STRUCTURE/FUNCTION OF CARBOXYPEPTIDASE M

这一建议是对羧基肽酶M(CPM)研究的继续， 它在许多组织和细胞中都是膜结合的，可能调节多肽 细胞表面的激素活性。长期目标是 了解CPM的体内功能。将进行三个领域的调查 被强调。 A)黑石物理服务器的结构。野生型和突变型CPM(由3个素数产生 截短或定点突变)，在杆状病毒中表达 系统，将特征确定：1.G1n249是否为侧边- 链结合残基，介导其对Arg或Lys的特异性；2.如果 CPM的C-末端疏水区是一种信号 磷脂酰肌醇-葡聚糖(PI-G)锚定。生物化学研究 纯化的酶将确定Ser406是否是PI-G连接位点和 肾脏中是否存在CPM的另一种跨膜形式。 阐明cpm基因的结构将有助于识别可能的 监管区域。 B)物理物理模型的本土化。假设存在肾脏CPM。 在近端肾小管和远端肾单位，尤其是亚细胞 富含质膜上的小凹。CPM在中国的本土化 肾脏将通过免疫荧光光学显微镜和 电子显微镜免疫金染色法。肾的分布 用原位杂交法检测CPM基因的表达。使用生物化学 技术，将研究从MDCK细胞的小窝中富集CPM。 C)对CPM的监管。假设：释放CPM和其他 磷脂酰肌醇-葡聚糖(PI-G)通过一种 磷脂酶产生二甘油酯信号，使酶上调 通过蛋白激酶C的合成这一信号通路依赖于 质膜小凹的功能完整性，可能是 极化上皮细胞的顶端或基底侧域。MDCK细胞 将被用来确定：I.其他PI-G锚定蛋白是否 通过上调CPM的刺激来上调；2.刺激是否 和/或该反应是针对 MDCK细胞。3.是否需要小窝的完整性 上调黑石物理服务器。 这些研究应该有助于深入了解CPM的潜在功能 生理和病理生理过程。例如，在 肾脏CPM可能通过控制缓激肽的活性来调节盐分和 水的排泄。在炎症条件下，它可能会产生一种激动剂 (Des-Arg9-Bradykinin)对BI受体的作用 内毒素和白介素I通过从蛋白质或蛋白质中切割C末端精氨酸 多肽，它可能是一氧化氮的前体。监管 CPM和其他PI-G锚定蛋白可能与病理相关 导致它们增加释放到细胞外的条件 体液(如牛皮癣)。

This proposal is a continuation of studies on carboxypeptidase M (CPM), which is membrane-bound in many tissues and cells and may regulate peptide hormone activity at the cell surface. The long term objective is to understand the in vivo functions of CPM. Three areas of investigation will be emphasized. A) THE STRUCTURE OF CPM. Wild type and mutant CPM (generated by 3 prime truncation or site-directed mutagenesis), expressed in a baculovirus system, will be characterized to determine: 1. Whether G1n249 is the side- chain binding residue that mediates its specificity for Arg or Lys; 2. If the C-terminal hydrophobic region of CPM is a signal for phosphatidylinositol-glycan (PI-G) anchoring. Biochemical studies on purified enzyme will determine if Ser 406 is the PI-G attachment site and whether an alternate transmembrane form of CPM is present in kidney. Elucidation of the structure of the CPM gene will help identify possible regulatory regions. B) THE LOCALIZATION OF CPM. It is hypothesized that renal CPM is present in both the proximal tubules and the distal nephron and, subcellularly it is enriched in caveolae of the plasma membrane. The localization of CPM in kidney will be studied by both immunofluorescent light microscopy and immunogold staining with electron microscopy. The distribution of renal CPM mRNA will be determined by in situ hybridization. Using biochemical techniques, CPM enrichment in caveolae from MDCK cells will be studied. C) THE REGULATION OF CPM. Hypothesis: release of CPM and other phosphatidylinositol-glycan (PI-G) anchored proteins from the cell by a phospholipase generates a diglyceride signal that upregulates enzyme synthesis via a protein kinase C. This signalling pathway depends on the functional integrity of plasma membrane caveolae and may be specific to apical or basolateral domains in polarized epithelial cells. MDCK cells will be used to determine: I. whether other PI-G anchored protein are upregulated by stimuli that upregulate CPM; 2. whether the stimulus and/or the response is specific to the apical or basolateral domain in MDCK cells. 3. whether the integrity of caveolae is required for the upregulation of CPM. These studies should provide insight into potential functions of CPM in physiological and pathophysiological processes. For example, in the kidney CPM may control the activity of bradykinin to regulate salt and water excretion. In inflammatory conditions, it could generate an agonist (des-Arg9-bradykinin) for the BI receptor which is upregulated by endotoxin and interleukin I. By cleaving C-terminal Arg from proteins or peptides, it may provide the precursor of nitric oxide. The regulation of CPM and other PI-G anchored proteins may be relevant to pathological conditions that result in their increased release into extracellular fluids (e.g., psoriasis).