Activation of the β₂-Adrenergic Receptor Involves Disruption of an Ionic Lock between the Cytoplasmic Ends of Transmembrane Segments 3 and 6*

The movements of transmembrane segments (TMs) 3 and 6 at the cytoplasmic side of the membrane play an important role in the activation of G-protein-coupled receptors. Here we provide evidence for the existence of an ionic lock that constrains the relative mobility of the cytoplasmic ends of TM3 and TM6 in the inactive state of the β₂-adrenergic receptor. We propose that the highly conserved Arg-131^3.50 at the cytoplasmic end of TM3 interacts both with the adjacent Asp-130^3.49 and with Glu-268^6.30 at the cytoplasmic end of TM6. Such a network of ionic interactions has now been directly supported by the high-resolution structure of the inactive state of rhodopsin. We hypothesized that the network of interactions would serve to constrain the receptor in the inactive state, and the release of this ionic lock could be a key step in receptor activation. To test this hypothesis, we made charge-neutralizing mutations of Glu-268^6.30 and of Asp-130^3.49 in the β₂-adrenergic receptor. Alone and in combination, we observed a significant increase in basal and pindolol-stimulated cAMP accumulation in COS-7 cells transiently transfected with the mutant receptors. Moreover, based on the increased accessibility of Cys-285^6.47 in TM6, we provide evidence for a conformational rearrangement of TM6 that is highly correlated with the extent of constitutive activity of the different mutants. The present experimental data together with the recent high-resolution structure of rhodopsin suggest that ionic interactions between Asp/Glu^3.49, Arg^3.50, and Glu^6.30may constitute a common switch governing the activation of many rhodopsin-like G-protein-coupled receptors.