Engagement of heterotrimeric G proteins by Sonic hedgehog

描述（由申请人提供）：由Sonic hedgehog （Shh）实现的信号传导在脊椎动物发育和几种胚胎后干细胞群体的维持中起着至关重要的作用。Shh利用的途径的一个必要成分是Smoothened，一种7-跨膜蛋白。我们已经通过G蛋白激活的直接测定确定小鼠Smoothened与异源三聚体G蛋白的Gi家族的所有成员明确结合。我们还证明，百日咳毒素破坏Gi与受体的偶联，抑制NIH 3T3成纤维细胞中Shh对Gli转录因子的激活，这是一种常用的Shh作用模型。拟议研究的总体目标是更好地理解与Shh信号相关的转导机制，重点是通过G蛋白介导或与G蛋白一起介导的转导机制。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Gi家族中最重要成员的偶联强度差异，并报告Smo也可以与G12家族成员偶联。第二个目标是确定与Shh作用相关的Gi靶标。我们将评估Shh对可疑靶点的百日咳毒素敏感调节，模拟靶点在百日咳毒素处理细胞中的作用，并通过Gi评估2-骤停蛋白的募集。第三个目的是评估作为Shh靶点的原代细胞对Gi的需求，特别是原代成纤维细胞、小脑颗粒前体和新皮质少突胶质细胞祖细胞。第四个具体目标是确定除了Gi的活性之外，Smo还需要什么信号。我们感兴趣的是测试Gi和2-阻滞蛋白是否都是激活Gli转录因子所必需的和充分的。Sonic hedgehog是一种在胚胎发育和组织再生中起重要作用的蛋白。这种蛋白的信号传导缺陷导致发育缺陷，而不受抑制的信号传导导致几种癌症。对Sonic hedgehog信号机制的理解将为研究如何在治疗环境中操纵该信号提供重要的线索。

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.