We determined that the rate of insulin-stimulated autophosphorylation of the insulin receptor is independent of receptor concentration and thus proceeds via an intramolecular process. This result is consistent with the possibility that ligand-dependent autophosphorylation may be a means by which cells can distinguish occupied from unoccupied receptors. We employed dithiothreitol to dissociate tetrameric receptor into αβ halves in order to further elucidate the structural requirements for the receptor-mediated kinase activity. Dithiothreitol had a complex biphasic effect on insulin-stimulated receptor kinase activity. Marked stimulation of kinase activity was observed at 1-2 mM dithiothreitol when the receptor was predominantly tetrameric and kinase activity diminished when dimeric αβ receptor halves predominate (>2 mM dithiothreitol). N-Ethylmaleimide inhibits insulin-stimulated receptor kinase activity. We suggest that the tetrameric horroreceptor is the most active kinase structure and this structure requires for maximal activity, a reduced sulfhydryl group at or near the active site. We treated receptor preparations with elastase to generate receptor proteolytically 'nicked' in the β subunit. This treatment completely abolishes insulin-dependent autophosphorylation and histone phosphorylation with essentially no effects on insulin binding as determined by affinity labeling of the receptor α subunit. We suggest such treatment functionally uncouples insulin binding from insulin-stimulated receptor kinase activity. The possible physiological significance of these findings is discussed.
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1983|