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 Gi 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.