REGULATION OF AP ADAPTOR FUNCTION

已知存在胞吐作用和胞吞作用的协调控制， 细胞保持相对恒定的表面积， 泡状膜向质膜的添加率。 的 抑制G蛋白活化的途径， 磷脂酰肌醇-4，5-二磷酸（PIP 2）裂解和钙 动员，后者参与刺激分泌， 好文件。 然而，人们对这种机制知之甚少， 内吞作用与分泌速率紧密相关。 检索 膜是通过受体介导的内吞作用进行的， 小坑和小泡 质膜的结构蛋白 结构包括网格蛋白和AP-2、装配衔接子或相关 蛋白 该实验室的研究最近发现了一种离散的 AP-2 α亚基上的高亲和力（Kd-10-8 M）结合位点， 在脂质双层的情况下，识别聚磷酸肌醇（PPI）。 该位点的占据抑制AP-1对网格蛋白的结合和被膜的组装。 2，表明这很可能是一个监管网站的主要 生理意义 这一发现以及 这些文献表明， 质膜伴随着PIP 2裂解的增加， 两个工作假设：1）PIP 2（主要位于或 仅在质膜的细胞质小叶中） 有助于AP-2在血浆中的募集或保留 2）PIP 2调节细胞膜的网格蛋白外壳组装活性; 细胞膜上的AP-2。 这里提出的实验设计 为了定位PPI在AP-2a亚基上的结合位点， 放射性标记的光亲和探针、肽分级分离和抗肽 抗体的 随后的定点诱变和体外 转录/翻译实验将鉴定aA的突变形式 其保留网格蛋白结合活性但缺乏功能性PPI位点。 突变型和野生型蛋白在哺乳动物细胞中的瞬时表达 然后将用于确定PPI研究中心在招募中的作用 AP-2的质膜，在包衣小窝组件和受体- 介导的内吞作用。 为了评估细胞中PIP 2和PIP 3之间的连接， 卵裂和质膜包被纹孔形成，定量时间- 组装网格蛋白水平的过程和剂量反应变化 将在小鼠中测量PIP 2和PIP 3的浓度 由免疫复合物表面刺激的腹膜巨噬细胞和由 血小板活化因子 类似的测量将在 完整和透化的大鼠肥大细胞， 选择性激活胞吐、PIP 2裂解或两者。 这些研究 对我们理解基本过程有着广泛的影响 调节真核细胞中的膜动力学，并用于 依赖于受体介导的潜在治疗测量 内吞作用用于将试剂递送至细胞内部。

Coordinate control of exocytosis and endocytosis is known to exist, as cells maintain a relatively constant surface area despite large variation in the rates of vesicular membrane addition tot he plasma membrane. The pathways of against activation of G-proteins that result in phosphatidylinositol-4,5-bisphosphate (PIP2) cleavage and calcium mobilization, and the latter's involvement in stimulated secretion are well document. However little is known about the mechanism by which endocytosis is tightly coupled to the rate of secretion. Retrieval of membrane is carried out by receptor-mediated endocytosis through coated pits and vesicles. The structural proteins of the plasma membrane coat structure include clathrin and AP-2, an assembly adaptor or associated protein. Studies in this laboratory have recently identified a discrete high affinity (Kd-10-8 M) binding site on the alpha subunit of AP-2 that recognized polyphosphoinositols (PPIs) in the context of a lipid bilayer. Occupancy of this site inhibits clathrin binding and coat assembly by AP- 2, indicating that this is likely to be a regulatory site of major physiological significance. This finding, and observations in the literature which indicate that increases in coated membrane assembly at the plasma membrane are accompanied by increases in PIP2 cleavage, lead to two working hypotheses: 1) that PIP2 (located predominantly or exclusively in the cytoplasmic leaflet of the plasma membrane) contributes to the recruitment or retention of AP-2 at the plasma membrane; 2) that PIP2 regulates the clathrin coat assembly activity of AP-2 at the plasma membrane. The experiments proposed here are designed to localize the site of PPI binding on the AP-2 a subunit using radiolabeled photoaffinity probes, peptide fractionation and anti-peptide antibodies. Subsequent site-specific mutagenesis and in vitro transcription/translation experiments will identify mutant forms of aA that retain clathrin binding activity but lack functional PPI sites. Transient expression of mutant and wild type proteins in mammalian cells will then be used to determine the role of the PPI site in recruitment of AP-2 to the plasma membrane, in coated pit assembly and in receptor- mediated endocytosis. To evaluate the linkage in cells between PIP2 cleavage and plasma membrane coated pit formation, quantitative time- course and dose-response changes in the levels of assembled clathrin coated membranes and of PIP2 and PIP3 will be measured in mouse peritoneal macrophages stimulated by an immune complex surface and by platelet activating factor. Similar measurements will be performed on intact and permeabilized rat mast cells under conditions that will selectively activate exocytosis, PIP2 cleavage, or both. These studies have broad implications for our understanding of basic processes regulating membrane dynamics in eukaryotic cells, and for development of potential therapeutic measurements that depend on receptor-mediated endocytosis for delivery of agents to the cell interior.