MEMBRANE LIPID EFFECTS ON THE INSULIN RECEPTOR

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The purpose of this project is to gain a better understanding of the molecular interactions between the insulin receptor and the membrane lipids with which it is in contact. Whether the localization of the receptor in different lipid microenvironments influences insulin binding will be assessed. All studies will be done with insulin receptors obtained from the turkey erythrocyte membrane, a widely used experimental model. Beta-Octylglucoside will be used to solubilize the receptor, and the solubilized material will be purified with respect to both membrane lipids and proteins by a series of column chromatographic steps in solutions containing this detergent. Insulin binding will be measured during the course of purification in order to determine whether the binding properties of the receptor are affected. Each successively purified preparation will be characterized with respect to protein as well as lipid content and composition. Reconstitution of the receptor in liposomes will be carried out with the best preparation that can be obtained which retains binding activity, probably 10-50% pure with respect to protein and containing less than 1 mol phospholipid and cholesterol per mol receptor (Mr 350,000). The effect of phospholipid head group composition, fatty acyl chain composition, cholesterol content, sphingomyelin content and glycosphingolipid content and composition on insulin binding to the reconstituted receptor will be determined. Attempts will be made to measure receptor phosphorylation in response to insulin binding in the reconstituted liposome preparation by trapping 32P-ATP in the aqueous core during liposome formation. The effect of liposomal lipid composition on insulin-stimulated phosphorylation will be tested. Phosphorylation in response to insulin binding also will be assessed in the detergent-solubilized preparation containing different added membrane lipids. This project will provide fundamental information regarding possible membrane lipid modulation of the insulin receptor. Such information will enhance our basic knowledge of the effects of membrane structure on the interaction between insulin and its receptor. This will provide information about hormonal regulation at the molecular level and may ultimately add to our understanding of the abnormalities associated with diabetes.