Historically, the signaling pathways used by G protein coupled receptors and receptor tyrosine kinases were considered very separate. However, current evidence indicates significant crosstalk between these pathways. Thus, occupation of G protein coupled receptors can stimulate the activity of tyrosine kinases, guanine nucleotide exchange factors and growth regulating pathways. G proteins markedly stimulate the p110-gamma isoform of phosphatidylinositol 3-kinase (Ptdlns 3-kinase) leading to production of phosphatidylinositol (3,4,5) trisphosphate. This lipid is an important signal that activates the phosphatidylinositol dependent protein kinase, PDK-1, leading to activation of protein kinase B and a host of signaling events. The focus of this project period is to understand the mechanisms by which G protein alpha and beta-gamma subunits activate the Ptdlns 3-kinase. This goal will be approached via 3 Specific Aims.
(Aim -1) To determine which G protein alpha and beta-gamma subunits are able to activate the Ptdlns 3-kinase. The broadly diverse members of the G protein family have differential activity on effectors. However, it is not known which members of the family activate this enzyme. In this aim, we will determine the ability of pure, recombinant G protein alpha subunits and beta-gamma dimers of defined composition to activate the purified p110-gamma isoform of Ptdlns 3-kinase.
(Aim -2) To understand the domains in the beta and gamma subunits that activate Ptdlns 3-kinase. It is not known which regions of the Py dimer interact with the p110-gamma form of Ptdlns 3-kinase. In this aim we will use beta-gamma dimers with selected point mutations in the beta subunit in combination with mutated and/or chimeric gamma subunits to evaluate the domains in the beta- gamma dimer which activate the Ptdlns 3-kinase.
(Aim -3) To understand how known regulatory mechanisms affect the activity of beta-gamma dimers on Ptdlns 3-kinase. We have demonstrated two novel regulatory mechanisms affecting the beta-gamma dimer. (a) Dimers containing the gamma11 subunit are not able to stimulate certain effectors such as adenylyl cyclase, and; (b) Dimers containing the gamma12 subunits can be phosphorylated by protein kinase C altering their activity. In this aim, we will examine how these and other regulatory mechanisms affect the ability of beta-gamma dimers to stimulate Ptdlns 3-kinase.
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