Transactivation within the AT1 angiotensin receptor homodimer: the role of the conserved DRY motif

Methods In this study we examined the possible functional consequence of transactivation within angiotensin AT1 receptor homodimers. To do this, we used the S109Y mutant of the AT1 receptor, in which the binding site of the nonpeptid AT1 receptor antagonist, candesartan, was destroyed. Expressing this mutant receptor together with the wildtype in CHO cells, in the presence of candesartan, we could examine the interaction between them by stimulating the S109Y mutant receptor, and following the activation of the wild-type receptor. To monitor the signaling of the receptor two parameters were measured: the conformational change of the receptor using an intramolecular sensor, and the binding of β-arrestin-2 to the activated receptors. In both cases the highly sensitive method of bioluminescence resonance energy transfer (BRET) was applied. Additionally, we performed a radioactive ligand binding assay to examine the cooperativity between receptor monomers.