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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.