A Ciliary SMOOTHENED-GRK2-PKA Signaling Pathway Initiates Hedgehog Signal Transduction

During Hedgehog (Hh) signal transduction in development, homeostasis, and cancer, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by directly binding the PKA catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous Hh signal transduction in the vertebrate primary cilium. Hh pathway activation triggers rapid GRK2 relocalization from the base to the shaft of the cilium, leading to SMO phosphorylation and ultimately formation of SMO / PKA-C complexes in this compartment. In vitro reconstitution experiments reveal that GRK2 phosphorylation is sufficient to trigger direct binding of the SMO active conformation to PKA-C, without participation from additional proteins. Lastly, the SMO-GRK2-PKA communication pathway operates during Hh signaling in a range of cellular and in vivo models. Our work highlights GRK2 phosphorylation of ciliary SMO as a key initiating event for the intracellular steps of the Hh cascade, enabling a deeper understanding of how Hh signals are transduced intracellularly in tissues and organs to orchestrate proliferative and differentiative decisions. More broadly, our study hints at an expanded role for GRKs in enabling direct GPCR interactions with a diverse array of intracellular effectors.

[1]  S. Christensen,et al.  Primary cilia as dynamic and diverse signalling hubs in development and disease , 2023, Nature Reviews Genetics.

[2]  P. Beachy,et al.  Cellular and molecular mechanisms of Hedgehog signalling , 2023, Nature Reviews Molecular Cell Biology.

[3]  Susan S. Taylor,et al.  Edmond Fischer's kinase legacy: History of the protein kinase inhibitor and protein kinase A , 2023, IUBMB life.

[4]  F. Gervasio,et al.  Subcellular location defines GPCR signal transduction , 2022, bioRxiv.

[5]  K. Hilgendorf,et al.  Ciliary control of adipocyte progenitor cell fate regulates energy storage , 2022, Frontiers in Cell and Developmental Biology.

[6]  W. Weis,et al.  PI(4,5)P2-stimulated positive feedback drives the recruitment of Dishevelled to Frizzled in Wnt–β-catenin signaling , 2022, Science Signaling.

[7]  J. Tesmer,et al.  G protein–coupled receptor interactions with arrestins and GPCR kinases: The unresolved issue of signal bias , 2022, The Journal of biological chemistry.

[8]  C. Robinson,et al.  Cryo-EM structure of human GPR158 receptor coupled to the RGS7-Gβ5 signaling complex , 2021, Science.

[9]  Shuying Yang,et al.  SAG therapy restores bone growth and reduces enchondroma incidence in a model of skeletal chondrodysplasias caused by Ihh deficiency , 2021, Molecular Therapy. Methods & Clinical Development.

[10]  Xuecai Ge,et al.  Control of the Hedgehog pathway by compartmentalized PKA in the primary cilium , 2021, Science China Life Sciences.

[11]  P. Andrews,et al.  Structures of rhodopsin in complex with G-protein-coupled receptor kinase 1 , 2021, Nature.

[12]  Chen Yang,et al.  The Role of Sonic Hedgehog Pathway in the Development of the Central Nervous System and Aging-Related Neurodegenerative Diseases , 2021, Frontiers in Molecular Biosciences.

[13]  Susan S. Taylor,et al.  A PKA Inhibitor Motif within Smoothened Controls Hedgehog Signal Transduction , 2021, bioRxiv.

[14]  G. Pazour,et al.  E3 ubiquitin ligase Wwp1 regulates ciliary dynamics of the Hedgehog receptor Smoothened , 2021, The Journal of cell biology.

[15]  Wan Li,et al.  Vertebrate cells differentially interpret ciliary and extraciliary cAMP , 2021, Cell.

[16]  S. Gygi,et al.  Time-resolved proteomics profiling of the ciliary Hedgehog response , 2021, The Journal of cell biology.

[17]  K. Kwan,et al.  Build me up optic cup: Intrinsic and extrinsic mechanisms of vertebrate eye morphogenesis. , 2021, Developmental biology.

[18]  S. Geley,et al.  Feedback control of the Gpr161-Gαs-PKA axis contributes to basal Hedgehog repression in zebrafish , 2021, Development.

[19]  Christina A. Kearns,et al.  Zebrafish spinal cord oligodendrocyte formation requires boc function , 2021, bioRxiv.

[20]  Qin Zhang,et al.  Control of , 2021, Agriculture Automation and Control.

[21]  A. Nager,et al.  Ubiquitin chains earmark GPCRs for BBSome-mediated removal from cilia , 2020, The Journal of cell biology.

