HORMONAL REGULATION OF MAP KINASE VIA RAP1

通过 RAP1 调节 MAP 激酶的激素

• 批准号: 6497690
• 负责人: PHILIP J.S. STORK
• 金额: $ 22.33万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6497690
• 关键词: G protein; Xenopus oocyte; cyclic AMP; dopamine; dopamine receptor; enzyme activity; hormone receptor; hormone regulation /control mechanism; isoproterenol; mitogen activated protein kinase; protein kinase A; somatostatin

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.

激素作用的主要功能之一是调节细胞生长。激素作用是在激素与将细胞外信号与细胞内信号耦合的特定受体结合后启动的。 对于大多数激素来说，这是通过异三聚体 G 蛋白实现的，异三聚体 G 蛋白由 α (Galpha) 和 β-gamma (Gbeta-gamma) 两种成分组成，可以独立发挥作用以触发细胞内信号。 现在已经清楚，G 蛋白可以通过激活细胞内磷酸化级联（将激素与 MAP 激酶级联连接起来）来调节细胞生长。 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 Gbeta-gamma 到 Ras 的信号，Ras 是一种激活 MAP 激酶激酶 Raf-1 的小 G 蛋白。 与这种普遍观点相反，我们发现了 Galpha 调节 ERK 的三种新的潜在途径。 其中两个涉及 Galpha-s 的激活，Galpha-s 是一种与刺激腺苷酸环化酶以增加细胞内 cAMP 相关的 G 蛋白。 cAMP 对细胞增殖和活化的作用具有细胞类型特异性。 我们认为这些作用取决于 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。 这些亚基因其对百日咳毒素的敏感性而闻名，但它们调节的细胞内信号尚不清楚。 我们提出所有三个 G α 亚基 Galpha-s、Galpha-i 和 Galpha-o 通过一种新型小 G 蛋白 Rap1 调节 ERK。 本提案的目的是阐明 Galpha 调节 Rap l 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种潜在机制的实验。具体目标 1 通过 cAMP 和 PKA 以及 Raf 同工型 B-Raf 对 Rap l 的作用来检查 Galpha-s 对 ERK 的激活。 具体目标 2 重点关注 Galpha-s 通过 Rap 1 拮抗 Ras 来抑制 ERK 信号传导。具体目标 3 将把我们的发现扩展到爪蟾卵母细胞成熟模型。 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）相互作用来调节 ERK 的新机制。