The overall goal of this application is to elucidate the mechanisms involved in the cross talk between G-protein-coupled receptors (GPCRs) and the MAP kinase (MAPK) pathway. The hypotheses to be tested are that 1) GPCRs signal through the MAPK pathway via molecules/kinases different from those involved in the EGF pathway, but converging on Ras/Raf-1/MEK and 2) the MAPK pathway, in turn, modulates calcium, cAMP, and cPLA2 signaling, providing a feedback mechanism for regulating intracellular messenger levels.
Specific Aims are:Identification and determination of the role of molecules/kinases involved in GPCR-mediated activation of MAPK: Molecules/kinases will be identified in cell lysates by Western blotting with specific antibodies. M3 receptor-mediated activation of MAPK will be assessed by determining the involvement of calcium, PKC, non-receptor tyrosine kinases (TYKs), P13-K, A-Kinase, G-kinase, and the epidermal growth factor receptor (EGFR), and by biochemical evidence of an interaction between these molecules. Kinase activation will be by phosphorylation and in-vitro assays. Beta-adrenergic receptor (Beta-AR)-mediated activation of MAPK will be assessed by determining: phosphorylation and association of Rap 1 with B-Raf and members of the MAPK pathway, an interaction with other molecules associated with Rap 1, and by transmodulation of EGFR. Determination of the role of the MAPK pathway in GFCR-induced Signaling: The mechanism(s) linking TYKs to GPCR-induced calcium signaling, and amylase release will be assessed by correlating M3-induced changes in calcium release/entry with tyrosine phosphorylation of Gq/l 1, PLCgamma, and 1P3 receptors, and store emptying. Beta-AR-induced cAMP signaling will be assessed by correlating cAMP-regulation of MAPK with stimulation of PDE4 isoenzymes which will be identified by immunoprecipitation and immunoblot analysis with specific PDE4D antibodies, and PDE assays, and amylase release. Determination of the role of the MAPK pathway in GPCR-induced cPLA2 activation and signaling: MAPK activation of cPLA2 will be determined by measuring phosphorylation of cPLA2. Mechanisms involved in AA-induced MAPK activation will be assessed by determining the involvement of calcium, PKC, TYKs and EGFR. A role for TYKs in PLA2-induced calcium signaling will be assessed by correlating AA/metabolite-induced calcium release/entry with tyrosine phosphorylation of Pyk2, Src kinases and P13-K. Mechanisms/pathways involved in AA/metabolite-mediated cAMP signaling will be assessed by correlating MAPK activation with cAMP synthesis and degradation.
