The hedgehog (Hh) signalling pathway has an evolutionarily conserved role in patterning fields of cells during metazoan development, and is inappropriately activated in cancer1,2. Hh pathway activity is absolutely dependent on signalling by the seven-transmembrane protein smoothened (Smo), which is regulated by the Hh receptor patched (Ptc). Smo signals to an intracellular multi-protein complex containing the Kinesin related protein Costal2 (Cos2), the protein kinase Fused (Fu) and the transcription factor Cubitus interruptus (Ci)3. In the absence of Hh, this complex regulates the cleavage of full-length Ci to a truncated repressor protein, Ci75, in a process that is dependent on the proteasome and priming phosphorylations by Protein kinase A (PKA)4. Binding of Hh to Ptc blocks Ptc-mediated Smo inhibition, allowing Smo to signal to the intracellular components to attenuate Ci cleavage. Because of its homology with the Frizzled family of G-protein-coupled receptors (GPCR)5, a likely candidate for an immediate Smo effector would be a heterotrimeric G protein. However, the role that G proteins may have in Hh signal transduction is unclear and quite controversial6,7,8,9,10, which has led to widespread speculation that Smo signals through a variety of novel G-protein-independent mechanisms. Here we present in vitro and in vivo evidence in Drosophila that Smo activates a G protein to modulate intracellular cyclic AMP levels in response to Hh. Our results demonstrate that Smo functions as a canonical GPCR, which signals through Gαi to regulate Hh pathway activation.
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This work was supported by National Institutes of Health grants CA82628 (D.J.R.) and HL074190 (J.H.). We are grateful to J. Knoblich for the Gαi antibody and Gαi flies; X. Yang and W. Chia for Gαi flies; D. Casso and T. Kornberg for Ptc antibody and UAS-Smo5A flies; J. Hooper for UAS-Gαi and UAS-GαiQ205L flies; K. Basler, D. Kalderon and P. Ingham for smo alleles; and L. Lum for Smo antibody. All additional fly stocks were provided by the Bloomington Stock Center. Gαi and Gαo cDNAs were obtained from the Drosophila Genomics Resource Center, Bloomington. E. Lee provided Gαs cDNA. We thank the Dartmouth microscopy core for their assistance. We would also like to thank K. Black for technical assistance and members of the Robbins laboratory, E. Lee, J. Hutchinson, R. Taussig, R. Ostrom and C. Pikielny for thoughtful discussion during the course of this work.
Author Contributions S.K.O. was involved in the design, execution and analysis of experiments, and in writing the manuscript. D.L.F. assisted in design and execution of dsRNA experiments and sequencing of Gαi alleles. J.H. was involved in design and execution of cAMP assays. N.S.S. assisted in execution of cAMP assays. Y.F.A. consulted on design and interpretation of genetic experiments. D.J.R. was involved in design and analysis of experiments and writing the manuscript.
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Ogden, S., Fei, D., Schilling, N. et al. G protein Gαi functions immediately downstream of Smoothened in Hedgehog signalling. Nature 456, 967–970 (2008) doi:10.1038/nature07459
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