Ethanol inhibits insulin-like growth factor-I activation in several cell types, including neuronal cells, leading to a reduction in cell proliferation and sensitization to apoptosis. We have further demonstrated that ethanol directly inhibits IGF-I receptor (IGF-IR) tyrosine autophosphorylation leading to the inhibition of tyrosine phosphorylation of key intracellular signaling intermediaries including IRS-1, Shc, phosphatidylinositol 3-kinase, and MAP kinase. We have identified the minimally defined tyrosine kinase domain of the IGF-IR and the closely homologous insulin receptor (1R) as inhibitory targets for ethanol. Our studies have further shown that intracellular factors affect the sensitivity of the IGF-IR to ethanol, and the activation of mediators downstream of the IGF-IR are differentially affected by ethanol. Biochemical studies and computer modeling allow us to hypothesize that ethanol and other n-alkanols interact within the highly conserved activation site, which contains the catalytic and activation loops. The effects of ethanol and other n-alkanols on IGF-IR and IR autophosphorylation and kinase activity will be assessed using recombinant expressed IGF-IR/IR kinase domains, including an active mutant kinase. Studies will focus on parameters of A-loop activation as they relate to differential peptide substrate phosphorylation. Mutations of key hydrophobic and polar residues within the activation cleft that alter hydrophobic and volumetric parameters will be tested for their effect on the kinetics of autophosphorylation and substrate phosphorylation and n-alkanol inhibition. Computer modeling will complement the observed biochemical effects. Direct structural analysis of the interaction of n-alkanols with the IGF-IR/IR kinases will be achieved by X-ray crystallography. We will investigate the intracellular factors of the IGF-IR that modulate sensitivity to ethanol. We hypothesize that cystolic tyrosine kinases, Src kinase in particular, transactivate the IGF-IR in the insensitive cells, thereby shifting the kinase equilibrium in the presence of ethanol towards the active mode. Taken together, these studies will provide an in depth characterization of the molecular mechanisms by which ethanol interferes with receptor tyrosine kinase, and may offer insights into future therapeutic modalities.
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