The rationale for this proposal is based upon similarities between regulation of Ras/mitogen-activated protein kinase (MAPK) pathway, and the regulation of electroneutral Na+/H+ exchangers, both of which have been implicated in growth regulation. Recent studies have shown that stimuli that rapidly activate the Ras-MAPK pathway also increase Na+/H+ exchanger activity in many cells, particularly in fibroblasts. Those stimuli include: 1) growth factors that modulate tyrosine phosphorylation cycles; 2) integrins; 3) hyperosmotic stress or cell shrinkage; 4) tyrosine phosphorylation cascades; 5) heterotrimeric G proteins; and 6) protein kinase C. Moreover, recent evidence suggests that RasMAPK may be direct proximal components of a Na+/H+ exchange regulatory pathway. They hypothesize that all stimulators of Na+/H+ exchange ultimately converge upon a single signaling pathway, and that the Ras-MAPK pathway serves as a """"""""funnel"""""""" for most signals that lead to activation of Na+/H+ exchange. The hypothetical pathway to be investigated would involve cell surface receptor - G protein subunits - tyrosine phosphorylation cycle including kinase(s) and/or phosphatases - docking & adaptor molecules Ras-GTP - Raf 1 kinase - MEK - and MAPK. Other likely upstream signaling molecules to be tested include phosphatidylinositol 3'-kinase, protein kinase C, and PTPlD phosphatase. In the interest of maintaining focus, the involvement of specific downstream effectors of MAPK, including S6 kinase, cytoskeleton, and cytoplasmic phospholipase A2, will not be tested unless specific advances strongly implicate them, and then only after the hypothesized convergence upon MAPK is confirmed. Recent technical advances, particularly in the areas of microphysiometry and molecular biology, have provided tools that will make feasible for the first time some of the studies proposed in this application. They will focus upon the signaling pathways linking G-alpha to Na+/H+ exchange.
The Specific Aim of this proposal is as follows: 1) to dissect the proximal signals that lead from activation of G proteins to stimulation of type 1 Na+/H+ exchangers expressed in CHO-KI fibroblasts and MDCK cells. In order to accomplish this aim, the investigator will use a combination of techniques derived from molecular biology, pharmacology, physiology, and the new technique of """"""""microphysiometry"""""""". The respective hypothetical roles of the following potential signaling molecules will be investigated: G alpha and G beta-gamma subunits, non- receptor tyrosine kinases of the Src family, multifunction docking proteins such as SHC and IRS-l, so-called adaptor proteins such as Grb2 and Sos, Ras-GTP, mitogen-activated serine-threonine and dual specificity kinase signaling cascade, and other related molecules, such as phosphatidylinositol 3'-kinase, protein kinase C, and PTP1D.
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