The objective is to contribute to our understanding of the molecular basis of insulin's action on hexose transport and other regulated membrane components. This work will attempt to define the biochemical processes that regulate the membrane dynamics and movements of three insulin-sensitive model membrane proteins--hexose transporters, IGF-II receptors, and transferrin receptors. Experimental procotols are designed to test a central hypothesis recently developed in our laboratory: Insulin action leads to rapid dephosphorylation or inhibited phosphorylation of these three membrane components in plasma membranes which in turn delays their rates of internalization, leading to increased numbers in the plasma membrane. A major effort will also be made to define the molecular pathway or pathways whereby insulin receptor activation leads to decreased phosphorylation of target membrane proteins. Thus, various steps in the membrane recycling of IGF-II receptors will be evaluated in respect to regulation by insulin using surface labeling techniques (e.g., lactoperoxidase) followed by immunoprecipitations in various membrane fractions with specific anti-receptor antibodies. We shall also evaluate the movement (and possible regulation) of these receptors from plasma membrane to coated pit regions using a rapid immunoadsorption method to isolate coated pits. Cellular kinases and phosphatases that modulate phosphorylation-dephosphorylation of IGF-II receptors and transferrin receptors will be identified in two ways. Antibodies already prepared in our laboratory against native, purified human erythrocyte transporters, and antibodies we will prepare against synthetic peptides identical to regions of the recently cloned human hexose transporter will be used. Highly insulin-sensitive rat tissues are best for biological studies, so we shall attempt to clone and sequence the rat transporter gene (or genes) in order to have structural information on this protein(s). cDNA and genomic libraries will be screened with oligonucleotide probes synthesized according to regions of the known human cDNA. These studies on transporter regulation by insulin should allow detailed molecular comparisons with mechanisms involved in IGF-II and transferrin receptor up-regulation.
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