Negative cooperativity in H2 relaxin binding to a dimeric relaxin family peptide receptor 1

Abstract

H2 relaxin, a member of the insulin superfamily, binds to the G-protein-coupled receptor RXFP1 (relaxin family peptide 1), a receptor that belongs to the leucine-rich repeat (LRR)-containing subgroup (LGRs) of class A GPCRs. We recently demonstrated negative cooperativity in INSL3 binding to RXFP2 and showed that this subgroup of GPCRs functions as constitutive dimers. In this work, we investigated whether the binding of H2 relaxin to RXFP1 also shows negative cooperativity, and whether this receptor functions as a dimer using BRET². Both binding and dissociation were temperature dependent, and the pH optimum for binding was pH 7.0. Our results showed that RXFP1 is a constitutive dimer with negative cooperativity in ligand binding, that dimerization occurs through the 7TM domain, and that the ectodomain has a stabilizing effect on this interaction. Dimerization and negative cooperativity appear to be general properties of LGRs involved in reproduction as well as other GPCRs.

Introduction

Relaxin was first isolated in 1926 by Frederik Hisaw (reviewed in Ziel and Sawin, 2000). After a period of neglect, recent research has given many new insights about the hormones of this peptide family, their receptors and the importance of relaxin family peptide receptors as new drug targets (reviewed in Bathgate et al., 2005b, Ivell et al., 2005, Ivell and Bathgate, 2002, Dschietzig et al., 2006, Van Der Westhuizen et al., 2007).

Relaxin belongs to the insulin/relaxin superfamily of peptides which in humans comprises insulin, IGF-I and IGF-II, relaxins (H1, H2 and H3), and INSL3–INSL6. H2 relaxin (which we will term relaxin from now on) was traditionally associated with pregnancy. It is synthesized in the corpora lutea of ovaries during pregnancy. Its physiological role appears to be species-specific (see Sherwood, 2004 and Dschietzig et al., 2006, for recent reviews). In nonhuman mammals, relaxin is highest in the days before birth (Hudson et al., 1983). Its major biological effect is to remodel the mammalian reproductive tract to facilitate the birth process (loosening of the pubic symphysis and relaxation of the cervix) (Ivell, 2002, Porter, 1972). Besides, relaxin also promotes the development of the mammary organs, thus enabling normal lactational performance. During pregnancy, relaxin inhibits uterine contractility and promotes the osmoregulatory changes of pregnancy in rats. In males, relaxin has been shown to be expressed in almost all parts of the male reproductive tract, with high levels in testis and vas deferens (Filonzi et al., 2007). In humans, relaxin is at its highest in the first trimester of pregnancy. Its involvement in decidualization and in preterm premature rupture of the fetal membranes has been extensively studied by Bryant-Greenwood's group (see Bryant-Greenwood et al., 2005, for recent review). Relaxin has also a number of nonreproductive actions (see Dschietzig et al., 2006, for recent review).

Relaxin-3 is not involved in reproduction but is believed to be a putative neuropeptide involved in appetite regulation (McGowan et al., 2005). Relaxin-3 is most likely the ancestor to the entire relaxin peptide family (Wilkinson et al., 2005). INSL3, expressed in the testis and ovary, and its receptor RXFP2 were shown to initiate oocyte maturation and to suppress male germ cell apoptosis. The administration of an RXFP2 antagonist resulted in an increased germ cell apoptosis, suggesting that INSL3 antagonists may have potential as novel contraceptive agents (Del Borgo et al., 2006). It has been proposed that INSL3 plays an important role in spermatogenesis as well as differentiation and maintenance of the male phenotype (reviewed in Ivell and Bathgate, 2002). In females, INSL3 has a role in the regulation of the oestrus cycle and possibly in follicular development, explaining the impaired fertility in INSL3 knockout mice (Nef and Parada, 1999). The functions of H1 relaxin and INSL4 to INSL6 remain unknown (Wilkinson et al., 2005).

Relaxin binds and activates RXFP1, which is assumed to be the cognate receptor of relaxin. It is also able to bind with lower affinity to RXFP2, although the significance of this binding in vivo is unknown (Svendsen et al., 2008). RXFP1 and RXFP2 belong to Type C LGRs (leucine-rich repeat-containing G-protein-coupled receptor) of the class A subtype of GPCRs (rhodopsin-like family). They are unique because they have N-terminal domains which have homology to the LDLa modules that constitute the ligand binding repeats found in the LDL receptor family (Scott et al., 2006). The ectodomain of RXFP1 and RXFP2 functions as the primary ligand-binding domain. Ligand binding leads to the activation of adenyl cyclase and the protein kinase A-dependent pathway in many target tissues (Bathgate et al., 2005a, Hsu et al., 2002).

There is increasing evidence that GPCRs are allosteric proteins (Springael et al., 2007). Allosterism is a property displayed by many oligomeric proteins. The binding of a molecule at one site induces a change in the binding properties of another site of the protein. In the case of negative cooperativity, the receptor sites do not have a fixed affinity, rather, the affinity of the receptors decreases as a function of the occupancy of the receptor population and is usually measured by ligand-accelerated tracer dissociation in an infinite dilution procedure (De Meyts et al., 1973). Negative cooperativity is a mechanism that increases the range of the effective concentrations of the ligands.

After negative cooperativity in ligand binding was demonstrated for the insulin receptor in the early seventies (De Meyts et al., 1973), it was also demonstrated for the β₂-adrenergic (Limbird et al., 1975) and TSH receptors (De Meyts, 1976), later found to be GPCRs. The homodimerization of the β₂-adrenergic receptor and the TSH receptor was subsequently established (Graves et al., 1996, Angers et al., 2000, Latif et al., 2001, Rapoport and McLachlan, 2007), and the link between homo- and heterodimerization and the existence of negative cooperativity in LGR receptors including the TSH receptor was recently demonstrated, confirming the earlier TSH receptor observations (Urizar et al., 2005). Similar findings with chemokine receptors suggest that dimerization and negative cooperativity may be the rule rather than the exception among GPCRs (Springael et al., 2005). The dimerization of receptors has been shown to have many physiological roles such as receptor maturation, regulation of ligand binding, G-protein selectivity, or internalization (reviewed in Terrillon and Bouvier, 2004), and therefore it is of importance to investigate this mechanism in any particular receptor/ligand system. The relevance of dimerization and allosterism for the physiological properties of GPCRs, and implications for drug design, has been recently reviewed (see Springael et al., 2007). However, no data were available on the RXFP group of receptors involved in human reproduction until we recently showed that INSL3 binding to RXFP2 shows negative cooperativity, and that RXFP2 forms homodimers, as well as heterodimers with RXFP1 (Svendsen et al., 2008).

In this study we investigated whether negative cooperativity and dimerization are general properties of the LGR class of GPCRs by investigating whether H2 relaxin binding to RXFP1 displays properties similar to those of INSL3 binding to RXFP2, and whether RXFP1 forms homodimers. Furthermore we have investigated in detail the binding kinetics of H2 relaxin binding to RXFP1. Earlier studies focused solely on the binding affinity while here we have also investigated in detail kinetic properties such as association/dissociation time, temperature-/pH dependence of association and dissociation, and dose–response curves for negative cooperativity.