Evaluating Smoothened as a G-protein-coupled receptor for Hedgehog signalling

doi:10.1016/j.tcb.2010.02.002

Trends in Cell Biology

Volume 20, Issue 5, May 2010, Pages 287-298

https://doi.org/10.1016/j.tcb.2010.02.002 Get rights and content

The Hedgehog signalling pathway controls numerous developmental processes. In response to Hedgehog, Smoothened (Smo), a seven-pass transmembrane protein, orchestrates pathway signalling and controls transcription factor activation. In the absence of Hedgehog, the receptor Patched indirectly inhibits Smo in a catalytic manner. Many questions surrounding Smo activation and signalling remain. Recent findings in Drosophila and vertebrate systems have provided strong evidence that Smo acts as a G-protein-coupled receptor. We discuss the role and regulation of Smo and reassess similarities between Smo and G-protein-coupled receptors. We also examine recently identified members of the invertebrate and vertebrate Smo signalling cascades that are typical components of G-protein-coupled receptor pathways. Greater understanding of the mechanisms of Smo activation and its signalling pathways will allow implementation of novel strategies to target disorders related to disruption of Hh signalling.

Section snippets

The Hedgehog signalling pathway and Smo

The Hedgehog (Hh) signalling pathway controls many aspects of tissue patterning during metazoan development (reviewed in Ref. [1]), and diminished Hh signalling results in developmental disorders such as holoprosencephaly and cyclopia [2]. Furthermore, aberrant activation of Hh signalling is implicated in several different cancers including basal cell carcinomas [3].

Maturation of the Hh protein is evolutionarily conserved, and displays unusual features including the covalent attachment of

Similarities in domain organisation

Initial hydropathy analysis revealed Smo to be an integral membrane protein containing seven membrane-spanning alpha helices, a long N-terminal extracellular peptide, and a C-terminal intracellular peptide [7] (Figure 3). Smo is most closely related to the Frizzled (Fz) family of GPCRs [7], with the Drosophila Smo (dSmo) heptahelical region showing 31% identity and 52% similarity to that of dFz. High homology is also found within the extracellular N-terminal cysteine-rich domains (CRD) of dFz

Concluding remarks

The Hh signalling pathway is vital in numerous aspects of cell biology and development, and its deregulation is the cause of several developmental disorders and cancers. Although this pathway has partially diverged during evolution (Box 3), the pivotal role of the GPCR-like protein Smo as a signalling gate-keeper is conserved. Although striking differences at the amino acid sequence level exist between Smo of different evolutionary lineages, the activation of Smo could use many of the same

Acknowledgments

We are grateful to F. Wendler and R. Hynynen for critical analysis of the manuscript and D.W. McConnell for his editing genius. K.L.A was supported by a Marie Curie early stage training scholarship as well as a grant from the Association pour la Recherche sur le Cancer (l’ARC). Our lab is supported by grants from La Ligue National Contre Le Cancer ‘Equipe labellisée 2008’ to P.P.T.

