Subunit structure of insulin receptor of rat adipocytes as demonstrated by photoaffinity labeling

Abstract

Isolated rat adipocytes were incubated in the dark with either 1 of 2 radioiodinated photoreactive insulin derivatives, N.epsilon.B29-(azidobenzoyl)insulin (B29-MABI) and N.alpha.B1-(azidobenzoyl)insulin (B1-MABI), and were then exposed to light. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and radioautography of the crude plasma membrane fraction after reduction showed that B29-MABI labeled specifically 3 proteins of MW 130,000, 90,000, and 40,000, whereas B1-MABI labeled specifically 2 proteins of MW 130,000 and 40,000. B1-MABI also variably labeled some bands of intermediate MW between 130,000 and 90,000. In contrast, the labeling of the 40-kilodalton [KD] protein was not observed in previous studies in which photolabeling was carried out on isolated plasma membrane preparations. Without reduction, a MW 300,000 band and a larger band which barely entered a 5-15% gradient gel were specifically labeled by both photoreactive insulins. Reduction of these 2 high MW bands gave rise to the 130-, 90-, and 40-kD bands. The labeling of these proteins was affected neither by the time or temperature of incubation nor by the addition of methylamine, chloroquine, bacitracin, phenylmethanesulfonyl fluoride, p-(chloromercuri)benzenesulfonic acid, Trasylol, N-ethylmaleimide, or benzamidine. The labeling of these proteins by the photoreactive insulin derivatives was inhibited by first incubating the adipocytes with a human autoimmune serum to insulin receptor. These proteins apparently are subunits of the insulin receptor in intact adipocytes.