Sonic Hedgehog-induced Proliferation Requires Specific Gα Inhibitory Proteins

Mercedes Barzi,Dorota Kostrz,Anghara Menendez,Sebastian Pons

doi:10.1074/jbc.m110.178772

Mercedes Barzi, Dorota Kostrz + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m110.178772

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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.

Highlights

BFU2008-02424/BFI. □S The on-line version of this article contains supplemental Table 1 and Figs. 1–5. 1 To whom correspondence should be addressed: Institute for Biomedical
It has been shown that, at least in Drosophila, Smo functions as a canonical G protein coupled receptor (GPCR) which signals through G␣i to regulate activation of the Hh pathway [18]
Its protein sequence clearly predicts that Smo belongs to the GPCR family, the evidence that this receptor uses heterotrimeric G proteins as downstream effectors is inconclusive

Summary

Introduction

CGNP cultures were transfected with mixtures containing Gi2UTR2/Gi3UTR2 and constructs to express wild-type (Fig. 4A) or active forms (Fig. 4B) of the different members of the G␣i/o class. These observations reinforce the notion that G␣i/o class molecules display rather specific expression patterns in the developing cerebellum, their function is interchangeable, at least, in transmitting Shh-induced proliferation of CGNPs under our experimental conditions.

Results

Conclusion