HORMONAL REGULATION OF MAP KINASE VIA RAP1

激素作用的主要功能之一是调节细胞生长。激素作用是在激素结合到将细胞外信号与细胞内信号偶联的特异性受体后启动的。 对于大多数激素，这是通过异源三聚体G蛋白实现的，异源三聚体G蛋白由两种组分组成，α（Galpha）和β-γ（Gbeta-gamma），它们可以独立地起作用以触发细胞内信号。 现在很清楚，G蛋白可以通过激活细胞内磷酸化级联反应来调节细胞生长，磷酸化级联反应将激素与MAP激酶级联反应联系起来。 G蛋白激活MAP激酶ERK的机制尚不清楚。 据认为，从G蛋白到MAP激酶的主要途径之一是通过从G β-γ到Ras的信号，Ras是一种激活MAP激酶Raf-1的小G蛋白。 与这种流行的观点相反，我们已经确定了三种新的潜在途径，通过这些途径，Galalpha可以调节ERK。 其中两个涉及Galpha-s的激活，Galpha-s是一种与腺苷酸环化酶刺激相关的G蛋白，可增加细胞内cAMP。 cAMP对细胞增殖和活化的作用是细胞类型特异性的。 我们认为这些作用依赖于MAP激酶激酶B-Raf的选择性激活和Raf-1的抑制。 我们建议测试激活Galpha-s的激素是否也以这种方式调节ERK。 我们还表明，Galpha-i和Galpha-s也可以调节ERK。 这些亚基对百日咳毒素的敏感性是众所周知的，但是它们调节的细胞内信号还没有很好地建立。 我们提出，所有三个G α亚基，Galpha-s，Galpha-i和Galpha-o，通过一种新的小G蛋白Rap 1调节ERK。 该建议的目标是阐明Ga调节Rap 1的分子机制，并确定这种调节对ERK的影响。 在四个具体目标中，我们提出了旨在检查每个潜在机制的实验。特异性目的1通过其经由cAMP和PKA和Raf同种型B-Raf对Rap 1的作用来检查ERK的Galpha-s活化。 具体目标2关注Galpha-s通过Rap 1对Ras的拮抗作用抑制ERK信号传导。具体目标3将我们的研究结果扩展到非洲爪蟾卵母细胞成熟的模型。 最后，Specific Aim 4探索了Galpha-i和Galpha-o通过与Rap 1 GAP（一种选择性抑制剂或Rap 1）相互作用进行ERK调节的新机制。

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.