Regulation of smoothened by Drosophila G-protein-coupled receptor kinases.
The Hedgehog (Hh) signaling pathway plays a conserved and essential role in regulating development and homeostasis of numerous tissues. Cytoplasmic signaling is initiated by Smoothened (Smo), a G-protein-coupled receptor (GPCR) family member, whose levels and activity are regulated by the Hh receptor Patched (Ptc). In response to Hh binding to Ptc, Ptc-mediated repression of Smo is relieved, leading to Smo activation, surface accumulation, and downstream signaling. We find that downregulation of Drosophila Smo protein in Hh-responding imaginal disc cells is dependent on the activity of G-protein-coupled receptor kinase 2 (Gprk2). By analyzing gain- and null loss-of-function phenotypes, we provide evidence that Gprk2 promotes Smo internalization subsequent to its activation, most likely by direct phosphorylation. Ptc-dependent regulation of Smo accumulation is normal in gprk2 mutants, indicating that Gprk2 and Ptc downregulate Smo by different mechanisms. Finally, we show that both Drosophila G-protein-coupled receptor kinase orthologues, Gprk1 and Gprk2, act in a partially redundant manner to promote Hh signaling. Our results suggest that Smo is regulated by distinct Ptc-dependent and Gprk2-dependent trafficking mechanisms in vivo, analogous to constitutive and activity-dependent regulation of GPCRs. G-protein-coupled receptor kinase activity is also important for efficient downstream signaling.
Institut de Recherches Cliniques de Montréal, 110 Pine Avenue West, Montreal, QC H2W1R7, Canada.
Shuofei
S
Cheng
Dominic
D
Maier
Dagmar
D
Neubueser
David R
DR
Hipfner
D000818:Animals; D019390:Arrestins / Q000502:physiology; D002478:Cells, Cultured; D029721:Drosophila Proteins / Q000235:genetics* / Q000502:physiology*; D004331:Drosophila melanogaster; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000502:physiology; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000502:physiology*; D005786:Gene Expression Regulation; D053823:Hedgehog Proteins / Q000502:physiology; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*; D015398:Signal Transduction; D000072086:Smoothened Receptor; D013696:Temperature; D000071557:beta-Arrestins
D019390:Arrestins; D029721:Drosophila Proteins; D053823:Hedgehog Proteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D000072086:Smoothened Receptor; D000071557:beta-Arrestins; C070844:ptc protein, Drosophila; C100664:smo protein, Drosophila; C077245:hh protein, Drosophila; D051606:G-Protein-Coupled Receptor Kinase 1; D054769:G-Protein-Coupled Receptor Kinase 2; C496574:Gprk2 protein, Drosophila