The overall aim of the project is to dissect the mechanism by which insulin increase the transport of glucose into cultured 3T3-F442A fat cells. This will be achieved by modifying certain structural features of the predominant insulin-sensitive glucose carrier in these cells (IRGT) and assessing effects on its functional mechanism and regulation. The transport and regulatory features unique to the IRGT will be determined by comparison to another type of glucose carrier also present in these cells, but which predominates in insulin-insensitive cell types, such as erythrocytes and brain. Initial studies will involve comparison of transport kinetics between the two carrier subtypes under conditions in which one subtype is alternatively absent or blocked. Selected structural modifications will then be made, and the resulting transport, biochemical behavior and responsiveness to insulin of the two subtypes contrasted. Methods for biochemically distinguishing between subtypes will include cell-surface labeling, Northern blot analysis with carrier-specific cDNA probes, and immunoprecipitation and immunoblotting with carrier-specific antibodies. The planned structural dissection of the IRGT will include 1) analysis of its transmembrane loop structure by susceptibility to proteolytic cleavage and by accessibility to membrane-impermeant labeling reagents; 2) investigation of whether the IRGT, like the erythrocyte carrier protein, is linked directly to the plasma membrane through a fatty acyl-thiol ester and if so, whether loss of such an anchoring mechanism affects its function or regulation by insulin; 3) determination of whether the IRGT-selective effects of the sulfhydryl reagent phenylarsine oxide are due to direct reaction with the IRGT, or to indirect effects on insulin-stimulated recruitment of carriers to the plasma membrane; and 4) assessment of whether and how phosphorylation of the IRGT or associated proteins affects insulin-stimulated transport. The specific components of insulin-stimulated transport to be clarified by this approach include: the relative contributions of intrinsic activation and translocation of the IRGT from internal sites to the plasma membrane in response to insulin, and whether unique structural features of the IRGT contribute substantially to its sensitivity to regulation by insulin and other agents. By increasing the understanding of how the structure of the IRGT contributes to its function and regulation, these results may also be relevant to its study in clinical situations including exercise, and insulin resistant and insulin-deficient states.
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