Engagement of heterotrimeric G proteins by Sonic hedgehog

描述(由申请人提供)：Sonic hedgehog(Shh)获得的信号在脊椎动物的发育和维持几个胚胎后干细胞种群中起着至关重要的作用。Shh利用的途径的一个必备成分是Smoothened，一种7-跨膜蛋白。我们已经用G蛋白激活的直接分析确定了小鼠明确地与异三聚体G蛋白的GI家族的所有成员进行了平滑配对。我们还证明了百日咳毒素，它破坏了Gi与受体的偶联，抑制了Shh在NIH3T3成纤维细胞中激活Gli转录因子，这是Shh作用的常用模型。这些研究的总体目标是更好地了解与Shh信号相关的转导机制，重点是那些通过或与G蛋白一起介导的转导机制。第一个目的是评估Smoothed对GI家族内外G蛋白的选择性。我们将跟踪GI家族中最重要的成员的耦合强度的差异，并报告Smo也可以耦合到G12家族的成员。第二个目标是确定与Shh的行动相关的GI的目标(S)。我们将评估Shh对可疑靶标的百日咳毒素敏感性调节，模拟百日咳毒素处理细胞中靶标的作用，并评估通过Gi招募2-拦阻剂。第三个目的是评估作为Shh靶点的原代细胞，特别是原代成纤维细胞、小脑颗粒前体细胞和新皮质少突胶质细胞前体细胞对GI的需求。第四个具体目标是确定除了GI的活动之外，SMO还需要哪些信号。我们感兴趣的是测试Gi和2-arrestins是否都是激活Gli转录因子所必需的，并且它们一起足以激活Gli转录因子。Sonic Hedgehog是一种在胚胎发育和组织再生中发挥重要作用的蛋白质。这种蛋白信号的缺失会导致发育缺陷，而不受抑制的信号会导致几种癌症。了解Sonic Hedgehog信号的机制将为如何在治疗环境中操纵该信号提供重要线索。

DESCRIPTION (provided by applicant): The signaling achieved by Sonic hedgehog (Shh) plays an essential role in vertebrate development and in the maintenance of several postembryonic stem cell populations. An obligatory component of the pathway utilized by Shh is Smoothened, a 7-transmembrane protein. We have determined using direct assays of G protein activation that mouse Smoothened couples unequivocally to all members of the Gi family of heterotrimeric G proteins. We have also demonstrated that pertussis toxin, which disrupts coupling of Gi to receptors, inhibits the activation of Gli transcription factors by Shh in NIH 3T3 fibroblasts, a commonly employed model of Shh action. The overall goal of the proposed studies is to understand better the mechanisms of transduction relevant to Shh signaling, with an emphasis on those mediated through, or in conjunction with, G proteins. The first aim is to evaluate the selectivity of Smoothened for G proteins within and beyond the Gi family. We will follow up differences in strength of coupling to the most important members of the Gi family and reports that Smo can couple to members of the G12 family as well. The second aim is to identify the target(s) for Gi relevant to the actions of Shh. We will evaluate pertussis toxin-sensitive regulation by Shh of suspected targets, mimic the actions of targets in pertussis toxin-treated cells, and evaluate recruitment through Gi of 2-arrestins. The third aim is to evaluate the requirement for Gi among primary cells serving as targets for Shh, specifically primary fibroblasts, cerebellar granule precursors, and neocortical oligodendrocyte progenitors. The fourth specific aim is to determine what signaling is required by Smo beyond the activity of Gi. We are interested in testing whether Gi and 2-arrestins are both required and together sufficient for activation of Gli transcription factors. Sonic hedgehog is a protein that plays an essential role in embryonic development and tissue regeneration. Deficits in signaling by this protein lead to developmental defects, while unrepressed signaling leads to several cancers. An understanding of the mechanisms by which Sonic hedgehog signals will provide important leads into how that signaling might be manipulated in a therapeutic context.