Structural requirements for the interaction of sheep insulin-like factor 3 with relaxin receptors in rat atria

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Abstract

Relaxin is a peptide with various reproductive and nonreproductive functions. The site for the peptide–receptor interaction contains two arginines (Arg) and an isoleucine (Ile) or valine (Val) residue in the B-chain with a configuration of -Arg-X-X-X-Arg-X-X-Ile/Val-X-. The sheep insulin-like peptide 3 (INSL3), a structural homologue of relaxin, also contains the n, n+4 arginines in the B-chain but they are displaced towards the carboxyl terminus by four residues (-X-X-X-X-Arg-X-X-Val-Arg-). Human INSL3 increases the activity of human relaxin in mouse bioassays. Here, we investigated whether sheep synthetic INSL3 affects the relaxin activity in rat atria. INSL3 lacked relaxin-like agonist activity but blocked the activity of relaxin and competed for relaxin binding sites at high concentrations. We also synthesized analogues of INSL3, with amino acid substitutions in the arginine-binding region. Analogues A, D and E, which have the arginines in positions identical to relaxin, showed weak relaxin-like agonist activity. These results suggest that other sites in the relaxin molecule are involved in high-affinity peptide–receptor interaction for the production of the relaxin biological responses.

Introduction

Relaxin is a structural homologue of insulin Bedarker et al., 1977, Isaacs et al., 1978, Eigenbrot et al., 1991 but it does not cross-react with insulin Osheroff et al., 1990, Tan et al., 1998. It is produced principally by the ovary during pregnancy, and acts via its own receptors to soften the cervix and the interpubic ligaments to facilitate birth (reviewed by Sherwood, 1994). It also has positive chronotropic and inotropic effects in the rat heart Kakouris et al., 1992, Ward et al., 1992, Tan et al., 1998, and acts on relaxin receptors Osheroff and Phillips, 1991, McKinley et al., 1997 located in the circumventricular organs to modulate blood pressure Mumford et al., 1989, Parry et al., 1990 and plasma osmolality (Weisinger et al., 1993), as well as receptors in the neurosecretory magnocellular hypothalamic nuclei to release the neuropeptides oxytocin and vasopressin Summerlee et al., 1984, Dayanithi et al., 1987, Way and Leng, 1992.

Although there is relatively little amino acid sequence homology between relaxins from various species (Sherwood, 1994), the primary active site consists of two arginine residues at positions n and n+4 and an isoleucine or valine residue within the B-chain α-helix in a configuration of -Arg-X-X-X-Arg-X-X-Ile/Val-X- (for example B13, B17 and B20 in human gene 2 (H2) (B29) relaxin) (Fig. 1). These residues are conserved in almost all relaxins, and studies have shown that substitution of these residues causes a reduction in or an abolition of the peptide activities Büllesbach and Schwabe, 2000, Büllesbach et al., 1992. The X-ray crystal structure of relaxin shows that the arginine residues are positioned in the first and second loops of the α-helix with their side chains project away from the core molecule into the surrounding water (Büllesbach et al., 1992) (Fig. 2). It has been suggested that these arginines, together with the side chain of isoleucine/valine, form a triangular contact with the receptor-binding sites (Büllesbach and Schwabe, 2000). The true dynamic of the peptide–receptor interaction is yet to be established as the relaxin receptors have only recently been identified as members of the orphan leucine-rich repeat-containing G protein-coupled receptor (LGR) family, LGR7 and LGR8 (Hsu et al., 2002).

A relaxin-like gene sequence has been found in the sheep genome but it is incapable of producing RNA that can be translated to a functional peptide (Roche et al., 1993). Nevertheless, a peptide structurally similar to relaxin and insulin is expressed in the sheep testicular Leydig cells and in female reproductive tissues (Roche et al., 1996). This peptide, described as Leydig cell insulin-like peptide (Ley I-L), relaxin-like factor (RLF) or insulin-like factor 3 (INSL3), is also expressed in cows (Bathgate et al., 1996) and other mammals including humans Burkhardt et al., 1994, Tashima et al., 1995, Ivell et al., 1997, pigs (Adham et al., 1993) and rodents Pusch et al., 1996, Zimmermann et al., 1997, Balvers et al., 1998, Spiess et al., 1999. The structural configuration of native INSL3 is not known as it has yet to be isolated from a biological source.

Like relaxin, INSL3 also has two arginine residues in the B-chain α-helix with the n, n+4 configuration, but they are offset towards the carboxyl terminus by precisely four residues (Roche et al., 1996). By means of circular dichroism spectroscopy, we showed that sheep synthetic INSL3 is structurally similar to relaxin (Dawson et al., 1999), suggesting that the side chain of the arginine residues in INSL3 would project out from the second and the third loops of the B-chain α-helix instead of the first and the second loops (Fig. 2). Sheep INSL3 also has a valine residue in the position corresponding to the B20 of H2 (B29) relaxin. The shift of the arginine residues, therefore, produced a different configuration (-X-X-X-X-Arg-X-X-Val-Arg-) compared to relaxin (Fig. 1). Theoretically, this configuration would interrupt the interaction of INSL3 with relaxin receptor but a study has shown that human synthetic INSL3 lacks relaxin-like bioactivity but it enhances the activity of H2 relaxin in elongating mouse pubic symphysis and competes with relaxin for binding sites in mouse brain and uterine homogenates at high concentrations (Büllesbach and Schwabe, 1995).