[22]  D. Nickerson,et al.  Mutations in GRK2 cause Jeune syndrome by impairing Hedgehog and canonical Wnt signaling , 2020, EMBO molecular medicine.

[23]  Xiaochun Li,et al.  Sterols in an intramolecular channel of Smoothened mediate Hedgehog signaling , 2020, Nature Chemical Biology.

[24]  Benjamin R. Myers,et al.  Smoothened transduces Hedgehog signals via activity-dependent sequestration of PKA catalytic subunits , 2020, bioRxiv.

[25]  J. Gaiarsa,et al.  Sonic Hedgehog Signaling Agonist (SAG) Triggers BDNF Secretion and Promotes the Maturation of GABAergic Networks in the Postnatal Rat Hippocampus , 2020, Frontiers in Cellular Neuroscience.

[26]  T. Caspary,et al.  Signaling in the primary cilium through the lens of the Hedgehog pathway , 2020, Wiley interdisciplinary reviews. Developmental biology.

[27]  G. Pazour,et al.  Ubiquitin links smoothened to intraflagellar transport to regulate Hedgehog signaling , 2019, bioRxiv.

[28]  M. von Zastrow,et al.  Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells , 2019, bioRxiv.

[29]  Jieun Song,et al.  Activation of sonic hedgehog signaling by a Smoothened agonist restores congenital defects in mouse models of endocrine-cerebro-osteodysplasia syndrome , 2019, EBioMedicine.

[30]  Naomi R. Latorraca,et al.  Smoothened stimulation by membrane sterols drives Hedgehog pathway activity , 2019, Nature.

[31]  G. Konopka,et al.  Derepression of sonic hedgehog signaling upon Gpr161 deletion unravels forebrain and ventricular abnormalities. , 2019, Developmental biology.

[32]  C. Siebold,et al.  Biochemical mechanisms of vertebrate hedgehog signaling , 2019, Development.

[33]  Rani K. Powers,et al.  Sequential specification of oligodendrocyte lineage cells by distinct levels of Hedgehog and Notch signaling. , 2018, Developmental biology.

[34]  A. Salic,et al.  Structural Basis of Smoothened Activation in Hedgehog Signaling , 2018, Cell.

[35]  T. Caspary,et al.  Hypomorphic Smo mutant with inefficient ciliary enrichment disrupts the highest level of vertebrate Hedgehog response. , 2018, Developmental biology.

[36]  F. D. de Sauvage,et al.  Grking the Smoothened signal , 2018, Science Signaling.

[37]  P. Ingham,et al.  G protein–coupled receptors control the sensitivity of cells to the morphogen Sonic Hedgehog , 2018, Science Signaling.

[38]  Z. Xuan,et al.  Basal Suppression of the Sonic Hedgehog Pathway by the G-Protein-Coupled Receptor Gpr161 Restricts Medulloblastoma Pathogenesis. , 2018, Cell reports.

[39]  D. Virshup,et al.  Oncogenic RAS-induced CK1α drives nuclear FOXO proteolysis , 2017, Oncogene.

[40]  J. Benovic,et al.  G protein-coupled receptor kinases: Past, present and future. , 2018, Cellular signalling.

[41]  Benjamin R. Myers,et al.  Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium , 2017, Proceedings of the National Academy of Sciences.

[42]  Ying Su,et al.  The emerging roles of phosphatases in Hedgehog pathway , 2017, Cell Communication and Signaling.

[43]  Qing Meng,et al.  PKA-mediated Gli2 and Gli3 phosphorylation is inhibited by Hedgehog signaling in cilia and reduced in Talpid3 mutant. , 2017, Developmental biology.

[44]  J. Reiter,et al.  Genes and molecular pathways underpinning ciliopathies , 2017, Nature Reviews Molecular Cell Biology.

[45]  S. Schulz,et al.  Emerging Paradigms of G Protein-Coupled Receptor Dephosphorylation. , 2017, Trends in pharmacological sciences.

[46]  J. Cheung,et al.  Structure-Based Design of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors Based on Paroxetine. , 2017, Journal of medicinal chemistry.

[47]  A. McMahon,et al.  Hedgehog Signaling: From Basic Biology to Cancer Therapy. , 2017, Cell chemical biology.

[48]  T. Hughes,et al.  Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics , 2016, Proceedings of the National Academy of Sciences.

[49]  A. Salic,et al.  Mechanism of inhibition of the tumor suppressor Patched by Sonic Hedgehog , 2016, Proceedings of the National Academy of Sciences.

