An immediate response of cells to stimulation by polypeptide growth-factors is an increase in the rate of uptake of essential macromolecular nutrients such as diferric transferrin and low density lipoproteins. This effect may be critically important in allowing an optimal mitogenic response of the cell to these growth factors. Recent results from our laboratory indicate that insulin and platelet-derived growth factor (PDGF) cause a rapid increase in the assembly of clathrin and of the plasma-membrane specific assembly polypeptide complex AP-2 (Corvera, S.(1990) J. Biol. Chem. 265, 2413-2416).This enhanced formation of endocytic structures is likely to underlie the increased uptake of nutrients observed in response to growth factor stimulation.The goal of this proposal is to answer the single specific question: What is the mechanism whereby polypeptide growth-factors induce de-novo clathrin-coated pit formation? Recent work in the field of signal transduction has led to the identification of a limited number of signaling events which in combination elicit the numerous cellular responses to polypeptide growth factors. We seek to gain insight on the molecular basis of clathrin-coated pit formation in-vivo by identifying the primary signaling event that leads to de-novo coated pit formation in response to growth-factor stimulation. These signaling events include the activation of PI-3 kinase and synthesis of lipids phosphorylated at the D3 position of the inositol ring, and the activation of rasGAP and p2lras-regulated pathways. Recent exiting preliminary results from our laboratory indicate that a significant amount of PI-3 kinase activity is found associated with clathrin-coated vesicles purified from growth factor-treated cells, supporting a possible role for this enzyme in growth-factor stimulated de-novo coated pit formation. Key elements in our strategy include the use of mutant constructs of the PDGF receptor each of which are defective in the stimulation of a specific primary signaling pathway. In addition, it has become clear that growth factors regulate specific proteins by inducing changes in their phosphorylation state. Cell lines generated in our laboratory that transiently or stably express an epitope-tagged construct of the clathrin beta light chain and that overexpress the alpha and beta subunits of casein kinase-2 will be employed to rigorously assess the role of phosphorylation in growth-factor stimulated coated-pit formation. These cells will also be employed to develop a sensitive and specific in-vitro assay system to biochemically measure coated pit assembly under conditions which accurately reflect the physiological requirements for coated pit formation in intact cells. The elucidation of the primary signalling pathway involved in growth factor induced de-novo coated pit formation will represent an important advancement in our understanding of the molecular mechanisms that mediate this process in intact cells.
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