Regulation of Smoothened by Drosophila G-protein-coupled receptor kinases

Abstract

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.

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

“…Primary antibodies and their sources: guinea pig anti-Gprk2 (Cheng et al, 2010); mouse anti-Col (M. Crozatier, Centre de Biologie du Développement, Toulouse, France), anti-myc (9E10; Santa Cruz Biotechnology), anti--Tubulin (12G10), anti-En (4D9), anti-Ptc (Apa I), anti-SuFu (25H3), and anti-Smo (20C6) (Developmental Studies Hybridoma Bank); rabbit anti-G i (J. Knoblich), anti-Fu and anti-Cos2 (P. Therond, Université Nice, France), anti-GFP (Torrey Pines Scientific); rat anti-Smo (S. Cohen), anti-Ci 155 (T. Kornberg, University of California, San Francisco, USA).…”

“…In flies, the Drosophila ortholog of mammalian GRK4/5/6, called Gprk2, promotes Smo phosphorylation, internalization, and downregulation in Hhresponding cells. Loss-of-function mutations in gprk2 impair highthreshold Hh target gene expression in wing imaginal discs, the epithelial precursor to the adult wing (Chen et al, 2010;Cheng et al, 2010;Molnar et al, 2007).…”

“…As these kinases typically function to limit GPCR signaling, their loss often leads to receptor hypersensitivity and too much activity rather than too little (Premont and Gainetdinov, 2007). Furthermore, strong accumulation of Smo at the cell surface, as is observed in gprk2-mutant Hh-responding cells (Cheng et al, 2010), is usually correlated with high-level signaling (Denef et al, 2000;Zhu et al, 2003). To explain the positive role of GRKs in Hh signaling, it has been suggested that they may be doing something more than just downregulating Smo.…”

“…For example, there is evidence that GRKs can directly regulate the activation state of Smo (Chen et al, 2010;Chen et al, 2011). Misregulation of G-protein signaling and second messenger production in GRK mutants could also contribute to signaling impairment (Cheng et al, 2010). To distinguish between these and other possibilities, we set out to assess how GRK activity affects signaling downstream of Smo by analyzing the activity of canonical and G-protein-dependent signaling outputs in gprk2 mutant flies.…”

“…To distinguish between these and other possibilities, we set out to assess how GRK activity affects signaling downstream of Smo by analyzing the activity of canonical and G-protein-dependent signaling outputs in gprk2 mutant flies. , gprk2 KO and UASGprk2 (Cheng et al, 2010). All other strains were from the Bloomington Drosophila Stock Center.…”