CELL SIGNALING REGULATION BY G PROTEIN SUBUNITS

该项目的目标是了解G蛋白α和β γ亚基的多样性如何导致跨膜信号的特异性。 我们的主要努力集中在β γ亚基的多样性，并已证明，重组β γ二聚体的定义的亚基组合物具有不同的活动，在他们的能力耦合α亚基的受体在膜上或直接激活效应。这些令人兴奋的结果为完整细胞中的信号选择性提供了潜在的解释。 在下一个项目期间，我们计划扩大我们对重组β-γ二聚体功能差异的研究，以更好地了解细胞信号传导的特异性。 这些β-γ二聚体的功能将通过三个特定目的进行探索。 目的1 -探索含有不同β亚基如β 5的β γ二聚体的功能作用。 我们的结果与二聚体组成的beta5 gamma 2亚基表明选择性耦合到Gq α亚基和激活的一组有限的效应。 我们将研究一组二聚体的功能，这些二聚体含有β 5亚基和各种γ亚基，它们能够将受体偶联到α亚基并激活效应子。 我们还将检查变异体（β 3S二聚体，在高血压患者中鉴定，缺失一个WD-40重复序列）的功能。 目的2 -确定γ亚基的哪些结构域与受体和效应子相互作用。使用一系列嵌合γ亚基含有开关和C-末端结构域从一对活性和非活性γ亚基，我们将确定这些结构域的作用，使用受体相互作用和效应器功能的测定。 目的3 -检查γ亚基的异戊烯基参与激活β-γ二聚体的可能性。 含有用C15法呢基修饰的γ亚基的二聚体的活性通常低于含有用C20香叶基香叶基修饰的γ亚基的二聚体。 我们将使用最近的观察，异戊二烯基团直接与β亚基相互作用，以测试的可能性，这两种脂质导致不同的构象变化的β亚基。 该方法将是制造异戊烯基无法折叠回β亚基的突变β亚基，并在受体相互作用和效应器功能的测定中测试它们的活性。

The goal of this project has been to understand how the diversity of G protein alpha and betagamma subunits leads to specificity in transmembrane signaling. Our major effort has centered on the diversity of the betagamma subunits and has demonstrated that recombinant betagamma dimers of defined subunit composition have different activities in their ability to couple alpha subunits to receptors in the membrane or to directly activate effectors. These exciting results provide a potential explanation for signaling selectivity in the intact cell. During the next project period we plan to expand our study of the functional differences of recombinant betagamma dimers to better understand the specificity of cell signaling. The function of these betagamma dimers will be explored via three Specific Aims. Aim 1 - To explore the functional role of betagamma dimers containing divergent beta subunits such as beta5. Our results with dimers composed of the beta5gamma2 subunits indicate selective coupling to the Gq alpha subunit and activation of a restricted set of effectors. We will examine the function of a panel of dimers containing the beta5 subunit combined with various gamma subunits for their ability to couple receptors to alpha subunits and to activate effectors. We will also examine the function of the variant (beta3S dimer, identified in hypertensive patients, which is missing one WD-40 repeat. Aim 2 - To determine which domains of the gamma subunit interact with receptors and effectors. Using a series of chimeric gamma subunits containing switched and C-terminal domains from a pair of active and inactive gamma subunits, we will determine the role of these domains using assays of receptor interaction and effector function. Aim 3 - To examine the possibility that the prenyl group of the gamma subunit participates in activating the betagamma dimer. Dimers containing gamma subunits modified with the C15 farnesyl group are usually less active than dimers containing gamma subunits modified with the C20 geranylgeranyl group. We will use the recent observation that the prenyl group interacts directly with the beta subunit to test the possibility that the two lipids cause different conformational changes in the beta subunit. The approach will be to make mutant beta subunits in which the prenyl group is not able to fold back into the beta subunit and test their activity in assays of receptor interaction and effector function.