Smoothened activates Galphai-mediated signaling in frog melanophores

The 7-pass transmembrane protein Smoothened was investigated for its ability to act as a G-protein-coupled receptor in Xenopus laevis melanophores. A plasmid containing the human Smoothened cDNA insert was transfected into immortalized frog pigment cells. Cells expressing the protein showed a phenotype of persistent pigment aggregation, a hallmark of constitutive Galpha(i) activation. Smoothened-mediated pigment aggregation was reversed by treatment with pertussis toxin or by co-expression with dominant negative Galpha(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 inhibitor of Smoothened. That the reconstituted Patched-Smoothened receptor complex functions normally in pigment cells was demonstrated by co-transfection with 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 findings demonstrate that the human Sonic hedgehog receptor complex can be functionally reconstituted in melanophores and that it is capable of transmembrane signaling by utilizing endogenous Galpha(i).