This proposal focuses on the role of the protein dynamin in mediating the vesicle fusion reactions underlying endocytosis. Previous work has demonstrated that two forms of endocytosis are observed in secretory cells. The first, rapid endocytosis (RE), was first observed during cell capacitance recordings from adrenal chromaffin cells. This process has been shown in work from the P.I.'s laboratory to differ significantly from receptor-mediated endocytosis (RME) in its strict requirement for calcium, the involvement of calmodulin, and dependence on GTP hydrolysis. The P.I. proposes to extend his studies on the role of dynamin in RE processes by now concentrating on PC12 cells as a model system. This cultured cell system offers a number of advantages over primary cultures of adrenal chromaffin cells. In order to set the stage for these studies, the first specific aim proposes to complete the characterization of the dynamin isoforms which are expressed in PC12 cells. Preliminary analysis results suggests that both dynamin1 and dynamin2 are present in PC12 cells. Studies will then investigate which isoforms are required for RE and RME by the production of isoform specific antibodies. Profusion or icroinjection of these antibodies will be used in attempts to selectively block each of these processes. The second specific aim focuses on which domains of dynamin are critical for the regulation of RE. Here the focus will be on PH domains since RE appears to be sensitive to the peptides representing the dynamin1 PH domain. Two approaches are proposed. First, PH domains representing chimeras between this region of dynamin1 and dynamin2 will be expressed in E. coli, purified, injected into PC12 cells, and RE in injected cells assessed. Alternatively, PC12 cells will be prepared which ectopically express each of the dynamin PH domains or specific mutant forms using powerful retroviral infection procedures which allow strong induction of each protein following removal of tetracycline. A second focus of these studies will be on the role of the proline-rich, C-terminal domain of the individual dynamin isoforms in either RE or RME. Again, tetracycline induced retrovirovitral systems will be used to express various forms of dynamin containing mutations or truncations of this C-terminal domain and then effect of overexpression of these proteins on each of these processes investigated. A second approach will involve the use peptides representing individual domains within this region to competitively block either processes. Finally, the role of phosphorylation of dynamin in R or RME will be investigated by determining the status of dynamin phosphorylation during each process, assessing which kinases and phosphatases are responsible for the phosphorylation state of dynamin and determining whether phosphorylation plays a critical role in either RME or RE.
