Analysis of Transmembrane Domains 1 and 4 of the Human Angiotensin II AT1 Receptor by Cysteine-scanning Mutagenesis

Abstract

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT 1 receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT 1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT 1 receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28( The octapeptide hormone angiotensin II (AngII) 4 is the active component of the renin-angiotensin system. It exerts a wide variety of physiological effects, including vascular contraction, aldosterone secretion, neuronal activation, and cardiovascular cell growth and proliferation (1, 2). Virtually all the known physiological effects of AngII are produced through the activation of the AT 1 receptor, which belongs to the G proteincoupled receptor (GPCR) superfamily (3, 4). The AT 1 receptor belongs to the rhodopsin-like family A of G protein-coupled receptors, which have a seven-transmembrane helix structure, an extracellular N-terminal tail, an intracellular C-terminal domain, and three extracellular and three intracellular loops.The seven transmembrane domains (TMDs) of GPCRs constitute structural support for signal transduction. Like other family A GPCRs such as rhodopsin and adrenergic receptors, the AT 1 receptor undergoes spontaneous isomerization between its inactive state and its active state (5). Movement of TMD helices through translational or rotational displacement is believed to be essential to achieve the active state (6 -8). These conformational changes would sustain GTP/GDP exchange on specific guanine nucleotide-binding proteins (G proteins) leading to activation of intracellular signaling cascades (5). For the AT 1 receptor, it has been proposed that TMD3, TMD5, TMD6, and TMD7 may participate in the activation process by providing a network of interactions throughout the AngII-binding pocket (9). The dynamics of this network would require that, following agonist binding, novel or existing interactions between the TMDs would either be created or broken, respectively.Based on homology with the recent high resolution structures of the ␤1 adrenergic, ␤2 adrenergic, and A 2A adenosine receptors (10 -12) it was expected that the binding site of the AT 1 receptor would be formed between its seven, mostly hydrophobic transmembrane domains and would be accessible to charged water-soluble agonists, like AngII. For this receptor, the binding site would thus be contained within a hydrophilic crevice, the binding pocket, extending from the extracellular surface of the receptor to the transmembrane portions. Because all crystallized structures to date suggest that TMD1 and TMD4 are somewhat removed from the binding pocket (13) The substituted-cysteine accessibility method (SCAM) (20 -22) is an ingenious ap...

Structure of the Human Angiotensin II Type 1 (AT1) Receptor Bound to Angiotensin II from Multiple Chemoselective Photoprobe Contacts Reveals a Unique Peptide Binding Mode

“…Other contact residues derived from this structure were also of particular interest. The residue Arg 167 , which was previously proposed to be important for AngII binding (52), was found to interact with the AngII Tyr 4 in a manner that is not influenced by the distance restraints imposed by the MPA results (Fig. 6g).…”

International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli

“…Activation of AT 1 receptor is shown to be associated with translation and rotation of TM helices, including TMII, TMIII, TMV, TMVI, and TMVII by two independent methods, reporter cysteine accessibility mapping and methionine proximity analysis Boucard et al, 2003;Miura et al, 2003b;Martin et al, 2004Martin et al, , 2007Domazet et al, 2009a,b;Arsenault et al, 2010a (Yan et al, 2010). Only subtle structural changes were identified between the AT 1 receptor and its constitutively active form (Clement et al, 2006).…”

Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

“…The basal activity of N111G-AT1R can be inhibited by AT1R-selective blockers, albeit at 3-315-fold higher concentrations compared with wild-type AT1R (16,19,20,22,23). The conformation of the TMD of the N111G-AT1R has been shown to be altered to the activated state (12,13,(32)(33)(34)(35)(36). The RCAM results in Fig.…”