Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A Structural Mechanism for Noncanonical GPCR Signal Transduction in the Hedgehog Pathway

ABSTRACT: The Hedgehog (Hh) signaling pathway is fundamental to embryogenesis, tissue homeostasis, and cancer. Hh signals are transduced via an unusual mechanism: upon agonist-induced phosphorylation, the noncanonical G protein-coupled receptor SMOOTHENED (SMO) binds the catalytic subunit of protein kinase A (PKA-C) and physically blocks its enzymatic activity. By combining computational structural approaches with biochemical and functional studies, we show that SMO mimics strategies prevalent in canonical GPCR and PKA signaling complexes, despite little sequence or secondary structural homology. An intrinsically disordered region of SMO binds the PKA-C active site, resembling the PKA regulatory subunit (PKA-R) / PKA-C holoenzyme, while the SMO transmembrane domain binds a conserved PKA-C interaction hub, similar to other GPCR-effector complexes. In contrast with prevailing GPCR signal transduction models, phosphorylation of SMO promotes intramolecular electrostatic interactions that stabilize key structural elements within the SMO cytoplasmic domain, thereby remodeling it into a PKA-inhibiting conformation. Our work provides a structural mechanism for a central step in the Hh cascade and defines a paradigm for disordered GPCR domains to transmit signals intracellularly.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Varun Venkatakrishnan

LAB HEAD: Benjamin Myers

PROVIDER: PXD067578 | Pride | 2026-03-26

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	230815_15uM_SMOPhospho_0sec_01.raw.zip	Raw
	230815_15uM_SMOPhospho_60sec_01.raw.zip	Raw
	230815_15uM_SMO_Phospho_0sec_02.raw.zip	Raw
	230815_15uM_SMO_Phospho_0sec_03.raw.zip	Raw
	230815_15uM_SMO_Phospho_300sec_01.raw.zip	Raw

Items per page:

1 - 5 of 62

Similar Datasets

Smoothened Transduces Hedgehog Signals via Activity-Dependent Sequestration of PKA Subunits

Project description:The Hedgehog (Hh) pathway is essential for tissue homeostasis, and misregulation of this cascade drives several cancers. To control expression of pathway target genes, the G protein-coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase 2 (GRK2)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking vertebrate Hh signal transduction at the membrane to transcription factor activation in the nucleus. In contrast to existing models, our work shows that this aspect of Hh signaling is fundamentally similar between species. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades.

2021-04-25 | PXD019346 | Pride

A Structural Mechanism for Transduction of Hedgehog Signals Across the Membrane

Project description:The Hedgehog (Hh) signaling pathway is fundamental to embryogenesis, tissue homeostasis, and cancer. Hh signals are transduced across the membrane via an unusual mechanism: upon agonist-induced phosphorylation, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) binds directly to the protein kinase A (PKA) catalytic subunit (PKA-C) and physically blocks its enzymatic activity. How SMO binds PKA-C, and how phosphorylation triggers this process, remain obscure. Here we show, using computational structural methods combined with biochemical, biophysical, and functional approaches, that SMO engages PKA-C by mimicking strategies prevalent in canonical GPCR and PKA signaling complexes, despite little primary sequence or secondary structural homology. A PKA decoy substrate motif and amphipathic helix in the intrinsically disordered SMO intracellular domain bind the PKA-C active site cleft and hinge region, forming a SMO/PKA-C interface resembling the PKA regulatory subunit (PKA-R)/PKA-C holoenzyme. Meanwhile, the intracellular face of the SMO membrane-spanning domain engages a conserved PKA-C interaction hub in a manner strongly reminiscent of canonical GPCR-effector complexes. In contrast with prevailing GPCR signal transduction models, phosphorylation of SMO promotes intramolecular electrostatic interactions that stabilize key secondary structural elements within its cytoplasmic domain, thereby remodeling it into a PKA-inhibiting conformation. Our work provides a structural model for a central step in the Hh cascade and defines a paradigm for disordered GPCR domains to transmit signals intracellularly.

