Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Phosphorylation bar-coding of Free Fatty Acid receptor 2 is generated in a tissue-specific manner

ABSTRACT: Free Fatty Acid receptor 2 (FFA2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using a Designer Receptor Exclusively Activated by Designer Drugs (DREADD) variant of human FFA2 we explored the activation and phosphorylation profile of the receptor, both in a heterologous cell line and in tissues from a transgenic knock-in mouse line expressing this DREADD. FFA2 phospho-site specific antisera targeting either pSer296/pSer297 or pThr306/pThr310 provided sensitive biomarkers of both constitutive and agonist-mediated phosphorylation as well as an effective means to visualise agonist-activated receptors in situ. In white adipose tissue phosphorylation of residues Ser296/Ser297 was enhanced upon agonist activation whilst Thr306/Thr310 did not become phosphorylated. By contrast, in immune cells from Peyer’s patches Thr306/Thr310 become phosphorylated in a strictly agonist-dependent fashion. This was also true in enteroendocrine cells of the colon. The concept of phosphorylation bar-coding has centred to date on the potential for different agonists to promote distinct receptor phosphorylation patterns. Here we demonstrate that this occurs for the same agonist-receptor pairing in different patho-physiologically relevant target tissues. This may underpin why a single G protein-coupled receptor can generate different functional outcomes in a tissue-specific manner.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sally Shirran

LAB HEAD: Graeme Milligan

PROVIDER: PXD042684 | Pride | 2023-11-28

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

Project description:Agonists for the nociceptin/orphanin FQ opioid peptide NOP receptor, a member of the opioid receptor family, are under active investigation as novel analgesics, but their modes of signaling are less well characterized than those for other members of the opioid receptor family. Therefore, we investigated whether different NOP receptor ligands show differential signaling or functional selectivity at the NOP receptor. Using newly developed phosphosite-specific antibodies to NOP receptor, we found that agonist-induced NOP receptor phosphorylation occurred primarily at four carboxyl-terminal serine (S) and threonine (T) residues, namely Ser346, Ser351, Thr362 and Ser363, and proceeded with a temporal hierarchy, with Ser346 as the first site of phosphorylation. G protein-coupled receptor kinases 2 and 3 (GRK2/3) cooperated during agonist-induced phosphorylation, which in turn facilitated NOP receptor desensitization and internalization. A comparison of structurally distinct NOP receptor agonists revealed dissociation in functional efficacies between G protein-dependent signaling and receptor phosphorylation. Furthermore, in vivo, in NOP-eGFP and NOP-eYFP mice, NOP receptor agonists induced multisite phosphorylation and internalization in a dose-dependent and agonist-selective manner that could be blocked by specific antagonists. Together, our study provides novel tools to study ligand-activated NOP receptor signaling in vitro and in vivo. Differential agonist-selective NOP receptor phosphorylation by chemically diverse NOP receptor agonists suggests that differential signaling by NOP receptor agonists may play a role in NOP receptor ligand pharmacology.

2020-05-26 | PXD012908 | Pride

ALLOSTERIC ACTIVATION OF A PLANT IMMUNE RECEPTOR KINASE COMPLEX

Project description:Receptor kinases (RKs) play fundamental roles in extracellular sensing to regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) function primarily as peptide receptors that regulate myriad aspects of plant development and response to external stimuli. Extensive phosphorylation of LRR-RK cytoplasmic domains is among the earliest detectable responses following ligand perception, and reciprocal transphosphorylation between a receptor and its co-receptor is proposed to activate the receptor complex. Originally conceived based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model RK, we set out to understand the steps critical for activating RK complexes. The EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo. Nevertheless, catalytically deficient EFR variants are functional in anti-bacterial immunity. These results reveal a non-catalytic role for the EFR cytoplasmic domain in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RK complexes with RD- versus non-RD-type protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD-type protein kinase may be activated by an allosteric mechanism triggered by activation segment phosphorylation and possibly involving conformational changes of the protein kinase domain of the ligand-binding receptor.

2021-10-27 | PXD025597 | Pride

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases.

