Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Dec 18;456(7224):967-70.
doi: 10.1038/nature07459.

G protein Galphai functions immediately downstream of Smoothened in Hedgehog signalling

Affiliations

G protein Galphai functions immediately downstream of Smoothened in Hedgehog signalling

Stacey K Ogden et al. Nature. .

Abstract

The hedgehog (Hh) signalling pathway has an evolutionarily conserved role in patterning fields of cells during metazoan development, and is inappropriately activated in cancer. 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). 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). 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), 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 controversial, 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 Galphai to regulate Hh pathway activation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Gαi is required for Hh signaling. a-b. Cl8 cells were transfected with ptc-luciferase, act-renilla, hh expression vector or empty vector control, and the indicated dsRNA and/or Gαi expression vectors. Percent reporter expression relative to maximal Hh activity for control dsRNA is shown. c. Cl8 cells were transfected with control or Gαi dsRNA and hh expression vector or empty vector control. Cell lysates were analyzed by immunoblotting with the indicated antibodies. The arrow marks the phosphorylation-induced mobility shift. Kinesin (Kin) serves as a loading control. Error bars indicate s.e.m.
Figure 2
Figure 2
Gαi expression results in ectopic Hh signaling. a-g. Longitudinal veins are numbered (a). Wings from (b) UAS-GαiG204A, (c) UAS-Gαi (d, e) UAS-GαiQ205L demonstrate ectopic veins (arrows) and LV3-4 widening (brackets), as compared to control (a). Wing discs from (f) dpp-lacZ, UAS-GαiQ205L and (g) dpp-lacZ, UAS-GαiQ205L, MS1096-Gal4 larvae were immunostained for Ci (red) and β-galactosidase (green). Overlays are shown in f* and g*. Anterior is left and dorsal is up. h-j. Expression of wild type Gαi partially rescues, and GαiQ205L fully rescues, the phenotype of induced by dominant negative UAS-Smo5A (compare i and j to h).
Figure 3
Figure 3
Gαi is required for Hh signaling in vivo. a-c. Mitotic clones were generated with the GαiP20 null (a, c) or GαiP8 hypomorphic (b) alleles. Wing discs were stained for the dpp-LacZ gene product β-gal (red). Loss of GFP expression (green) marks Gαi mutant clones. c. GαiP20 null clones were generated in MS1096-Gal4, UAS-Gαi wing discs. d-e. Gαi phenotypes are consistent with decreased Hh signaling. d. Homozygous GαiP8 mutant flies exhibit a small wing phenotype. e′. GαiP8 and e* pnr-Gal4, UAS-ptc flies have thoracic clefts (arrows), as compared to (e) wild type.
Figure 4
Figure 4
Hh regulates Gαi activity and association with Cos2. a. Cl8 cells were treated with control or HhN conditioned media. Error bars indicate s.e.m. b. Cl8 cells were transfected with control, Smo or Gαi dsRNA, and treated with HhN or control conditioned media. Wings from (c) hemizygous dnc1 (d) UAS-Smo5A, (e) dnc1, UAS-Smo5A and (f) smo3, UAS-Smo5A flies are shown. Introduction of dnc1 enhances the Smo5A phenotype in approximately 50% of flies (n=57). g. Cl8 cells were transfected with HA-Gαi and hh expression vectors, as indicated. Immunoprecipitations were performed from cell lysates using anti-Cos2 or IgG control antibodies.

References

    1. Mullor JL, Sanchez P, Altaba AR. Pathways and consequences: Hedgehog signaling in human disease. Trends Cell Biol. 2002;12:562–9. - PubMed
    1. Ingham PW, McMahon AP. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 2001;15:3059–87. - PubMed
    1. Robbins DJ, et al. Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein costal2. Cell. 1997;90:225–34. - PubMed
    1. Price MA, Kalderon D. Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by Glycogen Synthase Kinase 3 and Casein Kinase 1. Cell. 2002;108:823–35. - PubMed
    1. Alcedo J, Ayzenzon M, Von Ohlen T, Noll M, Hooper JE. The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal. Cell. 1996;86:221–32. - PubMed

Publication types

MeSH terms