A urotensin II (U-II) peptide analogue containing the photoreactive p-benzoyl-l-phenylalanine (Bz-Phe) in the sixth position was used to identify ligand-binding sites of the rat U-II receptor, also known as GPR14. [Bz-Phe6]U-II bound the receptor expressed in COS-7 cells with high affinity (IC50 0.7nM) and was as potent as U-II in the agonist-induced production of inositol phosphate. Photolabelling of the U-II receptor with 125I-[Bz-Phe6]U-II resulted in the specific formation of a glycosylated 125I-[Bz-Phe6]U-II—U-II receptor complex of 60kDa. Digestion of the 60kDa complex with endoproteinase Glu-C generated a fragment of 17kDa circumscribing the labelled fragment to residues 148—286. Digestion of the ligand—receptor complex with endoproteinase Arg-C produced a short peptide of 4kDa corresponding to fragments 125—148, 167—192 or 210—233. CNBr treatment of the endoproteinase-Glu-C and -Arg-C fragments yielded 2kDa fragments, defining the labelling site to methionine residues 184/185 of the fourth transmembrane domain. Photolabelling of two mutant receptors, M184L/M185L and M184A/M185A, led to a significant decrease in the overall yield of covalent labelling. Taken together, our results indicate that position 6 of U-II normally occupied by phenylalanine would interact with Met184 and/or Met185 of the fourth transmembrane domain of the U-II receptor. This information should be of significant value in the study of the interactions between U-II and its cognate receptor.
Abbreviations used: AngII, angiotensin II; Bz-Phe, p-benzoyl-l-phenylalanine; DMEM, Dulbecco's modified Eagle's medium; ECL, extracellular loop; Endo Arg-C, endoproteinase Arg-C; ET-1, endothelin-1; GPCR, G-protein-coupled receptor; M184L (etc.), Met184→Leu mutant (etc.); PNGase-F, peptide N-glycosidase F; U-II, urotensin II.