[50]  R. Segal,et al.  Hedgehog Signal Transduction: Key Players, Oncogenic Drivers, and Cancer Therapy. , 2016, Developmental cell.

[51]  M. Sansom,et al.  Structural basis for Smoothened regulation by its extracellular domains , 2016, Nature.

[52]  B. Wang,et al.  Regulation of Smoothened Phosphorylation and High-Level Hedgehog Signaling Activity by a Plasma Membrane Associated Kinase , 2016, PLoS biology.

[53]  P. Ingham,et al.  An essential role for Grk2 in Hedgehog signalling downstream of Smoothened , 2016, EMBO reports.

[54]  P. Jackson,et al.  Smoothened determines β-arrestin–mediated removal of the G protein–coupled receptor Gpr161 from the primary cilium , 2016, The Journal of cell biology.

[55]  Allis S. Chien,et al.  Proteomics of Primary Cilia by Proximity Labeling. , 2015, Developmental cell.

[56]  R. Luján,et al.  Orphan Receptor GPR158 Is an Allosteric Modulator of RGS7 Catalytic Activity with an Essential Role in Dictating Its Expression and Localization in the Brain*♦ , 2015, The Journal of Biological Chemistry.

[57]  H. Horiuchi.  Seven-transmembrane receptors , 2015 .

[58]  R. Ahrends,et al.  Gli protein activity is controlled by multisite phosphorylation in vertebrate Hedgehog signaling. , 2014, Cell reports.

[59]  Min Chen,et al.  Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification , 2013, Nature Medicine.

[60]  Benjamin R. Myers,et al.  Hedgehog pathway modulation by multiple lipid binding sites on the smoothened effector of signal response. , 2013, Developmental cell.

[61]  Susan S. Taylor,et al.  PKA: lessons learned after twenty years. , 2013, Biochimica et biophysica acta.

[62]  J. Briscoe,et al.  The mechanisms of Hedgehog signalling and its roles in development and disease , 2013, Nature Reviews Molecular Cell Biology.

[63]  N. Riobo,et al.  Smoothened Is a Fully Competent Activator of the Heterotrimeric G Protein Gi , 2013, Molecular Pharmacology.

[64]  L. Rangell,et al.  The Ciliary G-Protein-Coupled Receptor Gpr161 Negatively Regulates the Sonic Hedgehog Pathway via cAMP Signaling , 2013, Cell.

[65]  Ping Zhang,et al.  Assembly of allosteric macromolecular switches: lessons from PKA , 2012, Nature Reviews Molecular Cell Biology.

[66]  A. Schier,et al.  Attenuation of Notch and Hedgehog Signaling Is Required for Fate Specification in the Spinal Cord , 2012, PLoS genetics.

[67]  A. Mushegian,et al.  G protein-coupled receptor kinases: more than just kinases and not only for GPCRs. , 2012, Pharmacology & therapeutics.

[68]  P. Ingham,et al.  Hedgehog signaling. , 2012, Cold Spring Harbor perspectives in biology.

[69]  K. Anderson,et al.  Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube , 2011, Development.

[70]  D. Rowitch,et al.  A Small-Molecule Smoothened Agonist Prevents Glucocorticoid-Induced Neonatal Cerebellar Injury , 2011, Science Translational Medicine.

[71]  S. Angers,et al.  Gli proteins in development and disease. , 2011, Annual review of cell and developmental biology.

[72]  J. Briscoe,et al.  Sonic Hedgehog Dependent Phosphorylation by CK1α and GRK2 Is Required for Ciliary Accumulation and Activation of Smoothened , 2011, PLoS biology.

[73]  S. Pons,et al.  Sonic Hedgehog-induced Proliferation Requires Specific Gα Inhibitory Proteins* , 2011, The Journal of Biological Chemistry.

[74]  M. Serg,et al.  Smoothened agonist augments proliferation and survival of neural cells , 2010, Neuroscience Letters.

[75]  P. Thérond,et al.  Evaluating Smoothened as a G-protein-coupled receptor for Hedgehog signalling. , 2010, Trends in cell biology.

[76]  K. Anderson,et al.  The primary cilium: a signalling centre during vertebrate development , 2010, Nature Reviews Genetics.

[77]  M. Scott,et al.  The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins. , 2010, Genes & development.

[78]  S. Pons,et al.  Sonic-hedgehog-mediated proliferation requires the localization of PKA to the cilium base , 2010, Journal of Cell Science.

