Generation of oligomeric insulin receptor forms by intramolecular sulfhydryl-disulfide exchange. Involvement of masked sulfhydryl groups.
Insulin receptors from rat liver membranes were labelled with a 125I-labelled photoreactive insulin analogue or by iodination using lactoperoxidase and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Under nonreducing conditions different receptor forms with Mr 400,000 (alpha 2 beta 2), 360,000 (alpha 2 beta beta'), 330,000 (alpha 2 beta' beta'), 320,000 (alpha 2 beta), 280,000 (alpha 2 beta'), 240,000 (alpha 2), 210,000 (alpha beta), 165,000 (alpha beta') and 115,000 (alpha) were detected. The subunit composition of these receptor forms was determined by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence and presence of dithioerythritol. During denaturation in sodium dodecyl sulfate in the absence of reductants, the Mr 400,000 receptor form (alpha 2 beta 2) was converted into the Mr 320,000 (alpha 2 beta) and Mr 240,000 (alpha 2) receptor form. This conversion was prevented either by N-ethylmaleimide, oxidants, or low pH. In contrast, alkylation of the receptor with N-ethylmaleimide under non-denaturing conditions did not prevent the appearance of intermediate-sized receptor forms. Furthermore, the inhibition of receptor cleavage by N-ethylmaleimide during denaturation was also observed when the amount of free sulfhydryl groups was reconstituted by the addition of an unlabelled and non-alkylated receptor sample to the alkylated and photoaffinity-labelled receptor. These results suggest, that the generation of different oligomeric receptor forms detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis is due at least in part to the cleavage of one or both beta-subunits from the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
Krämer
H
H
Deger
A
A
Koch
R
R
Rapp
R
R
Hinz
M
M
Weber
U
U
D000345:Affinity Labels; D000818:Animals; D004220:Disulfides / Q000032:analysis*; D004586:Electrophoresis; D066298:In Vitro Techniques; D007328:Insulin; D007457:Iodine Radioisotopes; D007784:Lactoperoxidase; D008099:Liver / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis; D008970:Molecular Weight; D011233:Precipitin Tests; D011489:Protein Denaturation; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000032:analysis*; D013438:Sulfhydryl Compounds / Q000032:analysis*
D000345:Affinity Labels; D004220:Disulfides; D007328:Insulin; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; D007784:Lactoperoxidase; D011972:Receptor, Insulin