Project description:Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed two decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, MuSK agonist antibodies function independently of its co-receptor Lrp4. Recent reports suggest that MuSK agonist antibodies can delay the onset of muscle denervation in mouse models of amyotrophic lateral sclerosis (ALS). To get a deeper understanding of MuSK signaling, and potentially identify Lrp4-independent signaling downstream of MuSK activation, we performed phosphoproteome profiling of myotubes treated with each agonist. Our approach involved quantifying changes in site-specific phosphorylation in orthogonal dose response and time course experiments with each agonist using isobaric mass tags. We observed that both agonists elicited remarkably similar intracellular responses, as evidenced by highly correlating tyrosine phosphorylation on known MuSK signaling components and newly identified cytoskeletal and intracellular trafficking nodes. Among these was inducible phosphorylation on a conserved N-terminal phosphorylation site present on a family of endosomal Rab GTPases. We confirmed that suppression of MuSK signaling by a small molecule reduces Rab10 tyrosine phosphorylation. Importantly, Rab10 mutation at this site disrupts its association with adaptor molecules Mical1 and Mical3. Together these data provide an in depth characterization of the MuSK signaling pathway, describe two small molecule kinase inhibitors, and reveal a novel regulatory mechanism of small Rab GTPases that is poised to regulate intracellular trafficking downstream of receptor tyrosine kinase activation.

2020-06-04 | MSV000085547 | MassIVE

Expression data from Mus musculus white adipose tissue

Project description:We used microarrays to detail the gene expression profile during WAT -beige transition by treatment of beta adrenergic receptor agonist . Stromal vascular fractions (SVF) from mice (n = 3/group) that received vehicle or beta3 adrenergic receptor agonist, CL, treatment were served for RNA extraction and hybridization on Affymetrix microarrays. We are trying to find out angiogenic factors genes dynamics during white adipose tissues (WAT) - beige transition.

2014-03-15 | E-GEOD-55934 | biostudies-arrayexpress

miRNA expression profile in platelets after ex vivo activation

Project description:We performed microRNA sequencing (miRNA-seq) according to Illumina sequencing protocols. Platelet-rich plasma (PRP) has been obtained by centrifuging whole blood at 160 x g for 15 min at room temperature. Platelets have been activated with 3 different physiologic agonists, namely ADP (20uM), collagen (60ug/mL), or Thrombin Receptor Activating Peptide (TRAP 25uM) under continuous stirring. An aliquot of platelets has been obtained at 120 minutes following ADP, collagen and TRAP and then processed to determine miRNA expression profiles. Time 0, indicating samples before any agonist treatment, was used as baseline. Total RNA was extracted by using mirVana Paris kit (life technologies) and then was subjected to size selection library preparation and retrotranscribed. Obtained cDNA was sequenced with the Illumina HiSeq 1000 sequencer .

2015-04-27 | E-MTAB-2078 | biostudies-arrayexpress

Role of Adenosine 2A Receptors (A2AR) on regulatory T cells (Tregs)

Project description:The adenosine 2A receptor (A2AR) is expressed on regulatory T cells (Tregs), but the functional significance is currently unknown. We compared the gene expression between wild-type (WT) and A2AR knockout (KO) Tregs and between WT Tregs treated with vehicle or a selective A2AR agonist. FACS-sorted GFP positive Tregs from WT or A2AR KO FoxP3GFP mouse spleen and lymph nodes were incubated 18 hr with vehicle (DMSO), a separate set of WT Tregs were incubated with the selective A2AR agonist ATL1222 10 nM (Dogwood Pharmaceuticals, Inc.) for 18 hr prior to RNA isolation.

2012-05-31 | E-GEOD-34006 | biostudies-arrayexpress

Platelet RNA-Seq

Project description:Platelet-rich plasma (PRP) has been obtained by centrifuging whole blood at 160 g for 15 min at room temperature. Platelets have been activated with 2 different physiologic agonists, namely collagen (60ug/mL) or Thrombin Receptor Activating Peptide (TRAP 25uM) under continuous stirring. An aliquot of platelets has been obtained at 120 minutes following collagen and TRAP and then processed to determine mRNA expression profiles. Time 0, indicating samples before any agonist treatment, was used as baseline. Total RNA was extracted using the mirVana PARIS kit (LifeTechnologies), according to manufacturers instruction. Indexed libraries were prepared using 500 ng of total RNA as starting material and sequenced on HiSeq 1500 (Illumina, USA) at a concentration of 8pM for 2x100 plus 7 additional cycles for indexes sequencing. Standard workflow for quality control through RTA and bcl2fastq conversion tool was applied.

