Abstract
G protein-coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [13CϵH3]methionine-labeled α1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [13CϵH3]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α1A-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.
Highlights
G protein– coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site
Homology models of ␣1A-AR (Fig. 1) built on in inactive and active states of X-ray structures of 2-AR show that three of these methionines were interesting as conformational probes as they are located either within the adrenaline-binding site (Met2926.55), immediately adjacent to the highly conserved Ile1143.40 of the transmission switch (Met-1153.41), or sitting above Tyr-1253.51 of the DRY motif within the G protein-binding site (Met-2035.57)
Recent spectroscopic studies have demonstrated that different classes of GPCR ligands distinctly alter the population of receptor states within the GPCR conformational equilibrium [23]
Summary
G protein– coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Despite the reduced thermostability of ␣1A-AR-A4 (L312F) [32], we were able to [13C⑀H3]methionine label and record 1H-13C SOFAST-HMQC spectra for this receptor in the apo state and bound to adrenaline (full agonist), phenylephrine (full agonist), A-61603 (full agonist), and oxymetazoline (partial agonist) in addition to the inverse agonists and neutral antagonists tested on ␣1A-AR-A4 (Fig. 3A).
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