Control of cell growth and differentiation is regulated by a signal transduction cascade initiated by growth factor binding to transmembrane receptors containing tyrosine kinase activity. There is little mechanistic information available to explain the diverse mitogenic and non-mitogenic responses to epidermal growth factor administration in the intestine. The current understanding of the regulation of receptor kinase and substrate interaction is that src homologous domains designated SH2 and SH3 are regulatory sequences, determining the specificity of protein- protein interaction in the signal transduction process. Phosphotidylinositol specific phospholipase C gamma-1 (PLCgamma1) is the best characterized substrate of EGFR tyrosine kinase. We have detected protein expression, tyrosine phosphorylation and activity of PLCgamma1 in the rat small intestine to be greatest during weaning. The principal objective of this proposal is to characterize the expression, growth factor regulation and function of PLCgamma1 and other SH2/SH3 domain signal transduction proteins. In a unique, supportive environment, with access to all reagents and equipment necessary for these studies, the PI under the review and guidance of the sponsor will seek to study the significant regulatory mechanisms of PLCgamma1 activity in the intestine, the effect of PLCgamma1 on differentiation and function in an intestinal cell line and other SH2/SH3 domain proteins in the tyrosine kinase signaling pathway in the intestine. The model for this project will be the suckling and weanling rat small intestine with in vitro assays in IEC-6 and CaCo-2 cell lines. A combination of a baculovirus expression system, immunoprecipitation, cross-linking, activity assays, and Western and Northern blotting will be used to study potential binding proteins, tyrosine phosphorylation and dephosphorylation events and expression in growth factor regulation of PLCgamma1 and other SH2/SH3 domain proteins. A model will be developed for the study of PLCgamma1 function in the intestine using a hormonally induced expression vector containing antisense PLCgamma1 mRNA will be developed for transfection in the IEC-6 cell line. The successful development of this model will provide the mechanistic basis for the study of other signal transduction proteins in future studies.
