Complex cellular responses involving changes in the actin cytoskeleton, cell shape and migration involve the integration of signal transduction pathways regulated by seven transmembrane receptor (STMR) heterotrimeric G proteins and tyrosine kinases. A convergence point for the integration of STMR/G protein and tyrosine kinase signaling is the control of low molecular weight GTP binding proteins including Ras and Rho family members (Rho, Rac, Cdc42). Characterization of STMR/G protein integration with tyrosine kinases and Ras/Rho GTP binding proteins will be defined in two systems: i. chemokine receptor signaling in the neutrophil, and ii. gastrin releasing peptide (GRP) receptor regulation of Gq and G12,13 signaling in fibroblasts. Neutrophils are a primary source of tissue injury at inflammatory sites in diseases including respiratory distress syndrome and inflammatory bowel disease. Neutrophils migrate to an inflammatory site in response to chemokines such as IL8. The IL8 receptor and other chemokine receptors are STMR coupled to G proteins which in response to chemokine rapidly activate the tyrosine kinases Lyn, Fgr, Hck and Syk. We shall define the requirement of these kinases in neutrophil responses to chemokines using neutrophils isolated from mice having the triple targeted disruption of the Lyn, Fgr and Hck genes (Lyn-/-/Fgr-/-/Hck-/-) and the Syk gene (Syk- /-). The targeted disruption of the primary Src kinases (Lyn, Fgr, Hck) expressed in neutrophils and Syk will allow for the first time to define the involvement of these kinases in chemokine activation of the neutrophil and their role in chemokine stimulated inflammatory responses. Neuropeptides including GRP function in neuron-to-neuron communication, signaling in the immune system, epithelial cell proliferation and tissue restructuring. The GRP receptor is a STMR coupling the G proteins Gq,11 and G12,13. Peptide ligands have been characterized that activate the GRP receptor so that only G12,13 and not Gq,11 are activated. The peptide ligands function as """"""""biased agonists"""""""" that induce """"""""asymmetric signaling"""""""". G12,13 regulates the c-Jun kinase pathway and Rho family GTP binding proteins. Biased agonists provide a mechanism for the selective control of G protein signaling by STMRs. The amino acids in the GRPR primary sequence that are required for biased signaling will be defined. The consequence of biased agonism on cell growth, gene expression and regulation of the actin cytoskeleton will be defined. Cumulatively, the studies will define new strategies for the development of pharmacological ligands to regulate subsets of receptor functions that will have much more selective effects on target cells and tissues.
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