Characteristics of the binding of 32P-labelled human relaxins to the human fetal membranes

The two human relaxin genes termed H1 and H2 are expressed in the choriodecidua and placenta and have been proposed to act via specific receptors as local modulators of collagenolysis in the fetal membranes. Such receptors have been inferred, but not demonstrated, from studies of the effect of adding exogenous relaxin to these tissues. Thus conditions were optimized for the binding of 32 P-labelled human relaxin H2 to membrane-enriched particulate fractions of human fetal membranes, amnion and chorion, with adhering decidua. The membrane protein concentration was optimal at 250 μg, when incubated at 27 °C for 60 min, at pH 7.5 with Mn 2+ and Mg 2+ ion concentrations of 2.0 mM. Incubation of membrane particulate fractions with increasing amounts of labelled relaxin H2 suggested the presence of a single class of binding sites with an affinity constant (K a ) of 2.15 nM. The binding was primarily to the chorion and decidua with very little to the amnion layer. The competition for binding of the 32 P-labelled human relaxin H2 with unlabelled relaxin H2 gave an IC 50 of 28 pM, while an IC 50 of 60 pM and 280 pM was obtained for relaxin H1 and porcine relaxin respectively. In contrast, unlabelled guinea-pig relaxin inhibited this binding by only 10% even at a 1000-fold greater concentration than H2, and human recombinant insulin failed to inhibit even at a million-fold concentration of unlabelled relaxin H2. Relaxins H2 and H1 can readily displace the binding of either 32 P-labelled human relaxins H1 or H2 and gave very similar displacement curves. The binding affinity of relaxin H2, however, was fivefold higher than that of relaxin H1. These data provide evidence for the presence of a relaxin receptor that may preferentially bind relaxin H2.