Received for publication December 20, 2006.
Revised April 18, 2007.
Accepted for publication April 18, 2007.

ACTIVATION OF THE ANGIOTENSIN II TYPE 1 RECEPTOR LEADS TO MOVEMENT OF THE SIXTH TRANSMEMBRANE DOMAIN: ANALYSIS BY THE SUBSTITUTED CYSTEINE ACCESSIBILITY METHOD

Abstract

The role of transmembrane domain six (TMD6) of the angiotensin II type 1 receptor, which is predicted to undergo conformational changes following 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. Interestingly, 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 while 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 of the top (extracellular side) of TMD6 towards the binding pocket and simultaneously distancing 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.

Key words: Angiotensin, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, Mutagenesis/Chimeric approaches, Receptor binding studies