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Characteristics of Insulin and Epidermal Growth Factor Stimulation of Receptor Autophosphorylation in Detergent Extracts of Rat Liver and Transplantable Rat Hepatomas^*

PERRY J. BLACKSHEAR, RAPHAEL A. NEMENOFF and JOSEPH AVRUCH

Howard Hughes Medical Institute Laboratory, Harvard Medical School, the Diabetes Unit and Medical Services, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School Boston, Massachusetts 02114

Address requests for reprints to: Dr. P. J. Blackshear, Diabetes Unit, Massachusetts General Hospital, Boston, Massachusetts 02114.

Abstract

Insulin and epidermal growth factor (EGF) share a number of metabolic actions, including stimulation of protein synthesis and growth in certain tissues, and activation of apparent receptor tyrosine kinase activities. We have shown that insulin and EGF promote the phosphorylation of a number of intracellular proteins in common, suggesting that some of the shared metabolic actions of these hormones might be due to shared effects on protein kinases or phosphatases. We, therefore, compared the effects of these hormones on their respective membrane receptor autophosphorylation reactions in detergent extracts prepared from rat liver microsomes. Under appropriate conditions, ligand-promoted receptor autophosphorylation could be observed without further receptor purification. Insulin-and EGF-stimulated receptor autophosphorylation exhibited differing requirements for divalent cations and optimum ATP concentrations, and were enhanced by detergsnt extraction of the membranes. Insulin-promoted receptor autophosphorylation occurred on a tyrosine residue. In liver microsomal extracts prepared from rats exposed to various dietary conditions, insulinstimulated receptor autophosphorylation was enhanced in extracts prepared from starved or diabetic animals when compared to those prepared from fed or fasted-refed animals. Experiments with extracts from several transplantable rat hepatomas of varying degrees of differentiation indicated that both insulin binding and insulin-stimulated receptor autophosphorylation were remarkably preserved in all tumors; in contrast, EGF binding and EGF-induced receptor autophosphorylation were diminished or absent in all tumors studied. These studies suggest that the relationship between insulin-mediated receptor phosphorylation and subsequent metabolic events might be profitably studied in these tissues. (Endocrinology 114: 141, 1984)

Footnotes

^* This work was supported in part by NIH Grant AM-7776.

Associate Investigator, Howard Hughes Medical Institute.

Associate, Howard Hughes Medical Institute.

Investigator, Howard Hughes Medical Institute.

Received January 28, 1983.