In this study, we used an in vitro rat atrial bioassay (Tan et al., 1998) and quantitative receptor autoradiography with [33P]-labelled H2 (B33) relaxin (Tan et al., 1999) to investigate the interaction of sheep synthetic INSL3 with rat atrial relaxin receptors. Using the sheep INSL3 as a template, we then synthesized analogues with amino acid substitutions in the B-chain α-helix (Fig. 1) and tested their biological and binding activities to determine regions of the peptide molecule that are important for interaction with the relaxin receptor. We found that although the correct configuration and position of the n and n+4 arginines in the B-chain is essential for relaxin-like agonist activity, other region(s) must also be involved in determining the affinity of the relaxin peptide–receptor interactions.

Section snippets

Materials

Recombinant human gene 2 relaxins (B33 and B29) (Genentech and Connetics, San Francisco, USA), cyclic AMP-dependent protein kinase (Promega), [γ-33P]ATP (DuPont), HEPES (BDH), phenylmethylsulphonylfluoride (Sigma), other salts and organic solvents used were of analytical quality.

Peptide syntheses and characterization

The procedures for the synthesis of sheep INSL3 and analogues—the synthesis of A- and B-chains separately followed by chain combination—have been published elsewhere (Dawson et al., 1999). Overall, seven analogues of

Effects of sheep INSL3 and analogues on the rat atria and the chronotropic and inotropic responses produced by relaxin

Rat isolated atria were incubated in sheep INSL3 or the peptide analogues (all at 1 μM) for approximately 20 min, followed by H2 (B29) relaxin (cumulative 1 and 10 nM or 10 nM only), and (−)-isoprenaline (0.1 μM) to determine the maximum response of each preparation. INSL3 failed to increase the beating rate (chronotropic) of the isolated right atrial preparations or the force of contraction (inotropic) of the isolated left atrial preparations. The addition of H2 (B29) relaxin evoked changes in

Discussion

The rat atrial bioassay and quantitative receptor autoradiography have been used to study potential relaxin-like activity of sheep INSL3 and analogues. Although INSL3 lacked relaxin-like activity in rat isolated atria, it appeared to reduce the atrial responses to H2 (B29) relaxin. In the receptor autoradiographic studies, the results suggest that INSL3 binds to relaxin receptors at high concentrations (>1 μM) and may act as an antagonist. This conclusion differs with earlier studies

Acknowledgements

This work was supported by an Institute Block Grant to the Howard Florey Institute (#983001) from the National Health and Medical Research Council of Australia and by a grant from the National Heart Foundation of Australia.

References (51)

  • D.G. Ward et al.

    Relaxin increases rat heart rate by a direct action on the cardiac atrium

    Biochem. Biophys. Res. Commun.

    (1992)
  • M. Balvers et al.

    Relaxin-like factor expression as a marker of differentiation in the mouse testis and ovary

    Endocrinology

    (1998)
  • R. Bathgate et al.

    Relaxin-like factor gene is highly expressed in the bovine ovary of the cycle and pregnancy: sequence and messenger ribonucleic acid analysis

    Biol. Reprod.

    (1996)
  • S. Bedarker et al.

    Relaxin has conformational homology with insulin

    Nature

    (1977)
  • F.R. Boockfor et al.

    Relaxin-like factor (RLF) serum concentrations and gubernaculum RLF receptor display in relation to pre- and neonatal development of rats

    Reproduction

    (2001)
  • E.E. Büllesbach et al.

    Tryptophan B27 in the relaxin-like factor (RLF) is crucial for RLF receptor-binding

    Biochemistry

    (1999)
  • E.E. Büllesbach et al.

    The relaxin-like factor is a hormone

    Endocrine

    (1999)
  • E. Burkhardt et al.

    A human cDNA coding for the Leydig insulin-like peptide (Ley I-L)

    Hum. Genet.

    (1994)
  • Y.C. Cheng et al.

    Relationship between the inhibition constant Ki and the concentration of inhibitor which caused 50% inhibition (IC50) of an enzyme reaction

    Biochem. Pharmacol.

    (1973)
  • A.A. Claasz et al.

    Chemical synthesis and relaxin activity of analogues of ovine Insulin 3 containing specific B-chain residue replacements

  • N.F. Dawson et al.

    Solid phase synthesis of ovine Leydig cell insulin-like peptide—a putative ovine relaxin?

    J. Pept. Res.

    (1999)
  • G. Dayanithi et al.

    Relaxin affects the release of oxytocin and vasopressin from the neurohypophysis

    Nature

    (1987)
  • E. Gille et al.

    The affinity of (−)-propranolol for β1 and β2-adrenoceptors of human heart

    Naunyn-Schmiedeberg's Arch. Pharmacol.

    (1985)
  • I.P. Gorlov et al.

    Mutations of the GREAT gene cause cryptorchidism

    Hum. Mol. Genet.

    (2002)
  • S.Y. Hsu et al.

    Activation of orphan receptors by the hormone relaxin

    Science

    (2002)
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