Reversible inactivation of AT2angiotensin II receptor from cysteine-disulfide bond exchange

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Abstract
Dithiothreitol (DTT) treatment of angiotensin II (Ang II) type 2 (AT2) receptor potentiates ligand binding, but the underlying mechanism is not known. Two disulfide bonds proposed in the extracellular domain were examined in this report. Based on the analysis of ligand affinity of cysteine (Cys, C) to alanine (Ala, A) substitution mutants, we provide evidence that Cys35–Cys290 and Cys117–Cys195 disulfide bonds are formed in the wild‐type AT2 receptor. Disruption of the highly conserved Cys117–Cys195 disulfide bond linking the second and third extracellular segments leads to inactivation of the receptor. The Cys35–Cys290 bond is highly sensitive to DTT. Its breakage results in an increased binding affinity for both Ang II and the AT2 receptor‐specific antagonist PD123319. Surprisingly, in the single Cys mutants, C35A and C290A, a labile population of receptors is produced which can be re‐folded to high‐affinity state by DTT treatment. These results suggest that the free –SH group of Cys35 or Cys290 competes with the disulfide bond formation between Cys117 and Cys195. This Cys–disulfide bond exchange results in production of the inactive population of the mutant receptors through formation of a non‐native disulfide bond.