Mutation of a Highly Conserved Aspartic Acid in the β2Adrenergic Receptor: Constitutive Activation, Structural Instability, and Conformational Rearrangement of Transmembrane Segment 6

• DOI: 10.1124/mol.56.1.175
• Publication Date: 2018-01-08T20:36:12Z
• Authors: Søren G.F. Rasmussen; Anne D. Jensen; George Liapakis; Pejman Ghanouni; Jonathan A. Javitch; Ulrik Gether
• Keywords: Aspartic acid; Alanine; Conformational change; Structural motif

Movements of transmembrane segments (TMs) 3 and 6 play a key role in activation G protein-coupled receptors. However, the underlying molecular processes that govern these movements, accordingly control receptor activation, remain unclear. To elucidate importance conserved aspartic acid (Asp-130) Asp-Arg-Tyr motif β₂ adrenergic (β2AR), we mutated this residue to asparagine (D130N) mimic its protonated state, alanine (D130A) fully remove functionality side chain. Both mutants displayed evidence constitutive activation. In COS-7 cells expressing either D130N or D130A, basal levels cAMP accumulation were clearly elevated compared with wild-type β2AR. Incubation cell membranes purified at 37°C revealed also marked structural instability both mutant receptors, suggesting stabilizing intramolecular constraints had been disrupted. Moreover, obtained for conformational rearrangement by mutation Asp-130. D130N, cysteine TM 6, Cys-285, which is not accessible β2AR, became methanethiosulfonate ethylammonium, charged, sulfhydryl-reactive reagent. This consistent counterclockwise rotation tilting provides first time linking charge-neutralizing mutations DRY overall state receptor. We propose protonation leads release constraining interactions, resulting movements and, thus, conversion active state.