Solution structure of urotensin-II receptor extracellular loop III and characterization of its interaction with urotensin-II

Urotensin-II (U-II) is a vasoactive hormone that acts through a G-protein-coupled receptor named UT. Recently, we have shown, using the surface plasmon resonance technology that human U-II (hU-II) interacts with the hUT(281–300) fragment, a segment containing the extracellular loop III (EC-III) and short extensions of the transmembrane domains VI and VII (TM-VI and TM-VII). To further investigate the interaction of UT receptor with U-II, we have determined the solution structure of hUT(281–300) by high-resolution NMR and molecular modeling and we have examined, also using NMR, the binding with hU-II at residue level. In the presence of dodecylphosphocholine micelles, hUT(281–300) exhibited a type III β-turn (Q285–L288), followed by an α-helical structure (A289–L299), the latter including a stretch of transmembrane helix VII. Upon addition of hU-II, significant chemical shift perturbations were observed for residues located just on the N-terminal side of the β-turn (end of TM-VI/beginning of EC-III) and on one face of the α-helix (end of EC-III/beginning of TM-VII). These data, in conjunction with intermolecular NOEs, suggest that the initiation site of EC-III, as well as the upstream portion of helix VII, would be involved in agonist binding and allow to propose points of interaction in the ligand–receptor complex.