MEMBRANE TOPOLOGY OF INSULIN-RECEPTORS RECONSTITUTED INTO LIPID VESICLES

Insulin receptors were incorporated into liposomes by two different pr ocedures, one using dialysis and one using detergent removal by Bio-Be ads. Receptor incorporation was analyzed by gradient centrifugation an d electron microscopy. Reconstituted receptors projected up to 12 nm a bove the membrane and exhibited a T-shaped structure compatible with t hat previously described for the solubilized receptor. Insulin binding and autophosphorylation experiments indicated that approx. 50% of the receptors were incorporated right-side out. Such random orientation w as confirmed by immunogold labeling of the alpha- and the beta-subunit of the receptor. Immunogold labeling of the C-terminus of the beta-su bunit indicates that it resides about 6 nm off the membrane, while two alpha-subunit epitopes were labeled at about twice this distance, con firming that the alpha-subunit is harbored in the cross-bar of the T-s tructure.