Insulin and related growth factors such as IGF-1 and IGF-2 have recently been demonstrated to be important molecules in the regulation of cellular proliferation and in the development of the central nervous system (CNS). At the cellular level, for example, insulin promotes neuritic sprouting that is believed to be essential to initiate and maintain cellular contacts required for many physiologic functions including learning. Therefore, there is a need to better understand the molecular effects of ethanol on the IGF-1/insulin mediated signal transduction cascade as it relates to growth and development of the CNS. In order to accomplish these goals, it will be necessary to produce and characterize monoclonal and polyclonal antibody reagents to various structural and functional domains of the IRS-1 molecule as well as provide recombinant eukaryotic and prokaryotic proteins to be used in immunoprecipitation and kinase assays particularly in cellular extracts that have been prepared from tissues or neuronal cells exposed to ethanol. In this regard, our plans are as follows: 1) Develop cDNA expression constructs that contain various functional domains of the IRS-1 protein. 2) Characterize the functional properties of expressed recombinant proteins within transfected neuronal cells under the influence of IGF-1 and insulin stimulation 3) Produce monoclonal antibodies to functional domains and tyrosyl phosphorylation motifs of the IRS-1 molecule and characterize such antibodies with respect to: affinity constants, epitope specificity, immunoprecipitation and Western blot analysis, binding to the functional domains of the IRS-1 molecule, antiphosphotyrosine specificity and tissue and cellular distribution within cells and different regions of the human central nervous system. It is anticipated that the large library of monoclonal antibodies prepared against the various domains of the IRS-1 molecule will be made widely available to NIAAA funded investigators for cellular and molecular studies of IGF-1 and insulin mediated signal transduction processes in cells of CNS or other origin under the influence of acute and chronic ethanol exposure.
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