Photolabeling with N-Terminal Urotensin II Photoprobes Identifies Methionine 288 of Rat Urotensin Receptor as a Contact Point

摘要

Urotensin II (U-II) is a cyclic undecapeptide which has recently been shown to be the endogenous ligand of a G Protein Coupled Receptor (GPCR) termed UT. U-II has potent vasoactivity in the peripheral vasculature and it may be implicated in the pathogenesis of cardiovascular diseases such as essential hypertension and heart failure. Our laboratory is interested in defining the spatial orientation of biological ligands within the binding pocket of peptidergic GPCRs. By introducing spatial constraints between distant residues of the receptor and homology modelling, the number of possible structures is narrowed down to a very few. Spatial constraints can be experimentally determined by finding the points of contact between a peptide ligand and its receptor using multiple photoaffinity labeling e.g., with photoprobe radioligands incorporating para-benzoyl-L-phenylanaline (Bpa) at different positions within the peptide sequence. Using this approach, we have shown that the sixth residue of U-II is in close proximity to two adjacent Met residues within the fourth transmembrane domain of the rat UT (rUT). In order to determine ligand orientation and better define interactions within the binding pocket of UT, further contact points are needed. We have introduced Bpa into the N-terminal portion of U-II since this portion of the ligand is highly heterogeneous across species while still maintaining full biological properties.