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Research ArticleArticle

H2 Relaxin Is a Biased Ligand Relative to H3 Relaxin at the Relaxin Family Peptide Receptor 3 (RXFP3)

Emma T. van der Westhuizen, Arthur Christopoulos, Patrick M. Sexton, John D. Wade and Roger J. Summers
Molecular Pharmacology May 2010, 77 (5) 759-772; DOI: https://doi.org/10.1124/mol.109.061432
Emma T. van der Westhuizen
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Arthur Christopoulos
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Patrick M. Sexton
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John D. Wade
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Roger J. Summers
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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

  • ↵Embedded Image 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.

  • 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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Molecular Pharmacology: 77 (5)
Molecular Pharmacology
Vol. 77, Issue 5
1 May 2010
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Research ArticleArticle

H2 Relaxin Is a Biased Ligand Relative to H3 Relaxin at the Relaxin Family Peptide Receptor 3 (RXFP3)

Emma T. van der Westhuizen, Arthur Christopoulos, Patrick M. Sexton, John D. Wade and Roger J. Summers
Molecular Pharmacology May 1, 2010, 77 (5) 759-772; DOI: https://doi.org/10.1124/mol.109.061432

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Research ArticleArticle

H2 Relaxin Is a Biased Ligand Relative to H3 Relaxin at the Relaxin Family Peptide Receptor 3 (RXFP3)

Emma T. van der Westhuizen, Arthur Christopoulos, Patrick M. Sexton, John D. Wade and Roger J. Summers
Molecular Pharmacology May 1, 2010, 77 (5) 759-772; DOI: https://doi.org/10.1124/mol.109.061432
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