[79]  P. Beachy,et al.  Gli2 trafficking links Hedgehog-dependent activation of Smoothened in the primary cilium to transcriptional activation in the nucleus , 2009, Proceedings of the National Academy of Sciences.

[80]  James Briscoe,et al.  Establishing and interpreting graded Sonic Hedgehog signaling during vertebrate neural tube patterning: the role of negative feedback. , 2009, Cold Spring Harbor perspectives in biology.

[81]  P. Chuang,et al.  Smoothened Adopts Multiple Active and Inactive Conformations Capable of Trafficking to the Primary Cilium , 2009, PloS one.

[82]  M. Scott,et al.  Hedgehog signal transduction by Smoothened: Pharmacologic evidence for a 2-step activation process , 2009, Proceedings of the National Academy of Sciences.

[83]  James K. Chen,et al.  The decoupling of Smoothened from Galphai proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning. , 2008, Developmental biology.

[84]  Gord Fishell,et al.  The genetics of early telencephalon patterning: some assembly required , 2008, Nature Reviews Neuroscience.

[85]  K. Anderson,et al.  Intraflagellar transport, cilia, and mammalian Hedgehog signaling: Analysis in mouse embryonic fibroblasts , 2008, Developmental dynamics : an official publication of the American Association of Anatomists.

[86]  P. Cohen,et al.  The selectivity of protein kinase inhibitors: a further update. , 2007, The Biochemical journal.

[87]  M. Scott,et al.  Patched 1 Regulates Hedgehog Signaling at the Primary Cilium , 2022 .

[88]  G. Fishell,et al.  Morphogen to mitogen: the multiple roles of hedgehog signalling in vertebrate neural development , 2006, Nature Reviews Neuroscience.

[89]  N. Riobo,et al.  Activation of heterotrimeric G proteins by Smoothened , 2006, Proceedings of the National Academy of Sciences.

[90]  D. L. Stenkamp.  Multiple Roles for Hedgehog Signalling in Zebrafish Eye Development , 2006 .

[91]  Didier Y. R. Stainier,et al.  Vertebrate Smoothened functions at the primary cilium , 2005, Nature.

[92]  Qihong Zhang,et al.  Gli2 and Gli3 Localize to Cilia and Require the Intraflagellar Transport Protein Polaris for Processing and Function , 2005, PLoS genetics.

[93]  M. Zastrow.  Faculty Opinions recommendation of Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2. , 2005 .

[94]  Toyoaki Tenzen,et al.  Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia. , 2004, Genes & development.

[95]  P. Cohen,et al.  D4476, a cell‐permeant inhibitor of CK1, suppresses the site‐specific phosphorylation and nuclear exclusion of FOXO1a , 2004, EMBO reports.

[96]  M. Perron,et al.  Hedgehog signaling in vertebrate eye development: a growing puzzle , 2004, Cellular and Molecular Life Sciences CMLS.

[97]  R. Lefkowitz,et al.  Direct Binding of Activated c-Src to the β3-Adrenergic Receptor Is Required for MAP Kinase Activation* , 2000, The Journal of Biological Chemistry.

[98]  T. Jessell.  Neuronal specification in the spinal cord: inductive signals and transcriptional codes , 2000, Nature Reviews Genetics.

[99]  M. Scott,et al.  Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine , 2000, Nature.

[100]  R. Lefkowitz.  The superfamily of heptahelical receptors , 2000, Nature Cell Biology.

[101]  Philip A Beachy,et al.  Hedgehog-Regulated Processing of Gli3 Produces an Anterior/Posterior Repressor Gradient in the Developing Vertebrate Limb , 2000, Cell.

[102]  V. Wallace.  Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum , 1999, Current Biology.

[103]  Konrad Basler,et al.  Hedgehog Controls Limb Development by Regulating the Activities of Distinct Transcriptional Activator and Repressor Forms of Cubitus interruptus , 1999, Cell.

[104]  M. Scott,et al.  Control of Neuronal Precursor Proliferation in the Cerebellum by Sonic Hedgehog , 1999, Neuron.

[105]  A. Kasarskis,et al.  A phenotype-based screen for embryonic lethal mutations in the mouse. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[106]  T. Kornberg,et al.  Proteolysis That Is Inhibited by Hedgehog Targets Cubitus interruptus Protein to the Nucleus and Converts It to a Repressor , 1997, Cell.

[107]  M. Levine,et al.  Protein kinase A is a common negative regulator of Hedgehog signaling in the vertebrate embryo. , 1996, Genes & development.