Determining the Environment of the Ligand Binding Pocket of the Human Angiotensin II Type I (hAT1) Receptor Using the Methionine Proximity Assay

Abstract

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...

Mapping Peptide Hormone−Receptor Interactions Using a Disulfide-Trapping Approach

“…Importantly, although 24 positions were probed, only 4 cross-linked. This contrasts to the observation of many cross-linking sites detected due to the "methionine magnet effect" (14)(15)(16)(17)(18)(19). The novel sites of interaction identified by this study generated a molecular model that exhibits important differences from our previous model based on Bpa photoaffinity cross-linking data.…”

Methionine acts as a “magnet” in photoaffinity crosslinking experiments

“…This observation was recently used by Escher and coworkers in the design of a ''Methionine Proximity Assay'' (MPA) [20]. Scanning through three transmembrane domains of the angiotensin receptor, they identified the binding groove for Bpa in position 8 of angiotensin [20]. Our study demonstrates that the preferential reactivity of Bpa for methionine over other amino acids in a target domain can be more pronounced than previously appreciated.…”

“…The characteristic low MW band corresponding to free ligand makes detection straightforward. This observation was recently used by Escher and coworkers in the design of a ''Methionine Proximity Assay'' (MPA) [20]. Scanning through three transmembrane domains of the angiotensin receptor, they identified the binding groove for Bpa in position 8 of angiotensin [20].…”

Ligand binding properties of human galanin receptors

“…In docking to models without disul fide bond, all ligands under study were docked inside a binding pocket formed in between in the core of the receptor; large peptide ligands interact at the same time with the TM helices and extracellular loops. This characteristic binding is also postulated for other neuropeptides including NPY (Sautel et al 1995, Sjödin et al 2006, angiotensin (Clément et al 2005), and somatostatin (Strnad and Hadcock 1995). However, docking to models with this disul fide bond present yielded no good binding poses for any ligands, indicating that the reduced form must exist for ligand binding.…”