Clathrin-coated vesicles mediate selective movement of receptors between cellular membranes. This process contributes to efficient endocytosis and subsequent recycling of receptors for nutrients and for proteins which need to be degraded. It is critical for rapid down-regulation of hormone and signalling receptors. Clathrin-coated vesicles also participate in transport of proteins from the trans-Golgi to lysosomes and secretory clathrin from the cytoplasm. Cytoplasmic tails of receptors are trapped by this polymerization process which also contributes to membrane vesiculation. Incorporation of receptors into a clathrin-coated vesicle is selective. This selectivity depends both on the signals provided by the receptor tails and the characteristics of the coated vesicles proteins that recognize the tails and nucleate clathrin assembly. The recognition- nucleation function has been attributed to the adaptor proteins which together with clathrin comprise the major protein constituents of the coated vesicle. The goal of this proposal is to define the molecular basis for control of clathrin-coated vesicle formation within the cell. This will involve characterizing the dynamics of known coated vesicle components (Aim 1), establishing whether any additional molecules are required for receptor binding and nucleation of clathrin assembly (Aim 2) and analyzing the recognition sites which influence the interactions of the coated vesicle components (Aim 3). Two experimental systems will be used. An in vitro assay for assembly of clathrin onto fragments of plasma membrane has been established. Assembly can be achieved using purified coat proteins from coated vesicles or cytosol. Monoclonal antibodies specific for coated vesicle components, purified components themselves, and soluble proteins containing receptor tail sequences will be used to define the molecular requirements for clathrin assembly. The second experimental approach will analyze the binding between coated vesicle components, independent of cell membranes. Assays for interaction between soluble receptor tails and adaptors and between adaptors and clathrin will be used to determine whether these interactions are direct or whether additional proteins are required. These assays will also be used to identify the subunits and domains of adaptors and clathrin involved in recognizing each other and receptor tails. Analysis of the molecular processes which control clathrin-mediated endocytosis will undoubtedly lead to a better understanding of how to manipulate this important pathway for macromolecule uptake. This will ultimately contribute to design of receptor-mediated therapeutics and to new approaches to vaccination, since this pathway is utilized for the uptake and processing of foreign antigens.
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