The covalent tagging of the cell surface insulin receptor in intact cells with the generation of an insulin-free, functional receptor. A new approach to the study of receptor dynamics.
A new method is described in which the cell surface insulin receptor can be radioactively tagged in a specific manner with a small insulin-free probe. After protecting the amino groups of insulin essential for binding and bio-activity, insulin is coupled to the heterobifunctional, cleavable cross-linking reagent SASD (sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3'-dithiopropionate), via displacement of the N-hydroxysuccinimide moiety of SASD. Removal of the protecting groups results in the formation of 2-(p-azidosalicylamido)-1,3'-dithiopropionate (ASD)-insulin with insulin receptor binding activity equivalent to unmodified insulin. Iodination of ASD-insulin results in the incorporation of 125I into both the azidohydroxybenzoyl moiety of SASD and a tyrosine residue of insulin. Following binding of 125I-ASD-insulin to intact monolayers of 3T3-C2 cells, radiolabel is incorporated exclusively into a 135-kDa protein in a manner dependent upon the length of exposure of the cells to short wavelength ultraviolet light. This protein corresponds in molecular weight to the alpha subunit of the insulin receptor. Labeling of this protein can be inhibited by excess unlabeled insulin. Reduction of the disulfide bond of ASD with 10 mM glutathione causes the release of the 125I-insulin portion of the reagent from the receptor complex, with the iodinated photoactivated end of ASD covalently attached to the receptor. Insulin receptor labeled in this manner retains its ability to bind insulin. General metabolic processes of the intact cells do not appear to be perturbed by this labeling procedure, and the cellular processing of the insulin receptor does not appear to be modified by the covalent labeling of the receptor protein. This procedure therefore provides a way to specifically label the cell surface insulin receptor in a manner which does not perturb the normal functioning of the labeled cell and equally importantly, does not perturb the normal cellular processing of the insulin receptor itself.
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D003080:Cold Temperature; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005978:Glutathione / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D051379:Mice; D010782:Photolysis; D011972:Receptor, Insulin / Q000378:metabolism*; D013388:Succinimides / Q000494:pharmacology*
D000596:Amino Acids; D001386:Azides; D003432:Cross-Linking Reagents; D013388:Succinimides; C047257:sulfosuccinimidyl 2-(4-azidosalicylamido)ethyl-1,3'-dithiopropionate; D011972:Receptor, Insulin; D005978:Glutathione