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

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 ␤ 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 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 (1-3). The ␤ 2 -adrenergic receptor (␤ 2 AR) belongs to the subfamily of rhodopsin-like receptors and has been used as a prototype GPCR in numerous studies (1-3). Low-resolution structures of rhodopsin, resolved by Schertler and co-workers (4, 5), have demonstrated the presence of seven membrane-spanning ␣-helical segments and have provided important insights into the organization of the transmembrane bundle, allowing the development of tertiary structure models of GPCRs (6 -8). Importantly, a high-resolution structure of rhodopsin has now become available (9) making it possible to consider the functional roles of individual side chains from the perspective of an atomic resolution structure of a homologous GPCR.Understanding the function of GPCRs at a molecular level requires an understanding of how agonist binding to the receptor is converted into receptor activation (3). Studies based on EPR spectroscopy, fluorescence spectroscopy, alterations in cysteine accessibility, and engineering of metal-binding sites have altogether pointed to a key role for conformational changes of . The molecular mechanisms that underlie the movements of TM3 and TM6 and govern the transition of the receptor between its inactive and active states have nonetheless remained unclear. 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). Moreover, direct evidence has been obtained indicating that the photoactivation of rhodopsin is accompanied by the uptake of a proton b...

Structural Mimicry in Class A G Protein-coupled Receptor Rotamer Toggle Switches

“…The conformer selected from our CM results for inclusion in the CB 1 R bundle (Pro kink angle ϭ 53.1 o ) was chosen so that R3.50(215) and D6.30(339) could form a salt bridge at the intracellular ends of TMH3 and -6 in the CB 1 TMH bundle. An analogous salt bridge has been shown to be an important stabilizer of the inactive state of the ␤ 2 -adrenergic receptor, the 5HT-2a receptor (10), and to be present in Rho (3). Because of the extreme flexibility of TMH6 in CB 1 , we have proposed that an additional TMH3-6 salt bridge, K3.28(193)-D6.58(367), stabilizes the inactive state on the extracellular side of the TMH bundle (25).…”

“…At their intracellular ends, TMHs 3 and 6 in Rho are constrained by an E3.49(134)/R3.50(135)/E6.30 (247) salt bridge that limits the relative mobility of the cytoplasmic ends of TMH3 and TMH6 in the inactive state (3) and acts like an "ionic lock" (10,11). During activation, P6.50 of the highly conserved CWXP motif in TMH6 of GPCRs may act as a flexible hinge, permitting TMH6 to straighten upon activation, moving its intracellular end away from TMH3 and upwards toward the lipid bilayer (12).…”

Crystal structure of the ligand-free G-protein-coupled receptor opsin

“…The E(D)RY motif (in TM3) is part of a hydrogen bonded network -the so-called "ionic lock" [51,52,53,54] -which tethers Arg-135 (on TM3) to Glu-247 (on TM6). A second motif, the NPxxY(x) 5,6 F motif links Tyr-306 (on TM7) and Phe-313 (on H8).…”

“…and Phe 7.60 , respectively. Protein structural alterations and release of the constraints are induced by cis/trans isomerization of the chromophore following light absorption [51,52,53,54,77,129,130]. Due to retinal isomerization, the stabilizing salt bridge present in the inactive rhodopsin ground state between the protonated 11-cis-retinal Schiff base and the Glu-113 (on TM3) counterion is broken [25,31,58].…”

A Novel Mode for Integrin-mediated Signaling: Tethering Is Required for Phosphorylation of FAK Y397

“…The other large class of transmembrane receptors for soluble ligands are the 7-transmembrane family. A recent model proposes that ligand binding results in the disruption of a salt bridge between TM domains at the cytoplasmic face and a displacement of one of the TM domains opening a binding site on the cytoplasmic side (Ballesteros et al, 2001;Pierce et al, 2002). For some of the 7-transmembrane receptors, receptor dimerization appears to play a role perhaps in the bringing together of JAKs to generate a cross-phosphorylation as described for the EGF receptors (Mellado et al, 2001).…”