The long-term objective of the proposal is to further elucidate the molecular mechanism of regulated hormone release. Hormone release requires sequential steps of granule positioning at the cell cortex, docking to the plasma membrane, priming and fusion. Although many key components of this process have been identified, progress has been hindered because there have been no available in vitro assays that can measure docking of granules to the plasma membrane as a separate event. We have designed a novel in vitro assay that measures docking of granules to the plasma membrane. As one of the first results of the in vitro assay, it was found that docking is stimulated by Ca2+ and requires an interaction between SNAP-25 and Synaptotagmin 1. It is also known that other Synaptotagmins function in hormone release. We propose to determine, by using the in vitro assay in combination with immunoprecipitation experiments, whether the interaction of multiple Synaptotagmins with SNAP-25 is necessary for granule docking. SNAP-25 is expressed in neurons and endocrine cells, while Syndet/SNAP-23 is ubiquitous. Both SNAP-25 and Syndet/SNAP-23 are predominantly localized at the plasma membrane. The molecular mechanisms underlying differences in docking of granules and other vesicles of the regulated and constitutive pathway to the plasma membrane are unknown. By using the in vitro assay, we will determine the specifics of SNAP-25 and Sydet/SNAP-23 dependent docking. We will also study whether low molecular weight GTPases, or Rab3 proteins, and the putative Rab3 protein effectors, Rabphilin and Noc2, control the docking step. Furthermore, we will test the hypothesis that the Rab3-Rabphilin functions to position granules within the cortical actin by using immunofluorescence and electron microscopy analysis of transfected PC 12 cells. The molecular events involved in the recruitment of dense core granules near the cell surface are largely unknown. This study of hormone release will help elucidate possible defects underlying endocrine diseases such as Diabetes Mellitus and Cushing syndrome.

R01DK053293

1998-04-01

2008-03-31