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

Juan A Ballesteros,Anne D Jensen,George Liapakis,Søren G.F Rasmussen,Lei Shi,Ulrik Gether,Jonathan A Javitch

doi:10.1074/jbc.m103747200

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.

Highlights

The majority of hormones and neurotransmitters exerts its cellular effects by activating cell surface receptors belonging to the superfamily of G-protein-coupled receptors (GPCRs)1 [1,2,3]
It is well established that TM3 and TM6 play a role in GPCR activation, but the underlying mechanism has remained unclear
Previous studies have indicated a critical function of the adjacent aspartic acid (Asp3.49) for receptor activation [7, 14, 18, 20]

Summary

Introduction

The majority of hormones and neurotransmitters exerts its cellular effects by activating cell surface receptors belonging to the superfamily of G-protein-coupled receptors (GPCRs)1 [1,2,3]. It has been suggested that the protonation of the aspartic acid in the highly conserved (D/E)RY motif at the cytoplasmic side of TM3 leads to a release of constraining intramolecular interactions, thereby resulting in the movements of TM3 and TM6 and a conversion of the receptor to the active state [7, 14, 16] This hypothesis has been supported by the observation that charge-neutralizing mutations of the aspartic acid (or glutamic acid) in TM3 lead to increased agonist-independent activation of a number of GPCRs [7, 14, 17, 18]. Experiments using cysteine cross-linking and engineered metal ion-binding sites [10, 11, 21] suggest that the cytoplasmic ends of TM3 and TM6 are in close proximity Exploring these proposed proximities in a three-dimensional molecular model of the ␤2AR supports an orientation of the conserved Arg3.50 in TM3 facing a conserved Glu6.30 in TM6. Our hypothesis and results are remarkably consistent with the recent high-resolution structure of the inactive state of rhodopsin, which showed that Arg3.50 in this receptor is positioned to form an ionic interaction with both Glu3.49 and Glu6.30 in this receptor

Methods

Results

Conclusion