title: Methionine proximity assay, a novel method for exploring peptide ligand-receptor interaction.
abstract: Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,p'-nitrobenzoylphenylalanine (NO2Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT1 and AT2, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, 3001 12th Ave. N, Sherbrooke, QC JIH 5N4, Canada.
authors.forename: Lenka
authors.initials: L
authors.lastname: Rihakova
authors.forename: Maud
authors.initials: M
authors.lastname: Deraët
authors.forename: Mannix
authors.initials: M
authors.lastname: Auger-Messier
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Pérodin
authors.forename: Antony A
authors.initials: AA
authors.lastname: Boucard
authors.forename: Gaétan
authors.initials: G
authors.lastname: Guillemette
authors.forename: Richard
authors.initials: R
authors.lastname: Leduc
authors.forename: Pierre
authors.initials: P
authors.lastname: Lavigne
authors.forename: Emanuel
authors.initials: E
authors.lastname: Escher
mesh_terms: D000804:Angiotensin II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003488:Cyanogen Bromide / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008715:Methionine / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D019920:Photoaffinity Labels; D011485:Protein Binding; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D010649:Phenylalanine; C053764:benzoylphenylalanine; D008715:Methionine; D019204:GTP-Binding Proteins; D003488:Cyanogen Bromide