The longterm goal of this proposal is to understand the molecular basis of membrane functions regulated by clathrin-coated pits (CPs). The observation that rapid increases in CPs have been reported in response to some agents, e.g., EGF, provides an opportunity for dissecting mechanisms regulating CP assembly. To do this, cells plated on immobilized ligand will be studied by x-z confocal microscopy to determine whether receptor activation triggers only local CP formation or global assembly throughout the cell. The receptor domains responsible for CP induction will be determined using cells transfected with varied receptor constructs. To ascertain whether CP assembly takes place only at defined sites on the cell surface, the behavior of individual fluorescent-tagged LDL molecules bound to receptor on intact cells will be followed using confocal microscopy and computer-aided single particle tracking and by monitoring the behavior of fluorescent-tagged CP proteins microinjected into cells. The role of CPs in the desensitization and r e - s e nsitization of G-protein-coupled receptors will be studied by c h a r acterizing the interactions of clathrin with components of the sequestration machinery.
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