Dataset Information

Signal transduction pathways activated by insulin-like peptide 5 at the relaxin family peptide RXFP4 receptor.

ABSTRACT:

Background and purpose

Insulin-like peptide 5 (INSL5) is a two-chain, three-disulfide-bonded peptide of the insulin/relaxin superfamily, uniquely expressed in enteroendocrine L-cells of the colon. It is the cognate ligand of relaxin family peptide RXFP4 receptor that is mainly expressed in the colorectum and enteric nervous system. This study identifies new signalling pathways activated by INSL5 acting on RXFP4 receptors.

Experimental approach

INSL5/RXFP4 receptor signalling was investigated using AlphaScreen® proximity assays. Recruitment of G?_i/o proteins by RXFP4 receptors was determined by rescue of Pertussis toxin (PTX)-inhibited cAMP and ERK1/2 responses following transient transfection of PTX-insensitive G?_i/o C351I mutants. Cell proliferation was studied with bromodeoxyuridine. RXFP4 receptor interactions with ?-arrestins, GPCR kinase 2 (GRK2), KRas and Rab5a was assessed with real-time BRET. Gene expression was investigated using real-time quantitative PCR. Insulin release was measured using HTRF and intracellular Ca²⁺ flux monitored in a Flexstation® using Fluo-4-AM.

Key results

INSL5 inhibited forskolin-stimulated cAMP accumulation and increased phosphorylation of ERK1/2, p38MAPK, Akt Ser⁴⁷³ , Akt Thr³⁰⁸ and S6 ribosomal protein. cAMP and ERK1/2 responses were abolished by PTX and rescued by mG?_oA , mG?_oB and mG?_i2 and to a lesser extent mG?_i1 and mG?_i3 . RXFP4 receptors interacted with GRK2 and ?-arrestins, moved towards Rab5a and away from KRas, indicating internalisation following receptor activation. INSL5 inhibited glucose-stimulated insulin secretion and Ca²⁺ mobilisation in MIN6 insulinoma cells and forskolin-stimulated cAMP accumulation in NCI-H716 enteroendocrine cells.

Conclusions and implications

Knowledge of signalling pathways activated by INSL5 at RXFP4 receptors is essential for understanding the biological roles of this novel gut hormone.

Linked articles

This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

SUBMITTER: Ang SY

PROVIDER: S-EPMC5406383 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Signal transduction pathways activated by insulin-like peptide 5 at the relaxin family peptide RXFP4 receptor.

Ang Sheng Y SY Hutchinson Dana S DS Patil Nitin N Evans Bronwyn A BA Bathgate Ross A D RAD Halls Michelle L ML Hossain Mohammed A MA Summers Roger J RJ Kocan Martina M

British journal of pharmacology 20160713 10

<h4>Background and purpose</h4>Insulin-like peptide 5 (INSL5) is a two-chain, three-disulfide-bonded peptide of the insulin/relaxin superfamily, uniquely expressed in enteroendocrine L-cells of the colon. It is the cognate ligand of relaxin family peptide RXFP4 receptor that is mainly expressed in the colorectum and enteric nervous system. This study identifies new signalling pathways activated by INSL5 acting on RXFP4 receptors.<h4>Experimental approach</h4>INSL5/RXFP4 receptor signalling was i ...[more]

PMID: 27243554

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Project description:ObjectiveInsulin-like peptide 5 (INSL5) signalling, through its cognate receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), has been reported to be orexigenic, and the high fat diet (HFD) preference observed in wildtype mice is altered in Rxfp4 knock-out mice. In this study, we used a new Rxfp4-Cre mouse model to investigate the mechanisms underlying these observations.MethodsWe generated transgenic Rxfp4-Cre mice and investigated central expression of Rxfp4 by RT-qPCR, RNAscope and intraparenchymal infusion of INSL5. Rxfp4-expressing cells were chemogenetically manipulated in global Cre-reporter mice using designer receptors exclusively activated by designer drugs (DREADDs) or after stereotactic injection of a Cre-dependent AAV-DIO-Dq-DREADD targeting a population located in the ventromedial hypothalamus (RXFP4VMH). Food intake and feeding motivation were assessed in the presence and absence of a DREADD agonist. Rxfp4-expressing cells in the hypothalamus were characterised by single-cell RNA-sequencing (scRNAseq) and the connectivity of RXFP4VMH cells was investigated using viral tracing.ResultsRxfp4-Cre mice displayed Cre-reporter expression in the hypothalamus. Active expression of Rxfp4 in the adult mouse brain was confirmed by RT-qPCR and RNAscope. Functional receptor expression was supported by cyclic AMP-responses to INSL5 application in ex vivo brain slices and increased HFD and highly palatable liquid meal (HPM), but not chow, intake after intra-VMH INSL5 infusion. scRNAseq of hypothalamic RXFP4 neurons defined a cluster expressing VMH markers, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4VMH neural projections to nuclei implicated in hedonic feeding behaviour. Whole body chemogenetic inhibition (Di-DREADD) of Rxfp4-expressing cells, mimicking physiological INSL5-RXFP4 Gi-signalling, increased intake of the HFD and HPM, but not chow, whilst activation (Dq-DREADD), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and motivation to work for the HPM.ConclusionThese findings identify RXFP4VMH neurons as regulators of food intake and preference, and hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.

