Identification of important residues of insulin-like peptide 5 and its receptor RXFP4 for ligand–receptor interactions

Insulin-like peptide 5 (INSL5) is an insulin/relaxin superfamily peptide involved in the regulation of glucose homeostasis by activating its receptor RXFP4, which can also be activated by relaxin-3 in vitro. To determine the interaction mechanism of INSL5 with its receptor RXFP4, we studied their electrostatic interactions using a charge-exchange mutagenesis approach. First, we identified three negatively charged extracellular residues (Glu100, Asp104 and Glu182) in human RXFP4 that were important for receptor activation by wild-type INSL5. Second, we demonstrated that two positively charged B-chain Arg residues (B13Arg and B23Arg) in human INSL5 were involved in receptor binding and activation. Third, we proposed probable electrostatic interactions between INSL5 and RXFP4: the B-chain central B13Arg of INSL5 interacts with both Asp104 and Glu182 of RXFP4, meanwhile the B-chain C-terminal B23Arg of INSL5 interacts with both Glu100 and Asp104 of RXFP4. The present electrostatic interactions between INSL5 and RXFP4 were similar to our previously identified interactions between relaxin-3 and RXFP4, but had subtle differences that might be caused by the different B-chain C-terminal conformations of relaxin-3 and INSL5 because a dipeptide exchange at the B-chain C-terminus significantly decreased the activity of INSL5 and relaxin-3 to receptor RXFP4.