The long-term goal of this research program is a description at the molecular level of the structure and function of clathrin (CL) coated membranes in cells. Efforts in this laboratory have identified two discrete protein complexes, that are derived from coated vesicles, and which contain 100-110-kD, 47-50-kD, and 16- 19-kD components. These are termed """"""""assembly proteins"""""""" or AP by virtue of their ability to promote CL assembly into coat structures under physiological conditions. The involvement of the CL coated membrane in numerous cellular processes involving membrane dynamics, particularly receptor-mediated endocytosis, has been well documented morphologically. There is good reason to believe that the AP may perform important functions in these processes, possibly by driving CL assembly and subsequent membrane invagination; by bridging receptor proteins and the clathrin shell, resulting in receptor concentration in coated pit regions; or both. Experiments proposed here focus upon the structure and relationship between the two AP, denoted AP-1 and AP-2, their interactions with CL and with cell surface receptor proteins. The subunit and protomeric molecular weight of AP-1, and component assembly and phosphorylation domains that have been identified in AP-1 and AP-2, will be determined by low angle laser light scattering or analytical ultracentrifugation; secondary structure of these preparations will be studied by circular dichroism. Domains of the AP and CL molecules that are involved in their association will be identified using quantitative binding assays. In collaboration with J. Heuser (Washington Univ.) the AP, their functional domains, and their interaction with CL, will be studied by quick-freeze, deep-etch electron microscopy. Conditions will be identified that stabilize CL-cell surface receptor interactions in """"""""cytoskeletons"""""""". Using these conditions, direct AP interactions with radioloabeled receptors derived from cultured cells, or with other proteins that act to bridge the AP and receptors, will be detected and studied by co-assembly with carrier CL and AP. These studies will help to define the role of the CL coat structure in endocytosis, knowledge with direct application to our understanding of how hormones, growth factors and nutrients, as well as potential therapeutic agents such as immunotoxins, interact with and are internalized by cells.
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