2015-04-27 | E-MTAB-2355 | biostudies-arrayexpress

Characterization of receptor-associated protein complex assembly in Interleukin (IL)-2- and IL-15-activated T-lymphocytes

Project description:Despite extensive investigation, it remains a paradox that IL-2 and IL-15 can differentially modulate the immune response using the same signaling receptors. In our previous work, we dissected the phosphotyrosine-driven signaling cascades triggered by both cytokines in Kit225 T-cells unveiling subtle differences that may contribute to their functional dichotomy. In the present study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is fine tune orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared by the two cytokines, we defined the components of the early IL-2 and IL-15 receptor-associated complex discovering novel constituents such as FAM59A and SOCS2. Additionally, phosphopeptide-directed analysis allowed us to detect several cytokine-dependent and –independent phosphorylation events within the activated receptor complex including novel phosphorylated sites located in the cytoplasmic region of IL-2Rβ. We proved that the distinct phosphorylations induced by the cytokines serve for recruiting different types of effectors to the initial receptor/ligand complex. Whereas IL-2Rβ pS431 binds to clathrins, which are involved in the receptor internalization and subsequent signal attenuation, IL-2Rγ pY325 and pY357 attract phosphatases, adaptor proteins, kinases as well as the newly identified member of the interleukin receptor complex SOCS2. Overall our study sheds new light into the initial molecular mechanisms triggered by IL-2 and IL-15 and constitutes a further step towards a better understanding of the early signaling aspects of the two closely-related cytokines in T-lymphocytes.

2016-08-08 | PXD002386 | Pride

Mapping AT1R biased signaling using proximity-labeling and proteomics identifies diverse actions of biased agonists

Project description:Angiotensin II type 1 receptors (AT1Rs) are one of the most widely studied G protein-coupled receptors. To fully appreciate the diversity in cellular signaling profiles activated by AT1R transducer-biased ligands, we utilized peroxidase-catalyzed proximity labeling to capture proteins in close proximity to AT1Rs in response to six different ligands: Angiotensin II (full agonist), S1I8 (partial agonist), TRV055 and TRV056 (G protein-biased agonists), TRV026 and TRV027 (β-arrestin biased agonists) at 90s, 10min, and 60min after stimulation. We systematically analyzed the kinetics of AT1R trafficking and determined that distinct ligands lead AT1R to different cellular compartments for downstream signaling activation and receptor degradation/recycling. Distinct proximity labelling of proteins from a number of functional classes, including GTPases, adaptor proteins, and kinases were activated by different ligands suggesting unique signaling and physiological roles of the AT1R. Ligands within the same class, i.e. either G protein-biased or -arrestin-biased, shared high similarity in their labelling profiles. Comparison between ligand classes revealed distinct signaling activation such as greater labeling by G protein biased ligands on ESCRT-0 complex proteins that act as sorting machinery for ubiquitinated proteins. Our study provides a comprehensive analysis of AT1R receptor trafficking kinetics and signaling activation profiles induced by distinct classes of ligands.

2021-03-25 | PXD023814 | Pride

Phosphoproteomic and functional approaches reveal changes in sperm-specific proteins downstream KOR in human spermatozoa

Project description:G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptors superfamily that in response to external stimuli, transduce signals via G proteins to initiate different intracellular signaling pathways which culminate in specific cellular responses. Although in somatic cells the GPCR participate in a broad variety of physiological processes, the expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. Mature spermatozoa could present unique features in their molecular mechanisms downstream GPCRs due to the fact that they possess sperm-specific proteins and are transcriptionally and translationally silent. In order to decipher the signaling pathways engaged by this receptor superfamily in human spermatozoa, we selected the kappa-opioid receptor (KOR) as a study model and applied, for first time, phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses combined with functional studies using a specific agonist of KOR, U50488H.

2019-01-11 | PXD011290 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data