Unimpaired signal transduction by the adipocyte insulin receptor following its partial proteolytic fragmentation.

Abstract

Limited proteolytic digestion of adipocytes with tryp- sin leads to a stimulation of fat cell glucose transport and oxidation. This effect is dose-dependent and reaches half the maximal value due to insulin at ap- proximately 10 cg/ml of protease under the conditions of these experiments. Insulin binding studies reveal that adipocytes exposed to 10 pg/ml of trypsin for 30 min exhibit slightly decreased receptor number but increased affmity for hormone as compared to cells not exposed to the protease. This decrease in receptor num- ber is offset by the simultaneous increase in receptor affinity for insulin such that little change in receptor occupancy is observed at any insulin concentration when control and proteolyzed cells are compared. After treatment with tbie concentration of trypsin, the par- tially activated adipocytes respond further to increas- ing doses of insulin to give maximal glucose oxidation with the same dose-response relationship td hormone as is observed in untreated cells (EDa = 10-‘“~). Adipocytes treated with trypsin (2 to 60 pg/ml) were subjected to affinity cross-linking of ‘z%insulin to re- ceptor complexes (Pilch, P. F., and Czech, M. P. (1979) J Biol. Chem. 254,3375-3381 and (1980) 265,1722-1731) in order to visualize the extent of receptor proteolysis under these conditions. At least four major fragments of the insulin receptor are generated and a correlation between proteolysis of the native receptor, iV& = 350,000 and the insulinomimetic effect of trypsin is observed. Other plasma membrane proteins undergo no demon- strable proteolytic degradation under these conditions when the gels are stained for protein or carbohydrate. These data suggest that the insulinomimetic effect of trypsin is a result of direct proteolysis of the insulin receptor. When proteolysis is performed subsequent to affinity cross-linking, the labeled receptor fragments remain tightly associated with the plasma membrane and none are seen in the incubation medium. The ob- served limited tryptic digestion of receptor at low pro- tease concentrations (10 pg/ml) probably occurs at sites peripheral to the insulin binding domain. * This work was supported by Grants AM-17893 and HD-11343