Membrane boundaries define distinct structural and functional compartments between the eukaryotic cell and its environment, and within the cell itself. Movement of material and information between these compartments is mediated by vesicular transport, and protein coats are invariably associated with these processes. Multiple forms of coats are known, of which the clathrin-coated membrane is the paradigm. In addition to clathrin, these coats contain heterotetrameric complexes termed assembly, adaptor or associated proteins, or APs. The alpha subunit of the plasma membrane adaptor AP-2 has a regulatory high affinity polyphosphoinositide (PPI)-binding site. In the assembled coat, this site has greatest affinity for PI-3,4,5-P3, the product of PI-3 kinase that is recruited to clathrin coated pits by activated receptors and which has been implicated in regulation of receptor trafficking. The binding site has been mapped to a 75-residue region; it displays functional PPI binding similar to the intact protein. To test the hypothesis that interaction of AP-2 with PPIs, particularly PI-3,4,5-P3, regulates the properties of AP-2 and the processes in which it is involved, dominant inhibitory forms of mutant AP-2 alpha will be designed, generated and characterized, and subsequently expressed in intact cells. Under these conditions, as well as following pharmacologic and genetic manipulation of PI-3 kinase activity, alterations in coated membrane dynamics will be evaluated. Finally, the structure of the isolated prototypical PIP3 binding domain of the AP-2 alpha subunit will be studied using circular dichroism and nuclear magnetic resonance spectroscopy, x-ray crystallography and molecular modeling. In addressing the mode of regulation of coat function and vesicular transport in intact cells, this project focuses upon a central issue in cell biology with relevance for normal biological processes in all eukaryotic cells and derangements in disease.
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