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An angiotensin II (AngII) peptidic analogue in which the third residue (valine) was substituted with the photoreactive p-benzoyl-L-phenylalanine (Bpa) was used to identify ligand-binding sites of the human AT(1) receptor. High-affinity binding of the analogue, (125)I-[Bpa(3)]AngII, to the AT(1) receptor heterologously expressed in COS-7 cells enabled us to efficiently photolabel the receptor. Chemical and enzymatic digestions of the (125)I-[Bpa(3)]AngII-AT(1) complex were performed, and receptor fragments were analyzed in order to define the region of the receptor with which the ligand interacts. Results show that CNBr hydrolysis of the photolabeled receptor gave a glycosylated fragment which, after PNGase-F digestion, migrated as a 11.4 kDa fragment, circumscribing the labeled domain between residues 143-243 of the AT(1) receptor. Digestion of the receptor-ligand complex with Endo Lys-C or trypsin followed by PNGase-F treatment yielded fragments of 7 and 4 kDa, defining the labeling site of (125)I-[Bpa(3)]AngII within residues 168-199 of the AT(1) receptor. Photolabeling of three mutant receptors in which selected residues adjacent to residue 168 were replaced by methionine within the 168-199 fragment (I172M, T175M, and I177M) followed by CNBr cleavage revealed that the bound photoligand (125)I-[Bpa(3)]AngII forms a covalent bond with the side chain of Met(172) of the second extracellular loop of the AT(1) receptor. These data coupled with previously obtained results enable us to propose a model whereby AngII adopts an extended beta-strand conformation when bound to the receptor and would orient itself within the binding domain by having its N-terminal portion interacting with the second extracellular loop and its C-terminus interacting with residues of the seventh transmembrane domain.

The peptide hormone angiotensin II (AngII) binds to the AT 1 (angiotensin type 1) receptor within the transmembrane domains in an extended conformation, and its C-terminal residue interacts with transmembrane domain VII at Phe-293/Asn-294. The molecular environment of this binding pocket remains to be elucidated. The preferential binding of benzophenone photolabels to methionine residues in the target structure has enabled us to design an experimental approach called the methionine proximity assay, which is based on systematic mutagenesis and photolabeling to determine the molecular environment of this binding pocket. The octapeptide hormone angiotensin II (AngII) 1 (Fig. 1A) is the active component of the renin-angiotensin system. Virtually all known physiological effects of AngII are produced through the activation of the hAT 1 receptor, which belongs to the class A rhodopsin-like family of the heptahelical G proteincoupled receptor (GPCR) superfamily (1, 2). Elucidating the stereochemistry of the ligand-receptor interaction is vital for understanding the mechanism of ligand binding, GPCR activation, and, eventually, rational drug design.In the past, much effort was devoted to identifying the domains or individual residues of a given receptor that may interact with its ligand. Most experiments to address ligandreceptor interactions were performed with series of receptor mutants to identify specific residues critical to ligand binding (3-5). It is, however, speculative to deduce precise structures of ligand-receptor interactions through mutagenesis studies alone. More direct approaches have therefore been used to study ligand-receptor interactions. Among these is photoaffinity labeling, which allows covalent incorporation of the ligand within its binding site, presumably at the contact area of the photolabel in the receptor. This ligand-receptor contact can be identified by specific enzymatic or chemical digestion of the labeled receptor (6) or by mass spectrometry (7). The binding pockets within the transmembrane domains of several bioamine receptors have been identified using this kind of approach. The adenosine A 1 receptor (8) and the ␤ 2 adrenergic receptor (9, 10) are typical examples. Peptidergic receptors such as hAT 1 and hAT 2 (11, 12), neurokinin receptors (13), and several other receptors from the secretin GPCR family B (14) have been also studied using this approach. We previously identified ligandcontact points within the second extracellular loop (ECL) and the seventh transmembrane domain (TMD) of the hAT 1 receptor (12,15,16). Although photoaffinity labeling has been widely used to study peptidergic GPCR binding pockets, generally only a single contact point between a given ligand and its cognate receptor has been identified. The resulting information does not, however, induce sufficient restrictions to generate credible GPCR structures in the ligand-bound state using homology modeling.Labeling studies using benzophenone residues have identified many ligand-receptor contact points with a surpris...

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