Mechanisms of ERBB receptor activation by radiation

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DESCRIPTION (provided by applicant): The exposure of autocrine growth regulated human tumor cells to therapeutically applied doses of ionizing radiation activates ERBB and other receptor Tyr kinases. Details of these responses, including the simultaneous activation of the compliment of ERBB receptors on a cell and downstream signaling along major existing pathways, have been described by our group. The activation of ERBB1 is characterized by a biphasic increase in Tyr phosphorylation, the primary response occurring within 1-5 min, whereas the later phase is seen between 60 and 240 min, possibly mediated by TGF-alpha release as the result of the initial activation. Relative to growth factors, e.g., EGF as ligand for ERBB1, radiation induces a less intense response at the receptor level; however, at the level of cytoplasmic protein kinases the signals are of identical intensity. The biphasic activation of ERBB1 and the simultaneous activation of all ERBB receptors, expressed at widely varied concentration by tumor cells, represent fundamental differences in radiation- and growth factor-induced receptor activation and form the basis of the continuation application. We expect to unravel the unique mechanisms of radiation ERBB activation and downstream signaling by identifying the Tyr phosphorylation patterns of ERBBI using proteomics technology (mass spectroscopy phosphopeptide identification) and kinase and dominant negative receptor inhibitors of ERBB1 and its heterodimerization partner ERBB2. The overall objectives are addressed in three Specific Aims (SAs). SAI: The detailed ERBB1 Tyr phosphorylation pattern is defined in stably transfected CHO.ERBB1 cells after exposure to radiation and TGF-alpha. If feasible, ERBB1 is enriched in the plasma and the endosomal membranes to examine possible changes in Tyr phoshorylation during receptor processing. ERBB1 Tyr phosphorylation is examined as a function of receptor homo- and heterodimedzation through studies on interactions with ERBB2 and dominant negative ERBB1-CD533. SA2: ERBB1 Tyr phosphorylation is correlated with the formation of receptor protein signaling complexes, including studies with the Tyr-Phe ERBB1 mutation to affirm the functional importance of individual Tyr residues. SA3. This aim addresses the mechanisms underlying the immediate and simultaneous activation of ERBB receptors by investigating the potential role of protein Tyr phosphatases, known to interact with ERBBI, in controlling the quiescent steady state Tyr phosphorylation of ERBB1 and its processing with the cellular membranes.