Under the auspices of this grant, we established that the GTPase dynamin plays a critical role in receptor-mediated endocytosis. We proposed the first working model for dynamin function that integrates molecular events leading to coated vesicle formation. In addition, we suggested a novel role for dynamin as a mechanochemical-GTPase. However, our new results establish that dynamin does not function solely as a 'molecular pinchase'. Instead, or in addition, our data suggests that dynamin plays a central role in regulating other components of the molecular machinery required for receptor-mediated endocytosis. We propose a multi- disciplinary approach to understand dynamic function and to use dynamin GTPase as a probe to identify its functionally relevant upstream and downstream partners that together mediate endocytic coated vesicle formation. To accomplish these objectives we propose four aims.
The First Aim will correlate each step in dynamin's GTPase cycle with structurally-defined intermediates in coated vesicle formation using in innovative multi-disciplinary approach that combines high resolution electron microscopy, molecular genetics and biochemistry.
The Specific Aim, describes the first cell-free assay for dynamin in its physiological context of receptor-mediated endocytosis. Functional analysis of wild type and mutant and mutant dynamic molecules and individual domains of dynamin will provide important new mechanistic information.
The Third Aim proposes three complementary approaches to identified new factors that, together with dynamin, function in mediating coated vesicle formation.
The Fourth Aim describes a structure/function analysis of the mechanisms governing dynamin self- assembly and intramolecular regulation of dynamin GTPase activity. It includes a highly feasible proposal to solve the structure, by NMR, of a 13 kD domain we have identified as an intramolecular GAP responsible for coordinating self-assembly with GTPase activation, as well as our efforts to crystallize domains of dynamin. Together, results from these studies will provide important new insight into the regulation of receptor-mediated endocytosis, a process critical for cell growth (through nutrient acquisition), cellular homeostasis (through control of serum composition), cell proliferation (through the regulation of activated signaling receptors) and immunity (through antigen presentation and immunoglobulin transport). Thus, results from these studies will impact many broad areas of human health and disease.
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