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 alpha- and the beta-subunit of the receptor. Immunogold labeling of the C-terminus of the beta-subunit indicates that it resides about 6 nm off the membrane, while two alpha-subunit epitopes were labeled at about twice this distance, confirming that the alpha-subunit is harbored in the cross-bar of the T-structure.
Tranum-Jensen
J
J
Department of Medical Anatomy, Panum Institute, University of Copenhagen, Denmark.
Christiansen
K
K
Carlsen
J
J
Brenzel
G
G
Vinten
J
J
D006801:Humans; D007328:Insulin / Q000378:metabolism; D008081:Liposomes; D008567:Membranes, Artificial; D008854:Microscopy, Electron; D008954:Models, Biological; D011972:Receptor, Insulin / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure
D007328:Insulin; D008081:Liposomes; D008567:Membranes, Artificial; D011972:Receptor, Insulin