(Taken from application) This is a proposal to study the molecular mechanisms by which hepatocytes endocytose and process growth factors. It is well known that hepatocytes internalize multiple trophic factors which have profound effects on growth and differentiation during liver development and regeneration. While many of these factors are known to be sequestered within clathrin-coated pits, the mechanisms by which these receptor bearing pits are severed from the plasma membrane and targeted to the appropriate cytoplasmic compartment for degradation or recycling are unclear. Furthermore. how this important cellular process is regulated is totally undefined. In this study we focus on the role of the large GTPase, dynamin, which was originally predicted to regulate endocytic function in neurons. Through mutational analysis in fruit flies and cultured mammalian cells, dynamin has been implicated in severing vesicles from the plasma membrane during receptor-mediated endocytosis and membrane retrieval. In addition, dynamin has been shown to bind to microtubules, phospholipids, and several signaling molecules which possess SH3 domains. Thus. dynamin is believed to regulate endocytic processes through both mechanical and signaling functions. Recently, we have cloned and sequenced genes from rat liver encoding a novel dynamin (dynamin2) and a dynamin- like protein (p85-DLP). The identification of these novel hepatic dynamin genes provides strong support for the CENTRAL HYPOTHESIS of this proposal that multiple members of the dynamin family are expressed in the liver and regulate distinct endocytic and vesicular trafficking processes in the mature and developing hepatocyte. To pursue this hypothesis further, we will conduct novel molecular, biochemical, and morphological studies described in a single SPECIFIC AIM.
This aim i s focused on defining: first, the number of dynamin isoforms and related proteins in the hepatocyte; second, the expression of the dynamin family members during liver development; and third, the hepatocellular distribution of the dynamins during vesicle trafficking. The completion of this aim is crucial before mechanistic studies on the function of dynamin in the hepatocyte can be initiated. Based on our publication record and experimental expertise in hepatocyte vesicle trafficking and dynamin enzymes, we are confident that this proposal will lend important insight into how these cells regulate the internalization of growth factors and other endocytosed ligands.