2026-03-01 | PXD055272 | panorama

Ectopic Hedgehog signalling causes cleft palate and defective osteogenesis

Project description:In this study, we have investigated the molecular basis of Shh signalling during development of the secondary palate and how CNCC patterning and fate is influenced by the Shh signalling network. Using a gain-of-function mouse model to activate Smoothened (R26SmoM2) signalling in the palatal mesenchyme (Osr2-IresCre), we demonstrate ectopic Hh-Smo signalling results in fully penetrant cleft palate, disrupted oral-nasal patterning and defective palatine bone formation. We show that a series of Fox transcription factors, including the novel direct target Foxl1, function downstream of Hh signalling in the secondary palate. Furthermore, we demonstrate that Wnt/BMP antagonists, in particular Sostdc1, are positively regulated by Hh signalling, concomitant with down-regulation of key regulators of osteogenesis and BMP signalling effectors. Microarray analysis was performed on excised palatal shelves from Osr2-IresCre+/- (wild-type) and Osr2-IresCre;Smo+/M2 (mutant) embryos at embryonic day (E)13.5. Osr2-IresCre (PMID:17941042) and R26SmoM2 (PMID:15107405) mice have been described previously.

2018-10-02 | E-MTAB-5518 | biostudies-arrayexpress

Gene expression profiling of constitutive activation of Smoothened in the mouse uterus

Project description:In order to gain a better understanding of Ihh action during embryo implantation, we constitutively activated Smo in the murine uterus using the PRcre mouse model (PRcre/+SmoM2+; SmoM2). Female SmoM2 mice were infertile. They exhibited normal serum progesterone levels and normal ovulation, but ova failed to be fertilized in vivo and the uterus failed to undergo the artificially induced decidual response. SmoM2 mice exhibited uterine hypertrophy. The endometrium had a reduced number of uterine glands and the endometrial stroma lost its normal morphologic characteristics. Microarray analysis of 3 month old SmoM2 uteri demonstrated a chondrocytic signature and confirmed that constitutive activation of SmoM2 increased extracellular matrix production. Thus, constitutive activation of Smo in the mouse uterus alters the extracellular matrix which interferes with early pregnancy. Keywords: two group comparison We constitutively activated Hh signaling in the uterus by the expression of a mutant SmoM2 allele. We crossed these mice to the PRcre mouse model to constitutively activate Smo in the murine uterus (PRcre/+SmoM2+; SmoM2). High density DNA microarray analysis was performed on 3 month old control and SmoM2 uteri.

2010-04-13 | E-GEOD-17263 | biostudies-arrayexpress

An essential role for the Gai2 protein in Smoothened-stimulated mammary epithelial cell proliferation

Project description:Hedgehog (Hh) signaling is critical for organogenesis, tissue homeostasis, and stem cell maintenance. Smoothened (SMO), the primary effector of Hh signaling, is expressed ectopically in human breast cancer, as well as in other cancers. Constitutive activation of SMO in mouse mammary glands leads to paracrine stimulation of proliferation, as well as hyperplasia. In canonical signaling, SMO functions via GLI transcription factor activation. However, recent data from Drosophila and mammalian cell lines indicate that SMO can function non-canonically as a G-protein coupled receptor (GPCR) by coupling to heterotrimeric G proteins, particularly those in the pertussis toxin (PTX)-sensitive G-alpha-i (Gai) class. Whether SMO functions as a GPCR in mammalian tissues in vivo is not known. Using genetically modified mouse models, we demonstrate here that SMO-induced stimulation of proliferation is PTX sensitive, and requires Gai2, but not Gai1 or Gai3. Our findings provide evidence for a non-canonical GPCR function of activated SMO in vivo, a finding that may have clinical significance given that most SMO-targeted agents were selected based largely on their ability to block canonical GLI-mediated transcription. Primary mammary epithelial cell RNA was deep-sequenced from mT-mG/SmoM2;MMTV-Cre (EGFP), mT-mG/SmoM2;MMTV-Cre (tdTomato), and mT-mG/SmoM2;+ cells to examine the effects of SmoM2 overexpression in the mammary gland.

