Coupling ligand structure to specific conformational switches in the β2-adrenoceptor

Abstract

G protein-coupled receptors (GPCRs) regulate a wide variety of physiological functions in response to structurally diverse ligands ranging from cations and small organic molecules to peptides and glycoproteins. For many GPCRs, structurally related ligands can have diverse efficacy profiles. To investigate the process of ligand binding and activation, we used fluorescence spectroscopy to study the ability of ligands having different efficacies to induce a specific conformational change in the human beta2-adrenoceptor (beta2-AR). The 'ionic lock' is a molecular switch found in rhodopsin-family GPCRs that has been proposed to link the cytoplasmic ends of transmembrane domains 3 and 6 in the inactive state. We found that most partial agonists were as effective as full agonists in disrupting the ionic lock. Our results show that disruption of this important molecular switch is necessary, but not sufficient, for full activation of the beta2-AR.

Identification of two distinct inactive conformations of the β 2 -adrenergic receptor reconciles structural and biochemical observations

“…In simulations of several constitutively active mutants and of the ␤ 2 AR-T4L fusion construct, the equilibrium shifted toward conformations with the ionic lock broken and helices 3 and 6 farther apart. Our results thus suggest that an intact ionic lock is not a requirement for an inactive state of ␤ 2 AR, but rather that the wild-type receptor in the inactive state frequently adopts conformations with the ionic lock formed, in accord with biochemical observations indicating that the ionic lock stabilizes the inactive state (13,21).…”

“…Upon activation of rhodopsin, the intracellular ends of helices 3 and 6 move apart, breaking the Arg 3.50 /Glu 6.30 contact (14)(15)(16)(17). A variety of biochemical evidence has suggested that the homologous residues in many other GPCRs, including the ␤-adrenergic receptors, also form an ionic lock in the inactive state (13,(18)(19)(20)(21). Indeed, the term ''ionic lock'' was originally coined in a study of ␤ 2 AR to describe the interaction of Arg-131 3.50 with Glu-268 6.30 and Asp-130 3.49 (13).…”

“…Indeed, the term ''ionic lock'' was originally coined in a study of ␤ 2 AR to describe the interaction of Arg-131 3.50 with Glu-268 6.30 and Asp-130 3.49 (13). This interaction was believed to stabilize the inactive state, and its disruption was believed to be one of the critical events in the activation process (13,21).…”

Real-time conformational changes in LacY

“…The efficiency of bimane quenching is dependent upon proximity to Trp as well as other factors, such as stereochemistry and local environment. The magnitude of bimane quenching by Trp in membrane proteins is typically a 10-50% change in the emission spectra, as reported with visual rhodopsin (23,24), the cyclic nucleotide-gated ion channel (25), or the β-adrenoreceptor (26).…”

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

“…These studies showed that some key residues (e.g. Glu Previous mutagenesis, fluorescence and EPR studies [71,72,73,86,135,144] suggested that the disruption of the "ionic lock" as observed in the opsin* structure is induced by a rotational and translational movement of TM6. The opsin* crystal structure revealed that due to TM6…”