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Activation of the Angiotensin II Type 1 Receptor Leads to Movement of the Sixth Transmembrane Domain: Analysis by the Substituted Cysteine Accessibility Method

Department of Pharmacology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada

The role of transmembrane domain six (TMD6) of the angiotensin II type 1 receptor, which is predicted to undergo conformational changes after agonist binding, was investigated using the substituted-cysteine accessibility method. Each residue in the Lys240-Leu265 fragment was mutated, one at a time, to a cysteine. The resulting mutants were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). This treatment led to a significant reduction in binding of ¹²⁵I-[Sar¹,Ile⁸]AngII to the F249C, H256C, T260C, and V264C mutant receptors, suggesting that these residues orient themselves within the water-accessible binding pocket of the AT₁ receptor. It is noteworthy that this pattern of acquired MTSEA sensitivity was altered for TMD6 cysteines engineered in a constitutively active AT₁ receptor. Indeed, mutant F249C was insensitive to MTSEA treatment, whereas the sensitivity of mutant V264C decreased. Under these conditions, one other mutant, F261C, was found to be sensitive to MTSEA treatment. Our results suggest that constitutive activation of the AT₁ receptor causes TMD6 to pivot. This movement moves the top (extracellular side) of TMD6 toward the binding pocket and simultaneously distances the bottom (intracellular side) away from the binding pocket. Using this approach, we identified key elements within TMD6 that contribute to the activation of class A GPCRs through structural rearrangements.

Address correspondence to: Richard Leduc, Department of Pharmacology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12th Avenue North, Sherbrooke, Quebec, Canada, J1H 5N4.E-mail: richard.leduc{at}usherbrooke.ca