Dynamics of signaling during insulin-stimulated endocytosis of its receptor in adipocytes.

Insulin causes rapid insulin receptor autophosphorylation, receptor endocytosis, and phosphorylation of its principle substrate (IRS-1). Using rat adipocytes, we studied the dynamics of receptor autophosphorylation, the kinase activity, and the IRS-1 phosphorylation state relative to the subcellular localization of these proteins. After 2 min of insulin exposure, the specific phosphotyrosine content of the insulin receptor in the internal membranes (IM) peaks at a level 5-6-fold higher than the plasma membrane (PM) receptor and then declines after 5-8 min to a level similar to the PM receptor. The exogenous kinase activity of these receptors exactly mirrored their phosphotyrosine content. The distribution of IRS-1 is 80% cytosolic, 20% IM-associated, and essentially undetectable in the PM. The phosphorylation state of IRS-1 in the IM parallels that of the insulin receptor, but cytosolic IRS-1 phosphorylation remains constant. Insulin-dependent GLUT4 translocation to the PM occurs after the peak of IRS-1 phosphorylation. The data are consistent with the hypothesis that insulin action may be mediated by receptor internalization and interaction with its substrate(s) associated with internal membranes. A small fraction of phosphorylated insulin receptors is sufficient for signal transduction. The dephosphorylation of the insulin receptor and IRS-1 in the IM appears to be a concerted process, possibly mediated by the same enzyme.