Membrane topology of insulin receptors reconstituted into lipid vesicles

Insulin receptors were incorporated into liposomes by two different procedures, one using dialysis and one using detergent removal by Bio-Beads. Receptor incorporation was analyzed by gradient centrifugation and electron microscopy. Reconstituted receptors projected up to 12 nm above the membrane and exhibited a T-shaped structure compatible with that 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 was confirmed by immunogold labeling of the - and the -subunit of the receptor. Immunogold labeling of the C-terminus of the -subunit indicates that it resides about 6 nm off the membrane, while two -subunit epitopes were labeled at about twice this distance, confirming that the -subunit is harbored in the cross-bar of the T-structure.We thank Ms. Birthe Nystrøm, Lisette Hansen and Ulla Blankensteiner for excellent technical assistance and Ms. Birgit Risto for skillful work with the photographic prints.