STRUCTURE AND FUNCTION OF CARBOXYPEPTIDASE M

这一建议是羧肽酶M（CPM）研究的继续， 在许多组织和细胞中与膜结合， 细胞表面的激素活性。 长期目标是 了解CPM的体内功能。三个调查领域将 被强调。 一、CPM的结构。 野生型和突变型CPM（由3个引物产生 截短或定点诱变），在杆状病毒中表达 系统，将被表征以确定：1. G1n249是否是侧边- 介导其对Arg或Lys的特异性的链结合残基; 2.如果 CPM C-末端疏水区是 磷脂酰肌醇-聚糖（PI-G）锚定。 生化研究 纯化的酶将确定Ser 406是否是PI-G附着位点， 肾脏中是否存在CPM的交替跨膜形式。 CPM基因结构的阐明将有助于确定可能的 监管区域。 B）CPM的本地化。 假设存在肾脏CPM 在近端小管和远端肾单位中， 在质膜的小窝中富集。CPM的国产化 将通过免疫荧光显微镜和 免疫金染色电镜观察。 肾脏的分布 CPM mRNA将通过原位杂交测定。 使用生化 技术，CPM富集在小窝从MDCK细胞将进行研究。 C）CPM的监管。 假设：释放CPM和其他 磷脂酰肌醇-聚糖（PI-G）锚定蛋白从细胞中通过 磷脂酶产生甘油二酯信号， 通过蛋白激酶C合成。 这种信号通路依赖于 质膜小窝的功能完整性，可能是特定于 极化上皮细胞的顶端或基底外侧区域。 mdck细胞 将用于确定：I.其他PI-G锚定蛋白是否 通过上调CPM的刺激上调; 2.无论是刺激措施 和/或所述反应特异于顶侧或基底侧结构域， MDCK细胞。 3.是否需要完整的小窝 CPM的上调。 这些研究应该提供深入了解CPM的潜在功能， 生理和病理生理过程。 比如在 肾CPM可能通过控制缓激肽的活性来调节盐和 水排泄 在炎症情况下，它可以产生激动剂 （des-Arg9-缓激肽）的BI受体，其被 内毒素和白细胞介素I。 通过从蛋白质上切割C-末端Arg， 肽，它可以提供一氧化氮的前体。 调控 CPM和其他PI-G锚定蛋白可能与病理性 导致它们增加释放到细胞外的条件 流体（例如，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).