title: Synthesis of an agonistic, difluoro-azido photolabel of angiotensin II and labeling of the AT1 receptor: transmembrane domains 3, 6, and 7 form the ligand-binding pocket.
abstract: p-Azido-phenylalanine has been frequently used for photoaffinity labeling of target proteins such as the angiotensin receptors. However, chemical studies showed that simple aryl nitrenes first react intramolecularly, forming a semistable cyclic keteneimine and then reacting with nucleophile residues in the target structure like those of lysine and arginine. We synthesized 3,5-difluoro-4-azidophenylalanine where the formation of the keteneimine is prevented and where photoincorporation should be due to nonselective nitrene insertion only. This new amino acid was introduced in position 8 of angiotensin II and compared with the corresponding azidophenylalanine peptide using human AT1 receptor as target. The new photolabel maintained full agonist activity and a similar yield of photolabeling but without the previously observed gradual hydrolysis. Several selective proteolyses of the labeled receptor indicate that the new photolabel forms three simultaneous contact regions on the hAT1 receptor, suggestive of a nonselective behavior of the photolabel. A major contact was established in the sixth transmembrane domain but also in the third and seventh domain. Our results are in excellent agreement with those recently obtained from methionine proximity assay studies.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada.
authors.forename: Tommy
authors.initials: T
authors.lastname: Gagnon
authors.forename: Dany
authors.initials: D
authors.lastname: Fillion
authors.forename: Marie-Reine
authors.initials: MR
authors.lastname: Lefebvre
authors.forename: Emanuel
authors.initials: E
authors.lastname: Escher
mesh_terms: D000804:Angiotensin II / Q000819:agonists* / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002417:Cattle; D006801:Humans; D006868:Hydrolysis; D007097:Imines; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives*; D019920:Photoaffinity Labels; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry* / Q000378:metabolism
chemical_list: C517650:3,5-difluoro-4-azidophenylalanine; D001386:Azides; D007097:Imines; D008024:Ligands; D008565:Membrane Proteins; D019920:Photoaffinity Labels; D044140:Receptor, Angiotensin, Type 1; D000804:Angiotensin II; C017621:phenylnitrene; C027162:4-azidophenylalanine; D010649:Phenylalanine