Cholesterol modulates the binding properties of human relaxin family peptide receptor 3 with its ligands

Relaxin family peptide receptor 3 (RXFP3) is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. As an A-class G protein-coupled receptor, RXFP3 is an integral plasma membrane protein with seven transmembrane domains, yet influence of the membrane lipids on its function remains unknown. In the present study, we disclosed that cholesterol, an essential membrane lipid for mammalian cells, modulated the binding properties of human RXFP3 with its ligands. We first demonstrated that depletion of cholesterol from host human embryonic kidney (HEK) 293T cells by methyl-Beta-cyclodextrin altered ligand-binding properties of the overexpressed human RXFP3, such as increasing its binding potency with some antagonists and decreasing its binding affinity with a NanoLuc-conjugated R3/I5 tracer. Thereafter, we demonstrated that two B-chain residues, B5Tyr and B12Arg, were primarily responsible for the increased binding potency of these antagonists with human RXFP3 under the cholesterol depletion condition. Our results suggest that cell membrane cholesterol interacts with human RXFP3 and modulates its ligand-binding properties, providing new insights into the influence of membrane lipids on RXFP3 function.