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

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT₁) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT₁ 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-AT₁, N200C-AT₁, I201C-AT₁, G203C-AT₁, and F204C-AT₁ mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT₁ receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT₁ receptor background. Indeed, mutant I201C-N111G-AT₁ became more sensitive to MTSEA, whereas mutant G203C-N111G-AT₁ lost some sensitivity. Our results suggest that constitutive activation of AT₁ receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.