The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila - PubMed (original) (raw)

The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila

Cristina Molnar et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Signaling by Smoothened (Smo) plays fundamental roles during animal development and is deregulated in a variety of human cancers. Smo is a transmembrane protein with a heptahelical topology characteristic of G protein-coupled receptors. Despite such similarity, the mechanisms regulating Smo signaling are not fully understood. We show that Gprk2, a Drosophila member of the G protein-coupled receptor kinases, plays a key role in the Smo signal transduction pathway. Lowering Gprk2 levels in the wing disc reduces the expression of Smo targets and causes a phenotype reminiscent of loss of Smo function. We found that Gprk2 function is required for transducing the Smo signal and that when Gprk2 levels are lowered, Smo still accumulates at the cell membrane, but its activation is reduced. Interestingly, the expression of Gprk2 in the wing disc is regulated in part by Smo, generating a positive feedback loop that maintains high Smo activity close to the anterior-posterior compartment boundary.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Enhanced GPRK2 accumulation in regions of high-level Hh signaling in the wing disc. (A and B) In situ hybridization of third instar wing imaginal discs with Gprk2 antisense (as) (A) and sense (s) (B) probes. The expression of Gprk2 is detected at higher levels in cells abutting the A/P compartment boundary (white arrow in A). (C) Expression of β-gal (red) and Caupolican (Caup, green) in a third instar wing disc heterozygous for the P-lacZ insertion PZ-Gprk2[06936]. The white arrow indicates the stripe of β-gal accumulation. (D) Expression of β-gal/Gprk2 (Gprk2, red) and Engrailed (En, green) in wild-type wing disc (wt), showing that β-gal expression occurs in anterior cells expressing En (nuclei in orange). Individual channels are shown in D′ (Gprk2) and D″ (En). (E) Expression of β-gal/Gprk2 (Gprk2, red) and Patched (Ptc, green). Individual channels are shown in E′ (Gprk2) and E″ (Ptc). (F) Expression of β-gal/Gprk2 (Gprk2, red) and Blistered (Bs, green). Individual channels are shown in F′ (Gprk2) and F″ (Bs). (G) Expression of β-gal/Gprk2 (Gprk2, red) and Engrailed (En, green) in sal-Gal4/+; PZ-Gprk2[06936]/UAS-hh wing discs. The extent of β-gal and En expression is included in the bottom white line and appears as orange nuclei. (H) Expression of β-gal/Gprk2 (Gprk2, red) and Patched (Ptc, green) in _sal-Gal4/UAS-ptc; PZ-Gprk2_[06936]/+ wing discs, showing that β-gal expression is eliminated in the wing blade upon ectopic Ptc expression. (I) Individual red channel of H, showing that the expression of β-gal/Gprk2 in the thorax (where sal-Gal4 in not expressed) is not affected (white arrow). wt, wild type.

Fig. 2.

Gprk2 is required for Hh signaling. (A) Wild-type wing showing the position of the longitudinal veins L2 to L5. acv, anterior cross vein. (B–D) Adult wings resulting from ectopic expression of interference Gprk2 RNA in Gal4–638/UAS-Gprk2i flies. Mild phenotypes consist of a moderate reduction of wing size and the shortening of the distance between the L3 and L4 veins [B; Gprk2-RNAi (1)]. Medium phenotypes are characterized by partial fusion of the L3 and L4 veins [C; Gprk2-RNAi (2)]. Strong phenotypes consist in greater reductions of wing size and the disappearance of the L3 and L4 veins [D; Gprk2-RNAi (3)]. (E) Adult wing from Gal4-hh/UAS-Gprk2i flies, expressing interference Gprk2 RNA in the posterior compartment. (F) Adult wing from Gal4-ptc/UAS-Gprk2i flies, expressing interference Gprk2 RNA in anterior cells close to the A/P compartment boundary (compare to B and C). (G) Hh loss-of-function phenotype in 638-Gal4/UAS-hh-RNAi flies consists of wing size reduction, shortening of the distance between the veins L3 and L4, and loss of the anterior cross vein (acv in A). (H) Severe hh loss-of-function phenotype in 638-Gal4/UAS-hh-RNAi; UAS-Gprk2i/+; compare to the expression of only hh-RNAi (G) or only Gprk2-RNAi (B–D). (I) Severe hh loss-of function phenotype in Gal4–638/+; FRT42 smo2/FRT42 M (2)l2; UAS-FLP/+. These wings, formed by homozygous smo2 cells, are extremely reduced in size and have lost most pattern elements. (J) Hh loss-of-function phenotype caused by ectopic expression of ptc (sal-Gal4/UAS-ptc) is very similar to loss of Gprk2 function (compare to D). (K–K″) Expression of Knot (Kn; K and K′, green) and Engrailed (En; K and K″, red) in a third instar wild-type wing imaginal disc. (L–L″) Loss of Gprk2 reduces Knot expression (Kn; L and L′, green) and eliminates anterior expression of Engrailed (En; L, L″, red). The white lines in K″ and L″ delimit the anterior domain of En expression. (M and M′) Expression of Ptc in wild-type discs (wt; M) and in Gal4–638/UAS-Gprk2i (Gprk2-i; M′). Loss of Gprk2 reduces the expression of Ptc. (N and N′) Expression of Ci in wild-type discs (wt; N) and in Gal4–638/UAS-Gprk2i wing discs (Gprk2-i; N′). (O and O′) Expression of Bs in wild-type discs (wt; O) and in Gal4–638/UAS-Gprk2i wing discs (Gprk2-i; O′). Loss of Gprk2 eliminates the L3/L4 intervein domain of Bs expression. (P and P′) Expression of the Dpp target gene Sal in wild-type (wt; P) and Gal4–638/UAS-Gprk2i discs (Gprk2-i; P′). Overall, the level and anterior-posterior extent of the Sal domain is not affected. (Q and Q′) Expression of Dl in wild-type discs (wt; Q) and in Gal4–638/UAS-Gprk2i (Gprk2-i; Q′). Loss of Gprk2 reduces Dl expression in the L3 and L4 veins. (R and R′) Wing discs showing the expression of Caupolican (Caup) in wild-type (wt; R) and Gal4–638/UAS-Gprk2i wing discs (Gprk2-i; R′). Loss of Gprk2 expands Caup. (S and S′) In situ hybridization with a dpp RNA probe in wild-type (wt; S) and Gal4–638/UAS-Gprk2i (Gprk2-i; S′) wing discs. Upon a reduction in Gprk2 levels, dpp expression occurs at lower levels and in an expanded domain. (T–V) Clones of Df(3R)Gprk2 cells induced in hs-FLP1.22; FRT82 Df(3R)Gprk2/ FRP82 M (3)w Ubi-GFP discs at 48–72 h after egg laying. Homozygous _Gprk2_− cells are labeled by the absence of GFP and appear as black spots. In T and T′ and U and U′, the expression of En (En; red) is shown, and in V and V′, the expression of Ptc is shown in red. In anterior Gprk2 mutant cells located close to the A/P compartment boundary, the expression of En (T–U′) and Ptc (V and V′) is not detected. T′, U′, and V′ show the red channels of T, U, and V, respectively.

