Zhonghua Zhao1,2,¶, Raymond Teck Ho Lee1,¶, Ganesh V. Pusapati3, Audrey Qin Iyu1, Rajat Rohatgi3 and Philip W Ingham1,2,*
1 Developmental and Biomedical Genetics Laboratory, Institute of Molecular and Cell biology, Agency of Science, Technology and Research (A-STAR), Singapore
2 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore
3 Departments of Medicine and Biochemistry, Stanford University School of
Medicine, Stanford, California, USA
¶ equal contribution to this work
Published in EMBO reports on 4th April 2016.
The G-protein-coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock-down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but 4 remain responsive to inhibition of cAMP-dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase-dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C-terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho-null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.
Figure with legend
We generated a null allele of zebrafish grk2 by targeted mutagenesis and found that complete loss of Grk2 eliminates all responses to Hh signalling during embryogenesis. We also show that phospho-null mutants of Smo retain significant activity suggesting Smo is not the principal target of Grk2.
• Grk2 is essential for all responses to Hh signalling in the zebrafish embryo.
• Phosphorylation of Grk2/CK1 sites in the Smo CTT is neither necessary nor sufficient for its activation.
• Grk2 regulates Hh signalling downstream of Smo, possibly via an unidentified GPCR.
Phenotype of wild-type, MZgrk2- and grk2-GFP mRNA-injected MZgrk2 embryos at 24hpf (n = 20 for each sample). The white lines indicate the shape of the somites (middle panels) and the separation of the eyes (right hand panels). Scale bar, 200 um.
Prox1a and Eng expression in smo mutant embryos injected with mRNA encoding mSmo (n = 10), mSmoSA (n = 9) and mSmo14SA (n = 6). Note the full recovery of SSFs and MPs compared to the uninjected controls. Scale bar, 50 um.
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