Engagement of heterotrimeric G proteins by Sonic hedgehog

描述（由申请人提供）：Hedgehog家族分泌蛋白实现的信号传导在脊椎动物的发育和几种胚胎后干细胞群体的维持中起着重要作用。刺猬利用的途径的一个必要成分是Smoothened，一种7-跨膜（7- tm）蛋白。平滑配对明确的所有成员的异源三聚体G蛋白的Gi家族。虽然在Gli转录因子（Gli）的激活中可能需要Gi，但该建议的一个前提是，G通过“非规范”信号传导为刺猬提供了在其行动中扩展到远远超出Gli范围的能力。另一个前提是，除了Smoothened， Gli还可以通过7-TM受体进行控制。第一个目的是评估我们认为存在于规范和非规范信号之间的张力的决定因素。我们怀疑，并将测试Smo在初级纤毛中的隔离和/或抑制作用，除了促进隔离之外，是以牺牲非规范信号为代价的。后者的指标，即Gi活性，是RhoA的激活和腺苷酸环化酶的抑制。在这个目标中，我们还将评估在某些细胞中存在于Gi和规范信号之间的容许界面的性质。第二个目的是评估配体对典型和非典型信号的不同参与。我们将评估Gi在建立Smo对激动剂亲和力方面的作用，作为激动剂浓度的函数，规范和非规范信号传导顺序而不是同时参与的可能性，以及偏向激动作用的可能性。这项工作的基础是放射性标记位移、抑制蛋白募集、gli1和RhoA激活以及Smo易位的分析。第三个目的是评估除Gi外的G蛋白激活Gli的性质和后果，重点是G13。在这方面感兴趣的是G¿亚基的内在活动，G13控制Gli的机制，以及G13与刺猬在这种控制的性质和程度上的相似性。

DESCRIPTION (provided by applicant): The signaling achieved by the Hedgehog family of secreted proteins plays an essential role in vertebrate development and maintenance of several postembryonic stem cell populations. An obligatory component of the pathway utilized by Hedgehogs is Smoothened, a 7- transmembrane (7-TM) protein. Smoothened couples unequivocally to all members of the Gi family of heterotrimeric G proteins. While Gi can be required in the activation of Gli transcription factors (Gli), a premise of this proposal is that G provides to Hedgehogs, through 'noncanonical' signaling, the ability to expand well beyond the bounds of Gli in their actions. An additional premise is that Gli can be controlled through 7-TM receptors apart from Smoothened. The first aim is to evaluate determinants of a tension we believe to exist between canonical and noncanonical signaling. We suspect, and will test, that Smo's sequestration into the primary cilium and/or arrestin's actions apart from facilitating sequestration occurs at the expense of noncanonical signaling. Indices of the latter, i.e. Gi activity, are the activation of RhoA and inhibition of adenylyl cyclase. We will also evaluate in this aim the nature of a permissive interface, in certain cells, that exists between Gi and canonical signaling. The second aim is to evaluate differential engagement of canonical and noncanonical signaling by ligands. We will evaluate the role of Gi in establishing Smo's affinity for agonists, the possibility that canonical and noncanonical signaling are sequentially rather than simultaneously engaged as a function of agonist concentration, and the likelihood of biased agonism. The underpinnings of this work are assays of radiolabel displacement, arrestin recruitment, gli1 and RhoA activation, and Smo translocation. The third aim is to evaluate the nature and consequences of activation of Gli by G proteins apart from Gi, with a focus on G13. Of interest in this regard are the intrinsic activities of G¿ subunits, the mechanism by which G13 controls Gli, and the similarity of G13 to Hedgehogs in the nature and extent of this control.