Fig. 3.

GPRK2 function is required downstream of Hh. (A and B) Expression of Engrailed (En, red) in wing discs carrying clones of _Gprki_-expressing cells induced in hs-FLP; Ubx/abx Gal4-lacZ/UAS-Gprk2i (B) corresponds to the red channel, and the clones are labeled in green. The expression of En is lost in anterior cells. A′ and B′ show Z sections of the clones shown in A and B, respectively. (C) Expression of Engrailed (En, red) in wing discs carrying clones of Hh-expressing cells induced in hs-FLP; Ubx/abx Gal4-lacZ/UAS-hh larvae (, green). En is activated in anterior cells both within hh-expressing cells (green) and in the surrounding wild-type cells. Individual channels are shown for En (red, C″) and Hh-expressing cells (green, C″). (D–E) Expression of Engrailed (En, red) in wing discs carrying clones of cells expressing Hh and Gprk2i (<hh+Gprk2i>, green). En is activated in anterior cells surrounding the clones but not within the clone itself. Individual channels are shown below for En (D′ and E″) and (Hh+Gprk2i)-expressing cells (E′). E–E″ are higher magnifications of the clone shown in C.

Fig. 4.

Smo accumulation depends on Gprk2 activity. (A and B) Expression of Smo in wild-type (WT; A) and 638-Gal4/UAS-Gprk2i (GPRK2i; B) wing discs visualized by using mouse anti-Smo (red). Below each disc is a Z section through the middle of the wing blade. The apical side of the epithelium is up. (C and C′) Expression of GFP (green) and Smo (red) in ap-Gal4/UAS-Gprk2i UAS-GFP. Smo is accumulated in a broader domain in anterior-dorsal cells (white arrow). (D–E) Examples of Df(3R)gprk2 clones (labeled by the absence of green) showing increased expression of Smo (red) in anterior cells located close to the A/P compartment boundary. (F) Control S2 cells and S2 cells treated with dsRNAgprk2 for 4 days were plated on poly(lysine)-coated slides and incubated for 6 h with S2 or S2HhN-conditioned medium prior to fixation and immunoassayed for Smo.

Fig. 5.

Interactions between Gprk2 and the intracellular domain of Smo. (A) Phenotype caused by the expression of the Smo phosphomimic form SmoSD123 in the wing blade at 25°C. (B and C) Phenotype caused by ectopic expression of a Smo intracellular deletion (638-Gal4/UAS-Smo_Δ_C2; B) and severe loss-of-Hh signaling phenotype resulting from the coexpression of Gprk2 RNAi in this genetic background (638-Gal4/UAS-Smo_Δ_C2+UAS-Gprk2i; C). (D–F) Expression of Smo (D), En (E), and Ptc (F) in wing discs of 638-Gal4/+; UAS-SmoSD123 /+ genotype. Note the expansion of Smo, En, and Ptc domains of expression to the entire anterior compartment. (G–I) Expression of Smo (G), En (H), and Ptc (I) in wing discs of 638-Gal4/+; UAS-SmoSD123 /UAS-Gprk2i genotype. The anterior expression of En (H) and Ptc (I) is now suppressed upon a reduction in Gprk2 levels. Despite of the presence of the SmoSD123 protein in the cell membrane of anterior cells (G), the pathway does not activate the expression of the high-level targets En and Ptc.

References

1. Lum L, Beachy PA. Science. 2004;304:1755–1759. - PubMed
1. Hooper JE, Scott MP. Nat Rev. 2005;6:306–317. - PubMed
1. Tabata T, Kornberg TB. Cell. 1994;76:89–102. - PubMed
1. Zecca M, Basler K, Struhl G. Development (Cambridge, UK) 1995;121:2265–2278. - PubMed
1. Strigini M, Cohen SM. Development (Cambridge, UK) 1997;124:4697–4705. - PubMed

The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila - PubMed (original) (raw)