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Structural dynamics of Smoothened (SMO) in the ciliary membrane and its interaction with membrane lipids
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The Smoothened receptor (SMO, a 7 pass transmembrane domain, Class F GPCR family protein) plays a crucial role in the Hedgehog (HH) signaling pathway, which is involved in embryonic development and is implicated in various types of cancer throughout the animal kingdom. In the absence of HH signaling, SMO is inhibited by Patched 1 (PTC1; a 12 pass transmembrane domain protein), which is localized in the primary cilia. HH binding leads to the dislocation of PTC1 from the cilia, thus making way for SMO to localize in the primary cilia, as an essential prerequisite for its activation. We have carried out MARTINI coarse-grained molecular dynamics simulations of SMO in POPC and in ciliary membrane models, respectively, to study the interactions of SMO with cholesterol and other lipid molecules in the ciliary membrane, and to gain molecular-level insights into the role of the primary cilia in shaping the functional dynamics of SMO. We are able to identify the interaction of membrane cholesterols with definite sites and domains within SMO and relate them with known cholesterol-binding sequence and structure motifs. We show that cholesterol interactions with the transmembrane domain TMD, unlike those with the cysteine-rich domain (CRD) and the intracellular domain (ICD), are through residues belonging to known cholesterol-binding motifs. Notably, a few persistent interactions of cholesterol with lower TM cholesterol-binding domains are governed by the presence of multiple cholesterol-binding motifs. These analyses have also helped to identify and define a strict cholesterol consensus motif (CCM), which may well steer cholesterol into the hitherto identified binding sites within the TMD of SMO. We have also reported the interaction of phosphatidylinositol 4-phosphate with the intracellular region of transmembrane (TM) helices (TM1, TM3, TM4, and TM5), intracellular loop1, helix8, and Arg/Lys clusters of the ICD. Structural analysis of SMO domains shows significant changes in the CRD and ICD, during the course of the simulation. Further detailed analysis of the dynamics of the TMD reveals the movements of TM5, TM6, and TM7, linked with the helix8, which are possibly involved in shaping the conformational disposition of the ICD. The movement of these TM helices could possibly be a consequence of interactions involving the extracellular domain and extracellular loops. In addition, our analysis also shows that phosphatidylinositol-4-phosphate (PI4P), along with some ICD cholesterols, are implicated in anchoring SMO in the membrane.
Modulatory effects of G protein-coupled receptor in hepatocellular carcinoma
2022, Theranostics and Precision Medicine for the Management of Hepatocellular Carcinoma, Volume 1: Biology and Pathophysiology
Hepatocellular carcinoma is the major reason of death related to cancer around the globe. Inflammation is a significant factor in the progression of hepatocellular carcinoma. Two categories of inflammation-induced tissue injury have been reported. One is microbial attack or viral hepatitis which results in persistent inflammation and the other is drug-induced liver injury, nonalcoholic steatohepatitis and alcohol abuse. For 70% hepatocarcinoma patient’s, early diagnosis is not feasible and during later stages treatment is very difficult, reoccurrence of cancer and upsurge in metastatic rates which leads to increase in mortality rates. Hence, identification of novel molecular biomarkers of hepatocellular carcinoma and the elucidation of the molecular mechanisms underlying hepatocellular carcinoma is of great importance. The G protein–coupled receptors or GPCRs, also called seven receptors with transmembrane domains, are one of the biggest cell surface receptors. They are said to play an important function in migration of cells, growth, metastasis, invasion, and survival of cancer. GPCRs can regulate several signal transduction cascades by way of G protein–dependant or an independent pathway that comprises ion channels, G protein receptor kinases, and Src kinases otherwise β-arrestins. GPCRs and its cognate ligand’s role in the pathophysiological progression and development of hepatocellular carcinoma are complicated. Hence, GPCRs and their effectors that are regulated downstream deliver numerous drug targets for developing innovative strategies in hepatocellular carcinoma treatment and prevention.
Effect of mutations on drug resistance of smoothened receptor toward inhibitors probed by molecular modeling
2020, Chemical Physics Letters
The smoothened receptor (SMO) plays a critical role in embryonic development. Vismodegib was approved by the FDA to treat metastatic basal-cell carcinoma. However, The D473H mutation can cause obvious drug resistance toward Vismodegib. In this work, the drug-resistance mechanisms of three molecules were explored by theoretical calculations. The results indicate that the D473H mutation can disrupt the hydrogen-bonding network in the binding pocket, which can weaken the binding of Vismodegib. Whereas, for LY2940680 and L4 there are new hydrogen bond networks formed, which can stabilize their binding. This work provides a theoretical contribution to design more promising SMO inhibitors.
Sonic hedgehog enhances calcium oscillations in hippocampal astrocytes
2019, Journal of Biological Chemistry
Citation Excerpt :
These results suggest that Ca release not though ryanodine receptor but through IP3Rs is relevant to the Ca oscillations enhanced by SAG. SMO is a 7-transmembrane receptor that couples with a trimeric G protein, Gi (38, 39). We tested if the SMO enhancement of Ca oscillations requires Gi activation by incubating astrocytes with pertussis toxin (PTX), a Gi inhibitor.
Sonic hedgehog (SHH) is important for organogenesis during development. Recent studies have indicated that SHH is also involved in the proliferation and transformation of astrocytes to the reactive phenotype. However, the mechanisms underlying these are unknown. Involvement of SHH signaling in calcium (Ca) signaling has not been extensively studied. Here, we report that SHH and Smoothened agonist (SAG), an activator of the signaling receptor Smoothened (SMO) in the SHH pathway, activate Ca oscillations in cultured murine hippocampal astrocytes. The response was rapid, on a minute time scale, indicating a noncanonical pathway activity. Pertussis toxin blocked the SAG effect, indicating an involvement of a G_i coupled to SMO. Depletion of extracellular ATP by apyrase, an ATP-degrading enzyme, inhibited the SAG-mediated activation of Ca oscillations. These results indicate that SAG increases extracellular ATP levels by activating ATP release from astrocytes, resulting in Ca oscillation activation. We hypothesize that SHH activates SMO-coupled Gi in astrocytes, causing ATP release and activation of G_q/11-coupled P2 receptors on the same cell or surrounding astrocytes. Transcription factor activities are often modulated by Ca patterns; therefore, SHH signaling may trigger changes in astrocytes by activating Ca oscillations. This enhancement of Ca oscillations by SHH signaling may occur in astrocytes in the brain in vivo because we also observed it in hippocampal brain slices. In summary, SHH and SAG enhance Ca oscillations in hippocampal astrocytes, G_i mediates SAG-induced Ca oscillations downstream of SMO, and ATP-permeable channels may promote the ATP release that activates Ca oscillations in astrocytes.
Decoding Calcium Signaling Dynamics during Drosophila Wing Disc Development
2019, Biophysical Journal
The robust specification of organ development depends on coordinated cell-cell communication. This process requires signal integration among multiple pathways, relying on second messengers such as calcium ions. Calcium signaling encodes a significant portion of the cellular state by regulating transcription factors, enzymes, and cytoskeletal proteins. However, the relationships between the inputs specifying cell and organ development, calcium signaling dynamics, and final organ morphology are poorly understood. Here, we have designed a quantitative image-analysis pipeline for decoding organ-level calcium signaling. With this pipeline, we extracted spatiotemporal features of calcium signaling dynamics during the development of the Drosophila larval wing disc, a genetic model for organogenesis. We identified specific classes of wing phenotypes that resulted from calcium signaling pathway perturbations, including defects in gross morphology, vein differentiation, and overall size. We found four qualitative classes of calcium signaling activity. These classes can be ordered based on agonist stimulation strength Gαq-mediated signaling. In vivo calcium signaling dynamics depend on both receptor tyrosine kinase/phospholipase C γ and G protein-coupled receptor/phospholipase C β activities. We found that spatially patterned calcium dynamics correlate with known differential growth rates between anterior and posterior compartments. Integrated calcium signaling activity decreases with increasing tissue size, and it responds to morphogenetic perturbations that impact organ growth. Together, these findings define how calcium signaling dynamics integrate upstream inputs to mediate multiple response outputs in developing epithelial organs.
Activation of Smoothened in the Hedgehog pathway unexpectedly increases G<inf>s</inf>-dependent cAMP levels in Drosophila
2018, Journal of Biological Chemistry
Hedgehog (Hh) signaling plays a key role in the development and maintenance of animal tissues. This signaling is mediated by the atypical G protein–coupled receptor (GPCR) Smoothened (Smo). Smo activation leads to signaling through several well-characterized effectors to activate Hh target gene expression. Recent studies have implicated activation of the heterotrimeric G protein subunit Gα_i and the subsequent decrease in cellular cAMP levels in promoting the Hh response in flies and mammals. Although Hh stimulation decreases cAMP levels in some insect cell lines, here using a bioluminescence resonance energy transfer (BRET)-based assay we found that this stimulation had no detectable effect in Drosophila S2-R+ cells. However, we observed an unexpected and significant Gα_s-dependent increase in cAMP levels in response to strong Smo activation in Smo-transfected cells. This effect was mediated by Smo's broadly conserved core, and was specifically activated in response to phosphorylation of the Smo C-terminus by GPCR kinase 2 (Gprk2). Genetic analysis of heterotrimeric G protein function in the developing Drosophila wing revealed a positive role for cAMP in the endogenous Hh response. Specifically, we found that mutation or depletion of Gα_s diminished low-threshold Hh responses in Drosophila, whereas depletion of Gα_i potentiated them (in contrast to previous findings). Our analysis suggested that regulated cAMP production is important for controlling the sensitivity of cellular responses to Hh in Drosophila.

