Abstract
Relaxin family peptide 3 receptors (RXFP3) are activated by H3-relaxin to inhibit forskolin-stimulated cAMP accumulation and stimulate extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. In this study, we sought to identify novel signaling pathways coupled to RXFP3 and to investigate whether other members of the relaxin peptide family activated these pathways. Two patterns of signaling were observed in RXFP3-expressing Chinese hamster ovary (CHO)-K1 and human embryonic kidney (HEK)-293 cells (CHO-RXFP3 and HEK-RXFP3) and murine septal neuron SN56 cell lines: 1) strong inhibition of forskolin-stimulated cAMP accumulation, ERK1/2 activation and nuclear factor (NF)-κB reporter gene activation in cells stimulated with H3 relaxin, with weaker activity observed for H2 relaxin, porcine relaxin, or insulin-like peptide (INSL) 3 and 2) strong stimulation of activator protein (AP)-1 reporter genes by H2 relaxin, with weaker activation observed with H3 or porcine relaxin. Two distinct ligand binding sites were identified on RXFP3-expressing cells using two different radioligands. 125I-INSL5 A-chain/relaxin-3 B-chain chimera bound with high affinity to the RXFP3-expressing cells with competition by H3 relaxin or a H3 relaxin B-chain dimeric peptide, consistent with previous reports. Binding studies with 125I-H2 relaxin revealed a distinct binding site with potent competition observed with H2 relaxin, H3 relaxin, or INSL3 and weaker competition with porcine relaxin. Thus H3 relaxin potently activates all signaling pathways coupled to RXFP3, whereas H2 relaxin is an AP-1-biased ligand relative to H3 relaxin.
Footnotes
↵
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.This work was supported by the National Health and Medical Research Council (NHMRC) of Australia [Project Grants 436713 (to R.J.S.), 454375 (to J.D.W.), Program Grant 519461 (to R.J.S., A.C., P.M.S.)]; an NHMRC Dora Lush (Biomedical) Postgraduate Scholarship (to E.T.v.d.W.); an NHMRC Principal Research Fellowship (to P.M.S.); and NHMRC Senior Research Fellowship (to A.C.).
Portions of this work have appeared previously in the following forms: van der Westhuizen E.T., “Molecular characterization of human and mouse relaxin-3 receptors (RXFP3) in recombinant and endogenously expressing cell lines,” Ph.D. Thesis, Monash University, Australia; Experimental Biology 2007, Washington DC, 28 Apr–2 May 2007; Experimental Biology 2008, San Diego, CA, 5–9 April 2008; 5th International Conference on Relaxin and Related Peptides, Maui, HI, 18–23 May 2008; Australian Health and Medical Research Congress 2008, Brisbane, Australia, 16–21 Nov 2008; and the British Pharmacological Society's 3rd Focused Meeting on Cell Signalling, Leicester, UK, 20–21 April 2009.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.061432.
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ABBREVIATIONS:
- AP-1
- activator protein 1
- BSA
- bovine serum albumin
- CHO-RXFP3
- Chinese hamster ovary-K1 cells expressing RXFP3
- DMEM
- Dulbecco's modified Eagle's medium
- ERK
- extracellular signal-regulated kinase
- FBS
- fetal bovine serum
- GFP
- green fluorescent protein
- GPCR
- G protein-coupled receptor
- GTPγS
- guanosine 5′-O-(3-thio)triphosphate
- H2 relaxin
- human gene 2 relaxin
- H3 relaxin
- human gene 3 relaxin
- HEK-RXFP3
- human embryonic kidney 293 cells expressing RXFP3
- INSL
- insulin-like peptide
- JNK
- c-Jun N-terminal kinase
- MAPK
- mitogen-activated protein kinase
- MEK
- mitogen-activated protein kinase kinase
- NF-κB
- nuclear factor κB
- PD98059
- 2′-amino-3′-methoxyflavone
- PI3K
- phosphatidylinositol 3-kinase
- PKC
- protein kinase C
- PTX
- pertussis toxin
- RT-PCR
- reverse transcription-polymerase chain reaction
- RWJ67657
- 4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol
- RXFP
- relaxin family peptide receptor
- SB203580
- 4-(4-fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl)1H-imidazole
- SEAP
- secreted alkaline protease
- SN56
- mouse septal neuron-derived cell line
- SP600125
- anthra[1,9-cd]pyrazol-6(2H)-one
- TFA
- trifluoroacetic acid.
- Received October 1, 2009.
- Accepted February 4, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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H2 Relaxin Is a Biased Ligand Relative to H3 Relaxin at the Relaxin Family Peptide Receptor 3 (RXFP3)
