Drosophila G-protein-coupled receptor kinase 2 regulates cAMP-dependent Hedgehog signaling

Abstract

G-protein-coupled receptor kinases (GRKs) play a conserved role in Hedgehog (Hh) signaling. In several systems, GRKs are required for efficient Hh target gene expression. Their principal target appears to be Smoothened (Smo), the intracellular signal-generating component of the pathway and a member of the G-protein-coupled receptor (GPCR) protein family. In Drosophila, a GRK called Gprk2 is needed for internalization and downregulation of activated Smo, consistent with the typical role of these kinases in negatively regulating GPCRs. However, Hh target gene activation is strongly impaired in gprk2 mutant flies, indicating that Gprk2 must also positively regulate Hh signaling at some level. To investigate its function in signaling, we analyzed several different readouts of Hh pathway activity in animals or cells lacking Gprk2. Surprisingly, although target gene expression was impaired, Smo-dependent activation of downstream components of the signaling pathway was increased in the absence of Gprk2. This suggests that Gprk2 does indeed play a role in terminating Smo signaling. However, loss of Gprk2 resulted in a decrease in cellular cAMP concentrations to a level that was limiting for Hh target gene activation. Normal expression of target genes was restored in gprk2 mutants by stimulating cAMP production or activating the cAMP-dependent Protein kinase A (Pka). Our results suggest that direct regulation of Smo by Gprk2 is not absolutely required for Hh target gene expression. Gprk2 is important for normal cAMP regulation, and thus has an indirect effect on the activity of Pka-regulated components of the Hh pathway, including Smo itself.

A Broadly Conserved G-Protein-Coupled Receptor Kinase Phosphorylation Mechanism Controls Drosophila Smoothened Activity

“…We previously reported that Gprk2 affects phosphorylation of Smo at sites other than GPS1 and GPS2 [19]. Even when using a PKA- and CKI-phosphomimetic form of Smo, Smo SD [5] to circumvent the effects of gprk2 depletion on cAMP levels, there was a substantial reduction of Smo phosphorylation (detected by increased mobility in SDS-PAGE) upon dsRNA-mediated depletion of Gprk2 that could not be accounted for by the GPS sites (Figure S1A).…”

“…On the other hand, loss of gprk2 causes a reduction in cyclic AMP (cAMP) levels, affecting PKA-dependent Smo activation. Target gene expression can be largely rescued in gprk2 mutants by increasing cAMP levels, suggesting that this indirect effect of Gprk2 on Smo plays an important role in Hh pathway function [19].…”

Platelet-derived growth factor-A and sonic hedgehog signaling direct lung fibroblast precursors during alveolar septal formation

“…Our findings suggest that Shh signals through the noncanonical pathway in primary LF, which, as others have shown, is sufficient to increase fibroblast migration (35). Smoothened is regulated by other mechanisms: 1) it is dependent on intraflagellar transport proteins for entry and translocation within the primary cilium; 2) it is phosphorylated and activated by PKA, which is dependent on adequate intracellular cAMP; 3) smoothened is ubiquitinated and degraded, a process that is regulated by a ubiquitin-specific protease USP8; and 4) smoothened activity is also influenced by phosphorylation of suppressor of fused (SuFu) by PKA or glycogen synthase kinase-3␤ (7,19,48). In primary LF, negative regulation through one or more of these pathways could dampen signaling through the canonical pathway.…”

Gprk2 adjusts Fog signaling to organize cell movements in Drosophila gastrulation

“…Previous studies showed that Gprk2 acts in Hedgehog (Hh) signaling for imaginal disc patterning (Molnar et al, 2007;Chen et al, 2010;Cheng et al, 2012). In this process, Gprk2 phosphorylates a GPCR, Smoothened, and potentiates Hh signaling.…”

“…For instance, mouse Grk1 knockout retinal cells produce stronger and longer outputs upon receiving a flash of light (Chen et al, 1999), suggesting that GRK1 regulates the sensitivity and duration of signaling. Recently, it has been shown that a Drosophila GPCR kinase, Gprk2, plays diverse roles in various biological processes, such as egg morphogenesis, olfactory response and tissue patterning (Schneider and Spradling, 1997;Molnar et al, 2007;Tanoue et al, 2008;Chen et al, 2010;Cheng et al, 2012). However, the role of Gprk2 in embryonic development has remained unclear.…”