Overexpression of receptor tyrosine kinases (RTK's) is a common feature of human gliomas. Grb2 has a fundamental role in the signaling pathways of RTK's. It functions as an adapter protein where the SH2 domain binds to numerous RTK's while the SH3 domains mediate interaction with SOS, an exchange factor that upregulates ras activity. Recent discoveries by other laboratories and the principal investigator have shown that Grb2 interacts with several other potential effectors aside from SOS. Their previous work has found specific overexpression of such effectors in human gliomas. Several clones that bind to Grb2 have been isolated from a glioma cDNA expression library. It has been verified that one clone, named Gab1 (for Grb2 associated binder-1), is recognized by Grb2 and in turn can precipitate Grb2. It is highly conserved across species and a related cDNA has also been found. For these reasons, it is hypothesized that Gab1 has an important role in signal transduction. In this aim, the focus will be on cloning the full open reading frame of Gab1 and sequencing the cDNA in its entirety. A rabbit polyclonal antibody will be made and immunoprecipitation experiments will be performed to verify that Gab1 and Grb2 interact in vivo. Antibodies specific for two alternative splice forms of Gab1 also will be generated and this trio of antibodies will be used on Western blots to determine the size of the native protein, the distribution of this protein, and the relative levels of expression of the three forms. Northern blots will be used to determine if the splice variants are associated with a certain transcript size. The chromosome to which the Gab1 gene maps also will be determined. Determining the function of Gab1 will be key to understanding how it participates in the signal transduction pathway from RTK's. Clues could arise from homologies with known proteins. Overexpression of the protein in NIH-3T3 cells will reveal if Gab1 can affect cell growth or cause cellular transformation and if EGF augments any effect seen. The tyrosine or serine/threonine phosphorylation status of Gab1 will be ascertained. Information will also be gained by using cell fractionation and immunofluoresence studies to determine whether Gab1 associates with the particulate fraction or translocates to the nucleus upon growth factor addition. If these approaches are inconclusive, identifying what Gab1 associates with may yield insights into potential functions. Finally, the levels of expression of Gab1 in a series of high and low grade tumors will be examined to see if there is any correlation with ascending tumor grade and Southern blot analysis will be done to see if there are any deletions, rearrangements or amplifications. The affinity of Grb2 towards Gab1 will be determined, but the isolation of several clones that bind to Grb2 affords the opportunity to resolve several questions about the nature of the interaction beween the SH3 domains of Grb2 and their targets. Progressive amino and carboxy truncation mutants will be made in all the Gab clones and the affinity to Grb2 and those of the individual SH3 domains will be measured towards these substrates. The smallest region that still retains high affinity binding will be defined and from the evaluation of multiple clones a refined consensus binding site can be obtained. This analysis also will reveal if all the Gab clones possess more than one target site, whether the two SH3 domains have the same consensus binding site, if the two domains are necessary to give high affinity binding, and whether extended secondary structure of the target is necessary to confer high affinity binding. Other experiments will be done to confirm if the two domains result in co-operative binding to the effector. This information can be used to predict Grb2 interactions and to design novel inhibitors of Grb2.
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