Project description:G-protein coupled receptors (GPCR) transduce extracellular stimuli into the cell interior and are thus centrally involved in almost all physiological-neuronal processes. This essential function and association with many diseases or pathological conditions explain why GPCRs are one of the priority targets in medical and pharmacological research, including structure determination. Despite enormous experimental efforts over the last decade, both the expression and purification of these membrane proteins remain elusive. This is attributable to specificities of each GPCR subtype and the finding of necessary experimental in vitro conditions, such as expression in heterologous cell systems or with accessory proteins. One of these specific GPCRs is the leucine-rich repeat domain (LRRD) containing GPCR 7 (LGR7), also termed relaxin family peptide receptor 1 (RXFP1). This receptor is characterized by a large extracellular region of around 400 amino acids constituted by several domains, a rare feature among rhodopsin-like (class A) GPCRs. In the present study, we describe the expression and purification of RXFP1, including the design of various constructs suitable for functional/biophysical studies and structure determination. Based on available sequence information, homology models, and modern biochemical and genetic tools, several receptor variations with different purification tags and fusion proteins were prepared and expressed in Sf9 cells (small-scale), followed by an analytic fluorescence-detection size-exclusion chromatography (F-SEC) to evaluate the constructs. The most promising candidates were expressed and purified on a large-scale, accompanied by ligand binding studies using surface plasmon resonance spectroscopy (SPR) and by determination of signaling capacities. The results may support extended studies on RXFP1 receptor constructs serving as targets for small molecule ligand screening or structural elucidation by protein X-ray crystallography or cryo-electron microscopy.

Project description:BackgroundThe relaxin-like peptide family belongs in the insulin superfamily and consists of 7 peptides of high structural but low sequence similarity; relaxin-1, 2 and 3, and the insulin-like (INSL) peptides, INSL3, INSL4, INSL5 and INSL6. The functions of relaxin-3, INSL4, INSL5, INSL6 remain uncharacterised. The evolution of this family has been contentious; high sequence variability is seen between closely related species, while distantly related species show high similarity; an invertebrate relaxin sequence has been reported, while a relaxin gene has not been found in the avian and ruminant lineages.ResultsSequence similarity searches of genomic and EST data identified homologs of relaxin-like peptides in mammals, and non-mammalian vertebrates such as fish. Phylogenetic analysis was used to resolve the evolution of the family. Searches were unable to identify an invertebrate relaxin-like peptide. The published relaxin cDNA sequence in the tunicate, Ciona intestinalis was not present in the completed C. intestinalis genome. The newly discovered relaxin-3 is likely to be the ancestral relaxin. Multiple relaxin-3-like sequences are present in fugu fish (Takifugu rubripes) and zebrafish (Danio rerio), but these appear to be specific to the fish lineage. Possible relaxin-1 and INSL5 homologs were also identified in fish and frog species, placing their emergence prior to mammalia, earlier than previously believed. Furthermore, estimates of synonymous and nonsynonymous substitution rates (dN/dS) suggest that the emergence of relaxin-1, INSL4 and INSL6 during mammalia was driven by positive Darwinian selection, hence these peptides are likely to have novel and in the case of relaxin-1, which is still under positive selection in humans and the great apes, possibly still evolving functions. In contrast, relaxin-3 is constrained by strong purifying selection, demonstrating it must have a highly conserved function, supporting its hypothesized important neuropeptide role.ConclusionsWe present a phylogeny describing the evolutionary history of the relaxin-like peptide family and show that positive selection has driven the evolution of the most recent members of the family.

Dataset Information

Signal transduction pathways activated by insulin-like peptide 5 at the relaxin family peptide RXFP4 receptor.

Background and purpose

Experimental approach

Key results

Conclusions and implications

Linked articles

Publications

Signal transduction pathways activated by insulin-like peptide 5 at the relaxin family peptide RXFP4 receptor.

Similar Datasets

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