Activation of heterotrimeric G proteins by Smoothened

Abstract

The mechanisms by which the activation of Smoothened (Smo), a protein essential to the actions of the Hedgehog family of secreted proteins, is translated into signals that converge on the Gli transcription factors are not fully understood. The seven-transmembrane structure of Smo has long implied the utilization of heterotrimeric GTP-binding regulatory proteins (G proteins); however, evidence in this regard has been indirect and contradictory. In the current study we evaluated the capacity of mammalian Smo to couple to G proteins directly. We found that Smo, by virtue of what appears to be constitutive activity, activates all members of the G i family but does not activate members of the G s , G q , and G 12 families. The activation is suppressed by cyclopamine and other inhibitors of Hedgehog signaling and is enhanced by the Smo agonist purmorphamine. Activation of G i by Smo is essential in the activation of Gli in fibroblasts, because disruption of coupling to G i with pertussis toxin inhibits the activation of Gli by Sonic hedgehog and a constitutively active form of Smo (SmoM2). However, G i does not provide a sufficient signal because a truncated form of Smo, although capable of activating G i , does not effect activation of Gli. Rescue of pertussis toxin-inhibited activation of Gli by Sonic hedgehog can be achieved with a constitutively active Gα i -subunit. The data suggest that Smo is in fact the source of two signals relevant to the activation of Gli: one involving G i and the other involving events at Smo’s C -tail independent of G i .