HORMONAL REGULATION OF MAP KINASE VIA RAP1

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One of the major functions of hormone action is the regulation of cell growth. Hormone action is initiated upon hormone binding to specific receptors that couple extracellular signals to intracellular ones. For most hormones, this is achieved via heterotrimeric G proteins which are composed of two components, alpha (Galpha) and beta-gamma (Gbeta-gamma) that may function independently to trigger intracellular signals. It is now clear that G proteins can regulate cell growth by activating intracellular phosphorylation cascades that link hormones to the MAP kinase cascade. The mechanisms by which G proteins activate MAP kinase ERK are poorly understood. It is thought that one of the major pathways from G proteins to MAP kinase is via signals from Gbeta-gamma to Ras, a small G protein that activates the MAP kinase kinase kinase Raf-1. In contrast to this prevailing view, we have identified three novel potential pathways by which Galpha can regulate ERK. Two of these involve the activation of Galpha-s, a G protein linked to the stimulation of adenylyl cyclase to increase intracellular cAMP. The action of cAMP on cellular proliferation and activation is cell-type specific. We propose that these actions depend on the selective activation of MAP kinase kinase kinases B-Raf and inhibition of Raf-1. We propose to test whether hormones that activate Galpha-s also regulate ERKs in this way. We have also shown that Galpha-i and Galpha-s can also regulate ERKs. These subunits are well known for their sensitivity to pertussis toxin, but the intracellular signals they regulate are not well established. We propose that all three G alpha subunits, Galpha-s, Galpha-i, and Galpha-o, regulate ERKs via a novel small G protein Rap1. The goal of this proposal is to elucidate the molecular mechanisms by which Galpha regulates Rap l and to determine the consequence of this regulation on ERK. In four specific aims, we propose experiments designed to examine each potential mechanism. Specific Aim 1 examines Galpha-s activation of ERK through its actions on Rap l via cAMP and PKA and the Raf isoform B-Raf. Specific Aim 2 focuses on Galpha-s inhibition of ERK signaling via Rap 1's antagonism of Ras. Specific Aim 3 will extend our findings to a model of oocyte maturation in Xenopus. Finally, Specific Aim 4 explores a novel mechanism of ERK regulation by Galpha-i and Galpha-o through their interaction with Rap 1 GAP, a selective inhibitor or Rap 1.