This proposal represents a continuing effort by our laboratory to understand the molecular basis of endocrine and metabolic regulation of cellular sugar transport. Metabolic depletion appears to stimulate muscle and avian red cell (ARBC) transport by increasing the intrinsic activity of cell surface sugar carriers (i.e. by carrier activation). This contrasts with insulin stimulation of adipocyte and muscle transport by increased cell surface numbers. The broad goal of this proposal is to characterize the biochemistry of carrier activation and its relationship to insulin regulation of sugar transport. These studies will assist in our long term goal of understanding the molecular basis of protein mediated sugar transport and could ultimately be of value in the management of disordered states such as diabetes. The glucose transport protein contains a transport modulating ATP-binding site. The site has recently been specifically labeled using azido-ATP.
Specific AIM 1 is to identify this site by sequencing labelled peptides released on hydrolysis of labelled carrier.
Specific Aim 2 asks: since the carrier has recently been shown to be exquisitely susceptible to activation/inhibition by specific membrane bilayer lysophospholipids (LPLs), does carrier activation by metabolic depletion result from altered bilayer lipid composition? We employ reconstituted system and phospholipid analyses and manipulation of control and depleted ARBCs to answer this.
Specific Aim 3 asks: since stimulatory LPLs reduce and inhibitory LPLs increase the affinity of glucose carrier for ATP, does this synergism between ATP and LPLs act to amplify their effects on transport? We perform ligand binding and transport studies with reconstituted carrier to answer this.
Specific Aim 4 directly tests activation and recruitment hypotheses for transport regulation using recently developed antisera (exclusively reactive against an extracellular domain of the carrier) to quantitate cell surface carrier numbers in control and metabolically depleted ARBCs.
Specific Aim 5 extends Aims 2 to 4 to insulin stimulation of adipocyte sugar transport. If successful, these studies will provide the groundwork for future assessment of the role of the carrier ATP-binding site in transport regulation and for understanding the relationship between carrier activity and cellular metabolic status.
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