FIGURE 5.

Stimulation of cAMP production in LGR7- and LGR8-expressing cells by recombinant RLN2, RLN3, and INSL3. A, recombinant RLN3 and INSL3 selectively activated LGR7 and LGR8, respectively. In contrast, RLN2 activates both receptors at the nanomolar range. Unlike the wild type RLN2, substitution of Arg^B12 and Arg^B16 residues with alanine abolished the LGR7- and LGR8-activation activities of RLN2. Each data point represents the mean ± S.E. of quadruplicate samples. B, Western blotting analysis of recombinant wild type and mutant relaxin peptides including, RLN2 C-104, C-38, C-28, C-18, C-8 (upper left), WT RLN2, WT RLN3, WT INSL3 (upper middle), WT RLN2, RLN2 R^B12A, RLN2 R^B16A (upper right), WT RLN2, RLN2 T^A16A, RLN2^A19-20, RLN2^A22-23 (lower left), WT RLN2, RLN2 K^A17A, RLN2 R^A18A, RLN2 R^A22A, RLN2 F^A23A (lower middle), WT RLN2, RLN2 K^A17A, RLN2 K^A17G, RLN2 K^A17Q, and RLN2 K^A17D(lower right). Affinity column purified peptides (100 ng/lane) were resolved by 18% SDS-PAGE under nonreducing (upper panel) and reducing (lower panel) conditions. The positions of molecular mass markers are indicated on the left.