Engagement of heterotrimeric G proteins by Sonic hedgehog

描述（由申请人提供）：Sonic hedgehog（Shh）实现的信号传导在脊椎动物发育和几种胚后干细胞群的维持中起重要作用。Shh利用的途径的一个强制性组分是Smoothened，一种7-跨膜蛋白。我们已经使用G蛋白活化的直接测定确定小鼠Smoothened明确地与异源三聚体G蛋白的Gi家族的所有成员偶联。我们还证明，百日咳毒素，破坏耦合的Gi受体，抑制激活Gli转录因子的Shh在NIH 3 T3成纤维细胞，一种常用的模型Shh行动。拟议研究的总体目标是更好地了解与Shh信号相关的转导机制，重点是通过G蛋白介导或与G蛋白结合的转导机制。第一个目的是评估Smoothened对Gi家族内外G蛋白的选择性。我们将追踪与Gi家族最重要成员偶联强度的差异，并报告Smo也可以与G12家族成员偶联。第二个目标是识别与Shh的动作相关的Gi的目标。我们将通过Shh对疑似靶点的百日咳毒素敏感性调节进行评估，模拟百日咳毒素处理的细胞中靶点的作用，并通过Gi对2-arrestins的募集进行评估。第三个目的是评估作为Shh靶的原代细胞，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祖细胞对Gi的需求。第四个具体目标是确定Smo除了Gi的活性之外还需要什么信号传导。我们感兴趣的是测试是否Gi和2-arrestins都是必需的，并在一起足以激活Gli转录因子。Sonic hedgehog是一种在胚胎发育和组织再生中起重要作用的蛋白质。这种蛋白质的信号缺陷会导致发育缺陷，而未受抑制的信号会导致几种癌症。对Sonic hedgehog信号机制的理解将为如何在治疗背景下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.