This proposal represents a continuing effort by our laboratory to understand how certain cells stimulate sugar transport in response to increased cellular demand for glycolytic ATP. Skeletal muscle sugar transport and glycolysis, for example, are stimulated massively during exercise and anoxia. We will show that transport stimulation results from the controlled reversal of two, insulin-independent mechanisms that depress the activity of the sugar transport protein GLUT1. These inhibitory mechanisms (which can be studied in isolation in specific tissues) are RESTRAINT and REPRESSION. Restraint reduces whereas repression abolishes the catalytic activity of plasma membrane GLUT1. The broad goals of this proposal are to characterize the molecular basis of restraint, repression and their reversal and to understand how certain cells achieve coordinate regulation of transport and glycolysis. These studies will assist in our long term goal of defining the molecular basis of protein-mediated sugar transport and may ultimately be of value in the management of disordered states such as diabetes. Our studies show that GLUT1 restraint and its reversal and allosteric regulation of phosphofructokinase (the pacemaker of glycolysis, PFK) are functionally analogous. This could account for coordinate regulation of glycolysis and transport.
SPECIFIC AIM 1 addresses the mechanism of restraint and therefore asks: """"""""Does GLUT1 contain multiple regulatory domains for direct interaction with glycolytic species that also regulate PFK?"""""""" We propose biochemical reconstitutions using purified GLUT1 and allosteric activators of PFK to test this thesis. Thus far, we have discovered two GLUT1 regulatory domains. We will identify these domains by an affinity labeling/proteolysis/sequencing strategy. Finally, we will verify and extend these details and assess their significance to GLUT1 regulation by analysis of the effects of molecular mutagenesis of putative GLUT1 regulatory domains on GLUT1 regulation.
SPECIFIC AIM 2 addresses the mechanism of GLUT1 repression / derepression. This mechanism is only poorly resolved although we do know that cytosolic ATP is required for both transport repression and derepression. We will test the hypothesis that GLUT 1-repression is simply a more efficient form of restraint. If repression is a unique regulatory mechanism, we will determine: 1) the role of ATP in GLUT1 regulation (allosteric or covalent) and, 2) whether this regulation is intrinsic to GLUT1 or is mediated by other cellular proteins. If other proteins are involved, we ask: What are their identities, what are their roles in regulation, are these proteins expressed uniquely in cells where transport is regulated and if so, can we reconstitute transport regulation by appropriate transfections of non- regulated cells? If successful, these studies will establish whether restraint and repression are identical or dissimilar regulatory mechanisms and will significantly extend our understanding of the molecular basis of metabolic regulation of sugar transport.
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