Abstract
Human gene 3 relaxin (H3 relaxin) is a member of the relaxin/insulin family of peptides. Neuropeptides mediate behavioral responses to stress and regulates appetite; however, the cell signaling mechanisms that control these events remain to be identified. The relaxin family peptide receptor 3 (RXFP3, formerly GPCR135 or SALPR) was characterized as the receptor for H3 relaxin, functionally coupled to the inhibition of cAMP. We have identified that RXFP3 stably expressed in Chinese hamster ovary (CHO)-K1 (CHO-RXFP3) and human embryonic kidney (HEK) 293 (HEK-RXFP3) cells activates extracellular signal-regulated kinase (ERK) 1/2 when stimulated with H3 relaxin and an H3 relaxin B-chain (dimer) peptide. Using inhibitors of cellular signaling proteins, we subsequently determined the mechanism of ERK1/2 activation by RXFP3. ERK1/2 phosphorylation requires the activation of Gi/o proteins and seems to require receptor internalization and/or compartmentalization into lipid-rich environments. ERK1/2 activation also predominantly occurred via the activation of a protein kinase C-dependent pathway, although activation of phosphatidylinositol 3-kinase and Src tyrosine kinase were also involved to a lesser extent. The mechanisms underlying ERK1/2 phosphorylation were similar in both CHO-RXFP3 and HEK-RXFP3 cells, although some differences were evident. Phospholipase Cβ and the transactivation of endogenous epidermal growth factor receptors both played a role in RXFP3-mediated ERK1/2 activation in HEK293 cells; however, they were not involved in RXFP3-mediated ERK1/2 activation in the CHO-K1 cell background. The pathways identified in CHO- and HEK-transfected cells were also used in the murine SN56 neuronal cell line, suggesting that these pathways are also important for RXFP3-mediated signaling in the brain.
Footnotes
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E.T.v.d.W. is the recipient of a National Health and Medical Research Council (NHMRC) Dora Lush (Biomedical) Post Graduate Scholarship. P.M.S. is an NHMRC Principal Research Fellow.
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This work was presented in part was presented at British Pharmacological Society, 4th Focused Meeting on Cell Signaling, 15th World Congress of Pharmacology, International Union of Basic and Clinical Pharmacology 2006 and at Australian Health and Medical Research Congress 2006.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.032763.
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ABBREVIATIONS: INSL, insulin-like peptide; CHO, Chinese hamster ovary; RXFP3, relaxin family peptide receptor 3; CHO-RXFP3, Chinese hamster ovary-K1 cells stably expressing relaxin family peptide receptor 3; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; H3 relaxin, human gene 3 relaxin; H2 relaxin, human gene 2 relaxin; HEK, human embryonic kidney; HEK-RXFP3, human embryonic kidney 293 cells stably expressing relaxin family peptide receptor 3; NI, nucleus incertus; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; PDGF, platelet-derived growth factor; PTX, pertussis toxin; SALPR, somatostatin and angiotensin-like peptide receptor; SN56, murine septal neuron-derived cell line; GPCR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; FBS, fetal bovine serum; PMA, phorbol 12-myristate-13-acetate; PKC, protein kinase C; SH, sulfhydryl; FRT, Flp recombination target; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; ANOVA, analysis of variance; PLC, phospholipase C; Pyr, 2-pyridinesulfenyl; PD98059, 2′-amino-3′-methoxyflavone; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; AG1478, 4-(3-chloroanillino)-6,7-dimethoxyquinazoline; U73122, 1-(6-[17β-3-methoxyestra-1,3,5-(10)triene-17-yl]amino/hexyl) 1H-pyrroledione; Ro 31-8220, 3-[1-(3-(amidinothio)propyl-1H-indol-3-yl)]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX); AG370, 2-amino-4(1H-indol-5-yl)-1,1,3-tricyanobuta-13-diene.
- Received November 19, 2006.
- Accepted March 9, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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The Relaxin Family Peptide Receptor 3 Activates Extracellular Signal-Regulated Kinase 1/2 through a Protein Kinase C-Dependent Mechanism
