENSING RECEPTOR

Brown和他的同事(自然1993)克隆了一个新的钙敏感受体(CAR)，它是G蛋白偶联受体(GPCR超家族)的成员，特别是亚家族3，包括代谢性谷氨酸、GABA-B、味觉和推定的信息素受体。CAR表达于多种细胞类型，包括肾、脑、甲状腺C细胞，尤其是甲状旁腺细胞，并参与细胞外钙稳态。CAR cDNA预测了一个典型的GPCR的7个跨膜核心，但具有一个大的(约600个残基)N端胞外域(ECD)。我们正在研究受体的结构和功能，以了解钙与受体结合是如何导致G蛋白激活的。我们已经提出了针对与受体ECD序列相对应的合成肽的多克隆和单克隆抗体。这些抗体在免疫印迹、免疫细胞化学和流式细胞术的受体研究中被证明非常有用。我们还成功地表达和纯化了ECD，并成功地制备了抗纯化的ECD的单抗。这些化合物对汽车有有趣的功能影响，正在对它们的表位进行评估，以帮助确定受体的结构/功能。ECD的生化特征包括N末端测序以确定信号肽的切割位置、碳水化合物含量的定义、CD的二级结构和胰酶切割的位置。我们发现ECD是一种分子间二硫键连接的二聚体，这解释了完整受体的二聚性。ECD半胱氨酸的突变定义了哪些是受体表达所必需的，并确定了负责受体二聚化的半胱氨酸(129和131)。ECD的二硫键作图使用纯化的蛋白质的裂解和随后的质量。光谱分析正在进行中。我们还确定了对细胞表面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低钙血症受试者中发现的错义突变的功能效应。大多数这样的突变会导致受体对钙的敏感性增加。我们根据其与细菌周质结合蛋白的同源性对ECD结构进行了建模，已知的是一种双叶“金捕蝇器”结构，并正在使用突变和生化方法测试这一模型。我们已经证明，ECD中一个由~115-139残基组成的假定环形成了二聚体界面，这对受体激活是至关重要的。这个环是自然发生的、激活突变的“热点”。我们还表明，在ECD末端的一个富含半胱氨酸的区域形成了一个单独的结构域，该结构域在ECD捕蝇器结构域的钙激活和CAR的7跨膜结构域的激活之间起着关键作用。未来的研究方向是详细阐明钙激活受体的机制。

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 receptor's 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 cysteines (129 and 131) responsible for receptor dimerization. Disulfide mapping of the ECD using cleavage of the purified protein followed by mass. spectrometry 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. 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. We have shown that a putative loop in the ECD comprising residues ~115-139 forms the dimer interface and is critical for receptor activation. This loop is a "hotspot" for naturally occurring, activating mutations. We have also shown that a cysteine-rich region at the end of the ECD forms a separate domain that plays a critical role in linking calcium activation of the ECD venus flytrap domain to activation of the 7 transmembrane domain of the CaR. Future studies are directed at elucidating in detail the mechanism of receptor activation by calcium.