Smoothened activates G alpha(i)-mediated signaling in frog melanophores

The 7-pass transmembrane protein Smoothened was investigated for its abilit y to act as a G-protein-coupled receptor in Xenopus laevis melanophores. A plasmid containing the human Smoothened cDNA insert was transfected into im mortalized frog pigment cells. Cells expressing the protein showed a phenot ype of persistent pigment aggregation, a hallmark of constitutive G(alpha i ) activation. Smoothened-mediated pigment aggregation was reversed by treat ment with pertussis toxin or by co-expression with dominant negative G(alph a i). The ability of melanophores to express functional Smoothened was also determined by its co-expression with the twelve-pass transmembrane protein , Patched. Patched blocked Smoothened-mediated melanosome aggregation in a dose-dependent manner, consistent with its physiological role as an inhibit or of Smoothened. That the reconstituted Patched-Smoothened receptor comple x functions normally in pigment cells was demonstrated by co-transfection w ith the activating ligand, Sonic hedgehog, as well as by direct application of the recombinant Sonic hedgehog protein. Sonic hedgehog reversed Patched -mediated inhibition of Smoothened and induced pigment aggregation. The fin dings demonstrate that the human Sonic hedgehog receptor complex can be fun ctionally reconstituted in melanophores and that it is capable of transmemb rane signaling by utilizing endogenous G(alpha i).