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Trends in Cell Biology

ReviewEvaluating Smoothened as a G-protein-coupled receptor for Hedgehog signalling

Section snippets

The Hedgehog signalling pathway and Smo

Similarities in domain organisation

Concluding remarks

Acknowledgments

Dev. Cell

Cell

Cell

Cell

Mech. Dev.

Cell

J. Biol. Chem.

Mech Dev.

Curr. Biol.

J. Biol. Chem.

Dev. Biol.

Dev. Cell

Dev. Biol.

Pharmacol. Ther.

J. Biol. Chem.

Dev. Biol.

Dev. Biol.

Cell

Cell

Dev. Biol.

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Curr. Biol.

J. Biol. Chem.

Cell

Curr. Biol.

Curr. Biol.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Invest. Dermatol.

Curr. Opin. Cell Biol.

Curr. Top. Dev. Biol.

Curr. Top. Dev. Biol.

Curr. Top. Dev. Biol.

Hedgehog: functions and mechanisms

Genes Dev.

Hedgehog signalling in cancer

Cell. Mol. Life Sci.

Communicating with Hedgehogs

Nat. Rev. Mol. Cell Biol.

Mutations affecting segment number and polarity in Drosophila

Nature

Distinct pathways for autocrine and paracrine Wingless signalling in Drosophila embryos

Nature

smoothened encodes a receptor-like serpentine protein required for hedgehog signalling

Nature

Biochemical evidence that patched is the Hedgehog receptor

Nature

Biochemical characterization of Wnt–frizzled interactions using a soluble, biologically active vertebrate Wnt protein

Proc. Natl. Acad. Sci. U. S. A.

Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened

Genes Dev.

Small molecule modulation of Smoothened activity

Proc. Natl. Acad. Sci. U. S. A.

The third extracellular loop of G-protein-coupled receptors: more than just a linker between two important transmembrane helices

Biochem. Soc. Trans.

Activating Smoothened mutations in sporadic basal-cell carcinoma

Nature

A frequent activated smoothened mutation in sporadic basal cell carcinomas

Oncogene

Missense mutations in SMOH in sporadic basal cell carcinomas of the skin and primitive neuroectodermal tumors of the central nervous system

Cancer Res.

Hedgehog regulates smoothened activity by inducing a conformational switch

Nature

Targeting the hedgehog signaling pathway with small molecules

Anticancer Agents Med. Chem.

Patched acts catalytically to suppress the activity of Smoothened

Nature

Review
Evaluating Smoothened as a G-protein-coupled receptor for Hedgehog signalling