REGULATION OF AP ADAPTOR FUNCTION

已知存在胞吐和内吞的协调控制，如 尽管变化很大，细胞仍保持相对恒定的表面积 质膜加入囊泡膜的速率。这个 抗G蛋白激活的途径导致 磷脂酰肌醇-4，5-二磷酸(PIP2)裂解与钙 动员，以及后者参与刺激分泌是 很好的记录。然而，人们对这一机制知之甚少。 内吞作用与分泌速度密切相关。检索 膜是由受体介导的内吞作用通过涂层进行的 凹坑和水泡。质膜被膜的结构蛋白 结构包括笼状蛋白和AP-2、组件接头或相关联 蛋白。这个实验室的研究最近发现了一种离散的 AP-2α亚基上的高亲和力(KD-10-8M)结合位点 在脂质双层的背景下识别的多磷肌醇(PPI)。 这一位置的占据抑制了AP-1与笼蛋白的结合和被膜的组装。 2，表明这很可能是一个主要的监管网站 生理意义。这一发现，以及在 文献表明，涂层膜组件在 质膜伴随着PIP2裂解的增加，铅 根据两个工作假设：1)PIP2(主要位于或 仅在质膜的细胞质小叶中) 有助于AP-2在血浆中的募集或滞留 2)PIP2调节细胞膜蛋白的组装活性。 AP-2在质膜上的表达。这里提出的实验是设计的 要定位AP-2α亚基上的PPI结合部位，请使用 放射性标记光亲和探针、多肽分级和抗多肽 抗体。随后的定点突变和体外试验 转录/翻译实验将确定AA的突变形式 保留了笼状蛋白结合活性但缺乏功能性PPI结合位点。 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网站在招聘中的作用 AP-2在质膜、包被的凹坑组装和受体中的结合 介导的内吞作用。评价PIP2在细胞内的连接 卵裂和质膜被膜坑形成，定量时间- 组装网状蛋白水平的过程和剂量-反应变化 包被膜和PIP2和PIP3将在小鼠身上进行测量 免疫复合体表面和免疫复合物刺激的腹腔巨噬细胞 血小板活化因子。类似的测量将在 完整和通透性的大鼠肥大细胞在将 选择性地激活胞吐作用、PIP2裂解或两者兼而有之。这些研究 对我们对基本过程的理解有广泛的影响 调控真核细胞的膜动力学，并为发展 依赖于受体介导的潜在治疗措施 将药物输送到细胞内部的内吞作用。

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.