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

Abstract

BACKGROUND AND PURPOSEInsulin-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 APPROACHINSL5/RXFP4 receptor signalling was investigated using AlphaScreen® proximity assays. Recruitment of Gα 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 Gα i/o C351I 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 Ca 2+ flux monitored in a Flexstation® using Fluo-4-AM. KEY RESULTSINSL5 inhibited forskolin-stimulated cAMP accumulation and increased phosphorylation of ERK1/2, p38MAPK, Akt Ser 473 , Akt Thr 308 and S6 ribosomal protein. cAMP and ERK1/2 responses were abolished by PTX and rescued by mGα oA , mGα oB and mGα i2 and to a lesser extent mGα i1 and mGα i3 . 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 Ca 2+ mobilisation in MIN6 insulinoma cells and forskolin-stimulated cAMP accumulation in NCI-H716 enteroendocrine cells. CONCLUSIONS AND IMPLICATIONSKnowledge of signalling pathways activated by INSL5 at RXFP4 receptors is essential for understanding the biological roles of this novel gut hormone. LINKED ARTICLESThis 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 Abbreviations BrdU, 5-bromo-2′-deoxyuridine; GLP-1, glucagon-like peptide 1; GRK2, G protein receptor kinase 2; INSL5, insulin-like peptide 5; mTORC, mammalian target of rapamycin complex; PTX, Pertussis toxin; S6RP, S6 ribosomal protein

Real‐time examination of cAMP activity at relaxin family peptide receptors using a BRET‐based biosensor

“…The sustained cAMP activity seen at RXFP1 and RXFP2 is consistent with the finding that these receptors lack β‐arrestin‐mediated desensitization and show poor internalization in response to ligand stimulation . However, we also saw sustained cAMP inhibition at RXFP3 (particularly in HEK293T cells) and RXFP4, which have been shown to interact with β‐arrestins and internalize after stimulation with their endogenous ligands . Although the original characterization of CAMYEL in RAW 264.7 murine macrophage cells showed that CAMYEL can report substantial and rapid decreases in BRET signal, studies on some GPCRs, including the cannabinoid 1, dopamine D 2 L, and mu‐opioid receptors, have also shown relatively sustained changes in CAMYEL signal over extended periods of time.…”

“…(particularly in HEK293T cells) and RXFP4, which have been shown to interact with β-arrestins and internalize after stimulation with their endogenous ligands. 14,15 Although the original characterization of CAMYEL in RAW 264.7 murine macrophage cells showed that CAMYEL can report substantial and rapid decreases in BRET signal, 20 studies on some GPCRs, including the cannabinoid 1, 40 dopamine D 2 L, 19 and mu-opioid 34 Targeting RXFP receptors with drugs has potential for a range of therapeutic applications (reviewed in 4). Much effort has therefore been put into designing analogues of relaxin family peptides, including agonists and antagonists used in the present study.…”

“…Unusually, RXFP1 also couples to Gα i3 to increase cAMP downstream of Gβγ, PI3K, PKC‐ζ, and AC5 in a number of cell types . In contrast, RXFP3 and RXFP4 couple to Gα i/o proteins to inhibit the production of cAMP . However, patterns of cAMP activity also depend on the cell type being stimulated.…”

“…[10][11][12][13] In contrast, RXFP3 and RXFP4 couple to Gα i/o proteins to inhibit the production of cAMP. 14,15 However, patterns of cAMP activity also depend on the cell type being stimulated. For example, fibroblasts that natively express RXFP1 show little or no increases in cAMP activity when stimulated with an RXFP1 agonist.…”

Recent progress in the understanding of relaxin family peptides and their receptors

“…The key residues involved in interaction with RXFP4 receptors have been identified and a minimized analogue that is both easier to assemble and potent is now available. The paper by Ang et al , () is one of the few published to date that examines signalling pathways activated by RXFP4 receptors (Figure ). Not only do these receptors inhibit cAMP production but they also activate p38MAPK, ERK1/2, Akt and S6RP.…”