Reversible inactivation of AT₂ angiotensin II receptor from cysteine-disulfide bond exchange

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. Copyright (C) 2000 Federation of European Biochemical Societies.