Activation of Smoothened in the Hedgehog pathway unexpectedly increases G?_s-dependent cAMP levels in Drosophila.

ABSTRACT: Hedgehog (Hh) signaling plays a key role in the development and maintenance of animal tissues. This signaling is mediated by the atypical G protein-coupled receptor (GPCR) Smoothened (Smo). Smo activation leads to signaling through several well-characterized effectors to activate Hh target gene expression. Recent studies have implicated activation of the heterotrimeric G protein subunit G?_i and the subsequent decrease in cellular cAMP levels in promoting the Hh response in flies and mammals. Although Hh stimulation decreases cAMP levels in some insect cell lines, here using a bioluminescence resonance energy transfer (BRET)-based assay we found that this stimulation had no detectable effect in Drosophila S2-R+ cells. However, we observed an unexpected and significant G?_s-dependent increase in cAMP levels in response to strong Smo activation in Smo-transfected cells. This effect was mediated by Smo's broadly conserved core, and was specifically activated in response to phosphorylation of the Smo C-terminus by GPCR kinase 2 (Gprk2). Genetic analysis of heterotrimeric G protein function in the developing Drosophila wing revealed a positive role for cAMP in the endogenous Hh response. Specifically, we found that mutation or depletion of G?_s diminished low-threshold Hh responses in Drosophila, whereas depletion of G?_i potentiated them (in contrast to previous findings). Our analysis suggested that regulated cAMP production is important for controlling the sensitivity of cellular responses to Hh in Drosophila.

SUBMITTER: Praktiknjo SD 

PROVIDER: S-EPMC6120187 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature