Iron is an essential requirement for cellular proliferation and iron- deficiency is associated with numerous diseases. the major mechanism that accounts for the accumulation of iron by cells is the receptor- mediated endocytosis of diferric transferrin. This process is acutely regulated by several growth factors including epidermal growth factor, platelet- derived growth factor, insulin, and insulin- like growth factors. Treatment of cells with these growth factors causes an increase in the rate of transferrin receptor recycling and an inhibition of the rate of transferrin receptor internalization. At steady-state, the net result of these changes in the kinetics of cycling is an increase in the cell surface expression of transferrin receptors and a concomitant decrease in number of transferrin receptors located in an intracellular membrane compartment. The goal of this research project is to determine the mechanism of regulation of the cycling of the transferrin receptor. To achieve this goal, three Specific Aims are proposed: 1) The role of post- translation covalent modification of the transferrin receptor will be examined. Insulin action to inhibit transferrin receptor endocytosis is associated with increased palmitylation of the transferrin receptor. The hypothesis that the mechanism of regulation of transferrin receptor endocytosis is mediated by acute changes in the state of palmitylation of the transferrin receptor will be tested. 2) Cellular components that are required for the function of the transferrin receptor represent potential targets of growth factor action. Identification of these gene products is required in order to understand the mechanism of regulation of the transferrin receptor. The experimental strategy that will be employed is to perform a genetic analysis of transferrin receptor endocytosis. 3) A mutant transferrin receptor in which both Cys62 and Cys67 are replaced with Ala residues is expressed at the cell surface. Internalization of this mutant receptor is rapid, but it is not regulated by insulin. The hypothesis that distinct receptor structural elements are required for a) transferrin receptor endocytosis, and b) regulation of endocytosis by growth factors will be tested. The overall goal of this research project is to gain an understanding of the mechanism(s) of signal transduction that account for the regulation of the sub- cellular distribution of the transferrin receptor by growth factors. Achievement of this goal will add significant new information to two different areas of basic research in cell biology: 1) signal transduction by growth factor receptors, and 2) control of membrane receptor localization. GRANT=R01GM37677 DNA in both eukaryotic and prokaryotic chromosomes is organized into supercoiled topological domains. In prokaryotes about half the supercoils are equilibrated as unrestrained torsional tension in the DNA. Such strain has been shown to be important in mechanisms controlling gene expression. In eukaryotes, most supercoils are restrained by nucleosomes and, on average, the DNA helix is not wound with torsional tension. Although there is no direct evidence for unrestrained supercoiling in eukaryotic DNA, some current gene regulatory models suggest that a torsionally strained helix is involved. One model suggests that torsional tension in eukaryotic chromatin may be generated by the movement of RNA polymerase during transcription. Torsional tension could promote breathing the DNA, formation of cruciforms, formation of triple stranded DNA, or formation of left-handed Z-DNA. This proposal is designed to determine if torsional tension exists in eukaryotic DNA. Our original in vivo assay for unrestrained torsional tension involved measurement of rates of trimethylpsoralen photobinding to DNA. This provided an averaging measurement which would not detect torsional tension if present in a small fraction of the chromosome, such as in active genes or at a specific localized site in the DNA. This assay will be modified to quantitate rates of psoralen cross-linking to selected restriction fragments of DNA to measure torsional strain within a selected region of DNA. We will synthesize, characterize, and apply cruciform and Z-DNA """"""""torsionally tuned probes"""""""" for unrestrained supercoiling in vivo. This involves application of psoralen-based assays sequences in living cells. These alternate helical structures form at precise minimum (tuned) superhelical densities allowing measurement of the level of supercoiling in vivo at specific sites in the DNA. The cross-linking assay will be applied in vivo to determine if selected regions of SV40 minichromosomes or various Drosophila genes are wound with unrestrained torsional tension in living cells. In addition, we will insert the """"""""torsionally tuned probe"""""""" sequences into the control region of SV40 DNA, on either end of an inducible gene in a stable myc epichromosomal vector, and adjacent to Drosophila hsp70 heat shock genes to ask if unrestrained DNA supercoiling can be detected constitutively or in association with transcription in vivo.
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