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.
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