An essential role for Grk2 in Hedgehog signalling downstream of Smoothened.
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 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.
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore Developmental and Biomedical Genetics Laboratory, Institute of Molecular and Cell Biology, Agency of Science, Technology and Research (A-STAR), Singapore, Singapore.
Zhonghua
Z
Zhao
Developmental and Biomedical Genetics Laboratory, Institute of Molecular and Cell Biology, Agency of Science, Technology and Research (A-STAR), Singapore, Singapore.
Raymond Teck Ho
RT
Lee
Departments of Medicine and Biochemistry, School of Medicine, Stanford University, Stanford, CA, USA.
Ganesh V
GV
Pusapati
Developmental and Biomedical Genetics Laboratory, Institute of Molecular and Cell Biology, Agency of Science, Technology and Research (A-STAR), Singapore, Singapore.
Audrey
A
Iyu
Departments of Medicine and Biochemistry, School of Medicine, Stanford University, Stanford, CA, USA.
Rajat
R
Rohatgi
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore Developmental and Biomedical Genetics Laboratory, Institute of Molecular and Cell Biology, Agency of Science, Technology and Research (A-STAR), Singapore, Singapore pingham@ntu.edu.sg.
Philip W
0000-0001-8224-9958
PW
Ingham
D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D016000:Cluster Analysis; D004789:Enzyme Activation; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D005854:Germ Cells / Q000378:metabolism; D053823:Hedgehog Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation; D010641:Phenotype; D010766:Phosphorylation; D015398:Signal Transduction; D000072086:Smoothened Receptor / Q000378:metabolism*; D015027:Zebrafish
D053823:Hedgehog Proteins; D000072086:Smoothened Receptor; D054769:G-Protein-Coupled Receptor Kinase 2
Grk2; Hedgehog signalling; PKA; Smo; phosphorylation