There is a close association between the expression of the receptor for transferrin and cellular growth. The cell surface number of transferrin receptors is acutely regulated by polypeptide mitogens. We propose to study the biochemical mechanisms by which platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) cause a rapid (less than 2 min) redistribution of intracellular transferrin receptors that results in the increased expression of the transferrin receptor at the cell surface. We will test the hypothesis that the mechanism of action of the growth factors is to alter the rate of exocytosis or endocytosis of the transferrin receptor by directly investigating the kinetics of the cycling of the transferrin receptor between cell surface and endosomal membrane compartments. We will also investigate the cell-cycle dependence of the growth factor regulation of the transferrin receptor by flow cytometry. A central issue that we address is whether covalent post-translational modification (phosphorylation, palmit ylation) of the transferrin receptor is relevant to the regulation of the transferrin receptor cycling by growth factors. Data is available from this laboratory which shows that EGF causes the phosphorylation of the transferrin receptor at a site that we have identified as serine-24. We propose to investigate this phosphorylation in detail. The effect of PDGF on transferrin receptor phosphorylation will also be examined. A critical element of our strategy to investigate the role of the phosphorylation of serine-24 is to examine whether serine-24 is essential for the regulation (by EGF and PDGF) of the expression of the transferrin receptor at the cell surface. We propose to introduce point mutations into the human transferrin receptor cDNA at codon-24 by oligonucleotide-directed mutagenesis. The wild-type and mutanttransferrin receptors will then be expressed in NIH 3T3 cells using a retrovirus vector. The regulation of the endogenous (murine) and introduced (human) transferrin receptors by EGF and PDGF will be investigated using species-specific monoclonal antibodies. We hope to learn fundamental information from the studies described in this proposal about (1) the biochemical mechanisms that regulate the cycling of the transferrin receptor, (2) the signalling mechanisms involved in the actions of PDGF and EGF, and (3) the relationship between the regulation of the expression of the transferrin receptor and cellular growth.
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