Abstract
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.
Footnotes
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This work was supported by the Canadian Institutes of Health Research and is part of the M.Sc. thesis of S.S.M. R.L. is a Chercheur National of the Fonds de la Recherche en Santé du Québec. E.E. is recipient of the J.C. Edwards Chair in Cardiovascular Research. S.S.M. is the recipient of a studentship from the Heart and Stroke Foundation of Canada.
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ABBREVIATIONS: AngII, angiotensin II; AT1 receptor, angiotensin II type-1 receptor; TMD, transmembrane domain; SCAM, substituted-cysteine accessibility method; MTSEA, methanethiosulfonate-ethylammonium; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; MPA, methionine proximity assay; IP, inositol phosphate.
- Received December 20, 2006.
- Accepted April 18, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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