MECHANISM OF ACTION OF DYNAMIN

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Dynamin is a 100 kD GTPase identified in this laboratory which is involved in the initial stages of endocytosis. Mutations in dynamin and shibire,its Drosophila homologue, block the formation of coated and non-coated vesicles at the plasma membrane and the internalization of cell surface ligands and receptors. Mutations in shibire also block the reformation of synaptic vesicles at neuromuscular junctions and interneuronal synapses once the vesicles have discharged their contents. Dynamin has been found to interact with a number of macromolecular factors, most recently the SH3 domains of proteins involved in signal transduction, but which of these factors are involved in dynamin function in the cell remains uncertain. The long-term objective of this project is to understand the mechanism of action of dynamin, and, in particular, the steps in its GTPase cycle. To address this problem, we will take advantage of our extensive mutational analysis of the protein, and we will use a variety of molecular genetic, biochemical, ultrastructural and genetic approaches. We will seek to identify novel dynamin-interacting proteins and to characterize known dynamin interactions further. We will determine the dependence of dynamin interactions on the state of the guanine nucleotide in the dynamin active site, to help define the functional cycle of the protein. We will determine whether dynamin self-associates in the cell, and whether self- association activates the dynamin GTPase. We will characterize the association of dynamin with coated pits and other endocytic precursor structures by ultrastructural and biochemical means. Finally, we will attempt to identify a true dynamin homologue in yeast. An understanding of the dynamin functional cycle has a number of medical implications. This knowledge may ultimately lead to methods to control the entry of viruses, LDL, and other potentially deleterious agents into the cell. it may also provide a means to control the lifetime of activated growth factor receptors on the cell surface, and, therefore, a means to control normal and abnormal cell growth. Finally, because temperature-sensitive shibire mutations mimic certain conditional paralytic conditions of humans, an understanding of how dynamin functions could be of value in understanding and controlling these conditions.