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

TLDR

Transmembrane segments 3 and 6 of G‑protein‑coupled receptors move at the cytoplasmic side to drive receptor activation. The authors sought to determine whether an ionic lock between Arg‑131(3.50), Asp‑130(3.49), and Glu‑268(6.30) keeps the β2‑adrenergic receptor inactive and whether its disruption initiates activation. They introduced charge‑neutralizing mutations at Asp‑130(3.49) and Glu‑268(6.30) in the β2‑adrenergic receptor and measured cAMP production in COS‑7 cells. The mutations caused a significant rise in basal and pindolol‑stimulated cAMP and increased Cys‑285(6.47) accessibility, indicating TM6 rearrangement and supporting the ionic lock as a common activation switch in rhodopsin‑like GPCRs.

Abstract

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 beta(2)-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 beta(2)-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(6.30) may constitute a common switch governing the activation of many rhodopsin-like G-protein-coupled receptors.