Peptide hormones bind to and activate cell surface receptor tyrosine kinases (RTKs) that become auto-phosphorylated. Signal transducing molecules dock to phosphotyrosine residues within a specific sequence context through SH (src homology)-2 domains to initiate intracellular signaling. However, due to overlap of signaling molecules recruited to individual RTKs, these protein-protein interactions cannot, on their own, account either for the diversity of cellular responses to distinct stimuli or for cell-type specific responses to the same stimulus. Concomitant to signaling, growth factor binding triggers the rapid recruitment of activated RTKs in coated pits and their trafficking along the endocytic pathway to lysosomes: a process, termed 'down-regulation'. We have analyzed EGF-receptor (EGFR) signaling in cells made conditionally defective in receptor mediated endocytosis through expression of dominant- negative mutants of dynamin, a GTPase required for endocytic coated vesicle budding. We found that EGFR signaling continues as it traverses the endocytic pathway and that endocytosis is critical not only for attenuating signaling, but also for activation of selective signaling pathways. We propose to extend this analysis to define the role of endocytosis in signal transduction by 'stepping' activated EGFR through the endocytic pathway by the regulated expression of dominant-negative mutants of rab GTPases that control membrane trafficking between sequential endosomal compartments. These studies will involve interactions and collaborations with Drs. Balch, Emr, Gill, Farquhar and Hahn and will make extensive use of Cores B and C. By directly exploring the contribution of the spatial and temporal context of signaling interactions in modulating signal transduction and in determining cellular responses to peptide hormones, these studies should yield new insight into the control of cell proliferation and differentiation. In addition our results may provide new opportunities and novel targets for therapeutic intervention aimed at controlling unregulated cell proliferative signaling.
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