Mutation of a highly conserved aspartic acid in the beta(2) adrenergic receptor: Constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6

Movements of transmembrane segments (TMs) 3 and 6 play a key role in activa tion of G protein-coupled receptors. However, the underlying molecular proc esses that govern these movements, and accordingly control receptor activat ion, remain unclear. To elucidate the importance of the conserved aspartic acid (Asp-130) in the Asp-Arg-Tyr motif of the beta(2) adrenergic receptor (beta 2AR), we mutated this residue to asparagine (D130N) to mimic its prot onated state, and to alanine (D130A) to fully remove the functionality of t he side chain. Both mutants displayed evidence of constitutive receptor act ivation. In COS-7 cells expressing either D130N or D130A, basal levels of c AMP accumulation were clearly elevated compared with cells expressing the w ild-type beta 2AR. Incubation of COS-7 cell membranes or purified receptor at 37 degrees C revealed also a marked structural instability of both mutan t receptors, suggesting that stabilizing intramolecular constraints had bee n disrupted. Moreover, we obtained evidence for a conformational rearrangem ent by mutation of Asp-130. In D130N, a cysteine in TM 6, Cys-285, which is not accessible in the wild-type beta 2AR, became accessible to methanethio sulfonate ethylammonium, a charged, sulfhydryl-reactive reagent. This is co nsistent with a counterclockwise rotation or tilting of TM 6 and provides f or the first time structural evidence linking charge-neutralizing mutations of the aspartic acid in the DRY motif to the overall conformational state of the receptor. We propose that protonation of the aspartic acid leads to release of constraining intramolecular interactions, resulting in movements of TM 6 and, thus, conversion of the receptor to the active state.