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. 2006 Oct;26(20):7550-60.
doi: 10.1128/MCB.00546-06. Epub 2006 Aug 14.

Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2

Alison R Meloni  1 , Gregory B Fralish, Patrick Kelly, Ali Salahpour, James K Chen, Robert J Wechsler-Reya, Robert J Lefkowitz, Marc G Caron
Affiliations

Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2

Alison R Meloni et al. Mol Cell Biol. 2006 Oct.

Abstract

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many "conventional" 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened.

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Figures

FIG. 1.
FIG. 1.
Synergistic activation of Gli by GRK2 and active Smo. (A) C3H10T1/2 cells were transiently transfected with the indicated expression plasmids, a Gli luciferase reporter, and a βgal transfection control. Luciferase values were adjusted for transfection efficiency relative to that of a sample containing the reporter alone. Fold activation refers to the increase in luciferase activity of each sample relative to that of the sample containing the reporter alone. (B, top) Cells were transfected as for panel A with the addition of a GFP plasmid. Cells were sorted by flow cytometry, and lysates from equal numbers of cells were immunoblotted with Myc and actin antibodies. (B, bottom) Cells were transfected as for panel A. Extracts used for luciferase assays were normalized for transfection efficiency and immunoblotted with a GRK2 antibody. A representative blot of three independent experiments is shown. (C) Cells were transfected and assayed as for panel A. (D) Extracts from the experiments whose results are shown in panel C were adjusted for transfection efficiency and immunoblotted with a GRK2 antibody. (E) C3H10T1/2 cells stably expressing the indicated proteins were transfected with the Gli reporter and a βgal transfection control. Luciferase assays were performed as for panel A. (F) Cells were transfected as for panel A. After 24 h, the cells were split in half and treated with either the vehicle or cyclopamine for 48 h. Cells were then assayed for luciferase activity as for panel A. The data shown are the results of three independent experiments.
FIG. 2.
FIG. 2.
SAG and GRK2 synergy. C3H10T1/2 cells were transfected with the indicated expression plasmids. After 24 h, cells were split in half and treated with either the vehicle or SAG for 48 h. Cells were harvested and assayed for luciferase and βgal activities. Luciferase values were normalized for transfection efficiency with the βgal values of the sample containing the reporter alone. Fold activation refers to the increase in luciferase activity relative to that of the vehicle-treated sample containing the reporter alone. The data shown are the results of at least three independent experiments performed in duplicate.
FIG. 3.
FIG. 3.
Gli activation through SAG is GRK2 dependent. (A) C3H10T1/2 cells were transfected with the indicated shRNA and the Gli reporter vector. After 24 h, the cells were split in half and treated with either SAG or the vehicle for 48 h. Cells were then assayed for luciferase activity. Fold activation by SAG refers to the induction of luciferase activity in SAG-treated cells relative to that of vehicle-treated cells. (B) Cells were treated as for panel A and sorted by flow cytometry with a cotransfected GFP vector. Extracts from equal numbers of cells were immunoblotted with GRK2 and actin antibodies. (C) Cells were treated as for panel A with the additional transfection of pCDNA3 or an shRNA-resistant GRK2 2* expression vector. (D) Immunoblotting was performed as for panel B.
FIG. 4.
FIG. 4.
GRK2 contributes to oncogenic Smo M2 signaling and cooperates with Smo and Ras to transform C3H10T1/2 cells. (A) C3H10T1/2 cells were transfected with the Gli luciferase reporter, a βgal transfection control, and the indicated cDNA and shRNA plasmids. Fold activation refers to the induction of luciferase activity by the indicated cDNA relative to that of the control sample with the reporter alone containing the same shRNA. The data shown are the results of three independent experiments. (B) HEK 293 cells were transfected with the indicated expression plasmids. Cell extracts were normalized for transfection efficiency. Immunoblotting was performed with human Smo antibody. (C) Focus formation assays. C3H10T1/2 cells stably expressing the indicated proteins were transfected with Ras and stained for foci with crystal violet after 21 days. Representative fields are shown. At the bottom is a quantification of the number of foci per 60-mm plate (three independent experiments in duplicate).
FIG. 5.
FIG. 5.
Interaction between β-arrestin 2 and Smo. (A) Confocal images of Shh-LIGHT cells expressing β-arrestin 2-GFP and different Shh pathway components. The insets are magnifications of the boxed area of the membrane. (B) Quantification of experiments as for panel A with HEK cells with or without exogenous expression of GRK2. Percentage of cells with β-arrestin 2 translocation refers to the percentage of GFP-positive cells that show punctate β-arrestin 2-GFP localization at the plasma membrane. (C) Effect of GRK2 on Smo and β-arrestin 2 association. HEK cells were transfected with the indicated expression plasmids, and extracts were immunoprecipitated (IP) with Flag affinity gel. Immunoprecipitates and total lysates were immunoblotted (IB) with Myc and Flag antibodies. (D) Effect of cyclopamine on Smo and β-arrestin 2 association. Experiments were performed as for panel C, by treating HEK cells with either the vehicle or cyclopamine for 24 h.
FIG. 6.
FIG. 6.
Smo/GRK2 synergy is β-arrestin 2 dependent. (A) C3H10T1/2 cells were transfected with the indicated shRNAs and the Gli reporter with either Smo and GRK2 expression plasmids or a pCDNA3 control plasmid. Fold activation refers to the induction of luciferase activity in samples transfected with Smo and GRK2 relative to that of samples with pCDNA3. Luciferase values were adjusted for transfection efficiency with a cotransfected βgal vector. The data shown are the results of three independent experiments performed in triplicate. Significance (**, P < 0.001) was measured with the Student t test (Microsoft Excel). (B) Cells were transfected as for panel A and sorted by flow cytometry for a cotransfected GFP vector. Extracts from equal numbers of cells were immunoblotted with β-arrestin (A1CT) and actin antibodies.
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