title: Signal transduction pathways activated by insulin-like peptide 5 at the relaxin family peptide RXFP4 receptor.
abstract: 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.
  
  
  
  INSL5/RXFP4 receptor signalling was investigated using AlphaScreen® proximity assays. Recruitment of Gαi/oproteins by RXFP4 receptors was determined by rescue of Pertussis toxin (PTX)-inhibited cAMP and ERK1/2 responses following transient transfection of PTX-insensitive Gαi/oC351I mutants. Cell proliferation was studied with bromodeoxyuridine. RXFP4 receptor interactions with β-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.
  
  
  
  INSL5 inhibited forskolin-stimulated cAMP accumulation and increased phosphorylation of ERK1/2, p38MAPK, Akt Ser473, Akt Thr308and S6 ribosomal protein. cAMP and ERK1/2 responses were abolished by PTX and rescued by mGαoA, mGαoBand mGαi2and to a lesser extent mGαi1and mGαi3. RXFP4 receptors interacted with GRK2 and β-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.
  
  
  
  Knowledge of signalling pathways activated by INSL5 at RXFP4 receptors is essential for understanding the biological roles of this novel gut hormone.
  
  
  
  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.
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Sheng Y
authors.initials: SY
authors.lastname: Ang
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.|Department of Pharmacology, Monash University, Clayton, VIC, Australia.
authors.forename: Dana S
authors.initials: DS
authors.lastname: Hutchinson
authors.affiliation: The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, Australia.
authors.forename: Nitin
authors.initials: N
authors.lastname: Patil
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Bronwyn A
authors.initials: BA
authors.lastname: Evans
authors.affiliation: The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, Australia.|Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: Ross A D
authors.initials: RAD
authors.lastname: Bathgate
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Halls
authors.affiliation: The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, Australia.
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Hossain
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Summers
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Martina
authors.initials: M
authors.lastname: Kocan
mesh_terms: D000818:Animals; D016466:CHO Cells; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003412:Cricetulus; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D010766:Phosphorylation; D011506:Proteins / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism*; D038601:Ribosomal Protein S6 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013329:Structure-Activity Relationship; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins; C480239:RXFP4 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D038601:Ribosomal Protein S6; D000242:Cyclic AMP; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases