The rate of glucose transport can be regulated in a wide variety of cell types by oncogenic transformation, by treatment with mitogenic or anabolic hormones or by glucose starvation. Transformation of rat or chicken cells by the src oncogene results in an increase in the amount of the transporter protein in the cell. In chicken embryo fibroblasts, we have found that pp60 v-src causes a decrease in the rate of degradation of the glucose transported protein with little if any change in transported biosynthesis or mRNA levels. Surprisingly, expression of pp60 v-src in rat fibroblasts had the opposite result, with little effect on degradation of the transporter, but a pronounced increase in biosynthesis of the protein and expression of the gene. The overall aim of this proposal is to study the mechanisms whereby pp60 v-src and other factors regulate the glucose transporter. Since regulation of the stability of a cellular protein is a novel mechanism of action for a cytoplasmic oncogene, and an unusual control point for cellular regulation, considerable effort will go into analyzing the mechanism whereby pp60 v-src stabilizes the transporter in chicken embryo fibroblasts. We propose two parallel investigations, the first to analyze the molecular biology of the avian glucose transporter, with a particular view to identifying structural motifs necessary for src-induced stabilization; the second to examine the cellular and biochemical mechanisms involved in transporter stabilization. 1. Molecular analysis of glucose transporter protein stabilization and gene expression. (as) Clone and sequence the cDNA for the chicken fibroblast glucose transporter, to search for sequence differences between it and mammalian transporters and thus for potential """"""""stabilization signal sequences."""""""" (b) Construct vectors to express heterologous transporters in normal or src-transformed cells, to determine whether transporter stabilization is a consequence of the transporter sequence per se or of the cellular environment. (c) Construct chimeric transporters or perform mutagenesis experiments to localize the stabilization signal sequence. (d) Examine various tissues, cell types and physiological conditions to identify transporter isoforms which display differing regulatory behavior. (e) Utilizing avian fibroblasts, determine whether regulatory agents other than src utilize different regulatory modalities and regulate different isoforms. 2. Cellular and biochemical analysis of changes in glucose transporter stability induced by pp60 src. (a) Determine whether src expression affects the stability of other cellular proteins in addition to the transporter. (b) Analyze the pathway of transporter degradation. (c) Examine the effects of src expression on the intracellular distribution of the transporter. (d) Test for complex formation between the transporter and pp60 v-src or other cellular proteins.
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