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. 2011 Mar 11;286(10):8067-8074.
doi: 10.1074/jbc.M110.178772. Epub 2011 Jan 5.

Sonic Hedgehog-induced proliferation requires specific Gα inhibitory proteins

Mercedes Barzi  1 , Dorota Kostrz  1 , Anghara Menendez  1 , Sebastian Pons  2
Affiliations

Sonic Hedgehog-induced proliferation requires specific Gα inhibitory proteins

Mercedes Barzi et al. J Biol Chem. .

Abstract

Proliferation of cerebellar granular neuronal precursors (CGNPs) is mediated by Sonic Hedgehog (Shh), which activates the Patched and Smoothened (Smo) receptor complex. Although its protein sequence suggests that Smo is a G protein coupled receptor (GPCR), the evidence that this receptor utilizes heterotrimeric G proteins as downstream effectors is controversial. In Drosophila, Gα(i) is required for Hedgehog (Hh) activity, but the involvement of heterotrimeric G proteins in vertebrate Shh signaling has not yet been established. Here, we show that Shh-induced proliferation of rat CGNPs is enhanced strongly by the expression of the active forms of Gα(i/o) proteins (Gα(i1), Gα(i2), Gα(i3), and Gα(o)) but not by members of another class (Gα(12)) of heterotrimeric G proteins. Additionally, the mRNAs of these different Gα(i) members display specific expression patterns in the developing cerebellum; only Gα(i2) and Gα(i3) are substantially expressed in the outer external granular layer, where CGNPs proliferate. Consistent with this, Shh-induced proliferation of CGNPs is reduced significantly by knockdowns of Gα(i2) and Gα(i3) but not by silencing of other members of the Gα(i/o) class. Finally, our results demonstrate that Gα(i2) and Gα(i3) locate to the primary cilium when expressed in CGNP cultures. In summary, we conclude that the proliferative effects of Shh on CGNPs are mediated by the combined activity of Gα(i2) and Gα(i3) proteins.

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Figures

FIGURE 1.
FIGURE 1.
Shh-induced proliferation of CGNPs is strongly enhanced by the active forms of members of the Gαi/o class but not by G12. CGNP cells were plated in a saturating concentration of Shh (3 μg/ml) and transfected with different wild-type or active mutant forms of Gα cloned into pCIG, a nuclear GFP-expressing bicistronic vector. After 24 h, cells were washed and treated for an additional 48 h with the indicated amounts of Shh, and proliferation was analyzed by BrdU incorporation. A, comparison of different Gαi1-activating mutations at 0.25 μg/ml Shh. B, proliferation was studied in cells transfected with wild-type Gαi1, Gαi1Q204R, or mRI-PKA and treated with increasing amounts of Shh (0, 0.03, 0.06, 0.12, 0.25, and 3 μg/ml). C, comparison of the proliferation induced by expression of wild-type or active mutant forms of Gαi1, Gαi2, Gαi3, Gαo, and Gα12 in CGNPs maintained in 0.25 μg/ml Shh.
FIGURE 2.
FIGURE 2.
In situ hybridization analysis of Gαi/o mRNA at P7 in rat cerebellum. mRNA levels of Gαo, Gαi1, Gαi2, and Gαi3 were studied in P7 rat cerebellum by in situ hybridization using rat specific riboprobes. a–d, low magnification images reveal the unique expression patterns of the mRNAs of the various Gαi/o class members. e–h, high magnification photos of the mRNA levels in the different cerebellar layers: oEGL, inner external granular layer (iEGL), molecular layer (ML), Purkinje cell layer (PCL), and internal granular layer (IGL). i–l, high magnification photos of the in situ hybridization of the Gαi/o class members combined with immuno-histochemistry against the Purkinje cell marker, calbindin, to further delineate the extension of each cerebellar layer. Only Gαi2 and Gαi3 were significantly expressed at the oEGL were CGNPs proliferate.
FIGURE 3.
FIGURE 3.
Gαi2 and Gαi3 knockdowns significantly reduce Shh induced CGNP proliferation. Different DNA sequences encoding shRNA, targeting either the 3′UTR (UTR) or CS of the different Gαi/o class members, were cloned into pGHIN (a pSUPER-based GFP-expressing bicistronic vector). The number of shRNAs designed for each Gαi/o depended on the availability of adequate targeting motifs in the respective sequences (see “Experimental Procedures” for details). A, the shRNA expressing constructs were electroporated into freshly isolated CGNPs, which were then halved and grown in a saturating concentration of Shh (3 μg/ml) for 48 h. One culture was used to isolate mRNA for RT-PCR analysis and the other to measure proliferation by BrdU. B, the targeting efficiency of each shRNA was evaluated by RT-PCR. A direct correlation between targeting efficiency and proliferation reduction was observed only for shRNAs targeting Gαi2 and Gαi3. C, the most efficient shRNAs against Gαi2 and Gαi3 (Gi2UTR2 and Gi3UTR2) were assayed together or in combination with Gi2CS, a low efficiency shRNA which was used as a control. The total amount of DNA was equivalent for all transfections. Proliferation was significantly reduced compared with empty vector when either Gi2UTR2 or Gi3UTR2 were transfected (p < 0.01). Double transfection of Gi2UTR2 and Gi3UTR2 significantly reduced proliferation as compared with single transfections or to doubles with Gi2CS (p < 0.01).
FIGURE 4.
FIGURE 4.
Expression of different Gαi/o class members but not Gα12 rescues the impaired proliferation caused by Gαi2/Gαi3 knockdowns. Freshly isolated CGNPs were electroporated with mixtures of cDNA containing Gi2UTR2/Gi3UTR2 and constructs including wild-type (A) or active forms (B) of the different Gαi/o class members. Cells were cultured in a saturating concentration of Shh (3 μg/ml) for 48 h, and proliferation was measured by BrdU incorporation. Selected images of transfected cells are shown in C. A more complete panel of images is presented in supplemental Fig. 3.
FIGURE 5.
FIGURE 5.
The three Gαi members are distributed differently between the base and the shaft of the primary cilium. CGNP cultures were transfected with FLAG tagged constructs of Gαi1, Gαi2, and Gαi3 subunits cloned in pCIG, a nuclear-GFP expressing bicistronic vector that permitted us to compare the Gαi distribution in similarly transfected cells. Transfection of Smo-HA was included as a positive control. Cells were cultured for 48 h with 3 μg/ml of Shh, fixed, and double-stained with rabbit anti-ACIII (used as a cilium marker) and mouse anti-FLAG or anti-HA. The first column depicts images of direct GFP fluorescence combined with phase contrast pictures, demonstrating the transfection levels of each cell. ACIII staining was detected with anti-rabbit Cy5 (far red 675) but has been colored green for clarity. FLAG or HA tags were detected with anti-mouse Alexa Fluor 555 (red). The last column contains Z-stacks generated from the confocal images.
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