2015-09-08 | E-GEOD-70210 | biostudies-arrayexpress

An essential role for the Gai2 protein in Smoothened-stimulated mammary epithelial cell proliferation

2015-09-08 | GSE70210 | GEO

Smoothened Transduces Hedgehog Signals via Activity-Dependent Sequestration of PKA Catalytic Subunits

Project description:The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein-coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.

2021-04-25 | PXD023893 | panorama

Cell Lineage-Guided Microanalytical Mass Spectrometry Reveals Increased Energy Metabolism and Reactive Oxygen Species in the Vertebrate Organizer

Project description:The vertebrate body plan is established through inductive signaling during gastrulation via a signaling center, which is called the Spemann-Mangold organizer (SMO) in the developmental model Xenopus laevis, the South African Clawed Frog. Here, we performed a multiplexed quantitative proteomic screen of the SMO in contrast with the rest of the embryo (RE). Wild-type embryos were cultured to the 32-cell stage, where precursor cells of the SMO were injected with a fluorescent lineage tracing dye. These experimental embryos were cultured to stage 10 (beginning of gastrulation), whence the fluorescently labelled cells were dissected. The proteome of the SMO and the RE were quantified using multiplexing quantification. The tissue samples were processed using a bottom-up proteomic workflow and analyzed using LC-MS3. The resulting data revealed an upregulation of oxidative phosphorylation (OXPHOS) in the SMO tissues.

2024-01-10 | PXD043583 | Pride

Expression data from Hh signaling-deficient OP9 stromal cells

Project description:In the bone marrow B cell development occurs in initimate and essential association with stromal cells. Known effects of stromal cells on B cell development have been shown to be mediated through direct contact and by soluble factors produced by stromal cells. Our findings suggest that cell-nonautonomous Hh signaling may play an important role in B cell lymphopoiesis. We therefore interrogated OP9 stromal cells for Hh-dependent transcripts that may confer B lymphopoietic identity. We generated OP9 stromal cells deficient in Hh signaling through targeting the non-redundant, pathway-obligate GPCR Smoothened. OP9 cells were transduced with RNAi pLKO.1-Puro lentiviral particles expressing a scrambled control shRNA (NT) or shRNA targeting Smoothened (Smo-kd). RNA from stable NT and Smo-kd OP9 cells were isolated in triplicate and global changes in transcripts were analyzed using the Affymetrix Mouse Exon 1.0 ST gene chip.

2012-07-11 | E-GEOD-34421 | biostudies-arrayexpress

Transcription profiling of mouse gain- and loss-of-function Hedgehod (Hh) genetic models reveals Hedgehog signaling is dispensable for adult hematopoietic stem cell function

Project description:The Hedgehog (Hh) signaling pathway is a developmentally conserved regulator of stem cell function. Several reports suggested that Hh signaling is an important regulator of hematopoietic stem cell (HSC) maintenance and differentiation. Here we test this hypothesis in vivo using both gain- and loss-of-function Hh genetic models. Surprisingly, our studies demonstrate that conditional Smoothened (Smo) deletion or over-activation has no significant effects on adult HSC self-renewal and function. Moreover, they indicate a lack of synergism between the Notch and Hh pathways in HSC function, as RBPJ- and Smo-deficiency do not affect hematopoiesis. In agreement with this notion, detailed genome-wide transcriptome analysis reveals that silencing of Hh signaling does not significantly alter the HSC-specific gene expression âsignatureâ. Our studies demonstrate that the Hh signaling pathway is dispensable for adult HSC function and suggest that the Hh pathway can be targeted in future clinical trials addressing the effect of Hh inhibition on leukemia-initiating cell maintenance. Transcriptional consequences of inactivating Smo (hh loss-of-function) in LKS cells. Experiment Overall Design: Four samples were analyzed: wild-type (WT) control and Smo-deficient (SMO) Lin-ckit+Sca1+ (LSK) cells, as well as Lin-ckit+Sca1- myeloid progenitor (MP) cells, which served as a control for LSK-enriched/specific genes. Total bone marrow cells were pooled from four WT and four SMO mice before sorting LSK and MP populations.

2009-03-21 | E-GEOD-15194 | biostudies-arrayexpress