Photoaffinity labelling of the insulin receptor in intact rat hepatocytes, mouse soleus muscle, and cultured human lymphocytes

Using the photoreactive, biologically active insulin analogue, B2-(2 nitro, 4-azidophenylacetyl)des-PheB1 insulin, which can be covalently bound to receptor molecules upon photolysis, the insulin receptor has been studied in three different types of cells or tissues: isolated rat hepatocytes, intact murine soleus muscle and cultured human lymphocytes. When compared with native insulin, this analogue displayed a slightly reduced binding affinity. Accordingly, the biological potency of the photoreactive analogue was decreased by approximately 30% compared with native insulin when tested for its ability to stimulate amino acid transport in hepatocytes, and deoxyglucose uptake in soleus muscles. It was as effective as insulin, however, at maximally stimulating concentrations and therefore is a full insulin agonist. This photoprobe was used to specifically label the insulin receptor in the three tissues: after ultra-violet irradiation, sodium dodecyl sulphatepolyacrylamide gel analysis of extracts under reducing conditions revealed that most of the radioactivity was associated with a 130,000 dalton band. In isolated hepatocytes, two bands at 125,000 and 23,000 daltons were also specifically labelled. In three different cell types from three different animal species, the 130,000 dalton band appeared to be the major subunit of the insulin receptor.