The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation

Abstract

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT 1 ) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G proteincoupled receptors, the AT 1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT 1 , N200C-AT 1 , I201C-AT 1 , G203C-AT 1 , and F204C-AT 1 mutant receptors, which suggests that these residues orient themselves within the wateraccessible binding pocket of the AT 1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT 1 receptor background. Indeed, mutant I201C-N111G-AT 1 became more sensitive to MTSEA, whereas mutant G203C-N111G-AT 1 lost some sensitivity. Our results suggest that constitutive activation of AT 1 receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

Differential Roles of Cysteine Residues in the Cellular Trafficking, Dimerization, and Function of the High-Density Lipoprotein Receptor, SR-BI

“…Likewise, it was reported that treatment of COS-1 cells expressing human cannabinoid receptor (CB1) with MMTS or MTSEA leads to a dramatic reduction in agonist SR141716A binding to the receptor; such treatment, however, had no significant effect on agonist CP55940 binding to the CB1 receptor (69). Human AT 1 receptors (70) and P2X 2 (61) are shown to be insensitive to MTSEA and MTSET, respectively. On the other hand, it was reported that MTSET treatment of HEK cells transiently overexpressing glucagon receptor enhanced (instead of decreasing) glucagon potency for cAMP stimulation by two- to three-fold (71).…”

Current topics in angiotensin II type 1 receptor research: Focus on inverse agonism, receptor dimerization and biased agonism

“…Previous AT1R structure-function studies have suggested both rotational and translational motion of TM2, TM3, TM5, TM6 and TM7 in the constitutively active mutant N111G-AT1R, which mimics the active state of AT1R [59–62]. In the active state, formation of the Asn46-Asp74-Asn295 H-bond network is proposed, which involves additional interacting residues around Asn111 TM3 and Asn295 TM7 (see Section 5).…”

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.…”