The rate of glucose uptake into vertebrate cells is regulated by oncogenic transformation and by many other factors including mitogen, hormones and glucose starvation. It has been shown that regulation of glucose transporters can occur at different levels including transcription, mRNA stability, protein translocation and protein turnover. Glucose transporters provide a way of studying cellular regulation at these levels as well as the relationship of growth control to carbohydrate metabolism. In addition there are five transporter isoforms which are regulated differently, thus providing a useful model system for understanding how isoforms are regulated according to the metabolic needs of the cell. We are interested in chicken embryo fibroblasts since they express at least two glucose transporter isoforms and these are differentially regulated (type 1 is regulated by protein turnover and type 3 by the level of its mRNA). The goals of this proposal are to understand the molecular basis of this differential regulation.
AIM 1 : We propose to complete the cloning and sequencing of the cDNAs of glucose transporter isoforms expressed by chicken embryo fibroblasts.
AIM 2 : These will be used to measure the levels of the mRNAs for each isoform under various physiological conditions. The rate of transcription for each isoform and stability of its mRNA will also be measured.
AIM 3 : The molecular basis for the differential transcriptional control of the transporter isoforms will be determined by isolating genomic clones and analyzing their promoter regions.
AIM 4 : The control of transporters at the protein level will be investigated by generating specific antisera to each isoform. These will be used to quantitate the level of each isoform under various physiological conditions and to measure their rates of biosynthesis and turnover. It has already been established that the type 1 isoform is regulated at the level of turnover.
AIM 5 : Cellular and biochemical analysis of the molecular basis of this turnover control will be undertaken. This study will provide basic information about the regulation of different isoforms and about several levels of cellular control.
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