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.

Cell–cell communication via endogenous peptides and their receptors is vital for controlling all aspects of human physiology and most peptides signal through G protein-coupled receptors (GPCRs). Disordered peptides bind GPCRs through complex modes for which there are few representative crystal structures. The disordered peptide neurotensin (NT) is a neuromodulator of classical neurotransmitters such as dopamine and glutamate, through activation of neurotensin receptor 1 (NTS1). While several experimental structures show how NT binds NTS1, details about the structural dynamics of NT during and after binding NTS1, or the role of peptide dynamics on receptor activation, remain obscure. Here saturation transfer difference (STD) NMR revealed that the binding mode of NT fragment NT10-13 is heterogeneous. Epitope maps of NT10-13 at NTS1 suggested that tyrosine 11 (Y11) samples other conformations to those observed in crystal structures of NT-bound NTS1. Molecular dynamics (MD) simulations confirmed that when NT is bound to NTS1, residue Y11 can exist in two χ1 rotameric states, gauche plus (g+) or gauche minus (g–). Since only the g+ Y11 state is observed in all the structures solved to date, we asked if the g– state is important for receptor activation. NT analogues with Y11 replaced with 7-OH-Tic were synthesized to restrain the dynamics of the side chain. P­(OH-TIC)­IL bound NTS1 with the same affinity as NT10-13 but did not activate NTS1, instead acted as an antagonist. This study highlights that flexibility of Y11 in NT may be required for NT activation of NTS1.

Relaxin family peptide (RXFPs) 1‐4 receptors modulate the activity of cyclic adenosine monophosphate (cAMP) to produce a range of physiological functions. RXFP1 and RXFP2 increase cAMP via Gαs, whereas RXFP3 and RXFP4 inhibit cAMP via Gαi/o. RXFP1 also shows a delayed increase in cAMP downstream of Gαi3. In this study we have assessed whether the bioluminescence resonance energy transfer (BRET)‐based biosensor CAMYEL (cAMP sensor using YFP‐Epac‐Rluc), which allows real‐time measurement of cAMP activity in live cells, will aid in understanding ligand‐ and cell‐specific RXFP signaling. CAMYEL detected concentration‐dependent changes in cAMP activity at RXFP1‐4 in recombinant cell lines, using a variety of ligands with potencies comparable to those seen in conventional cAMP assays. We used RXFP2 and RXFP3 antagonists to demonstrate that CAMYEL detects dynamic changes in cAMP by reversing cAMP activation or inhibition respectively, with real‐time addition of antagonist after agonist stimulation. To demonstrate the utility of CAMYEL to detect cAMP activation in native cells expressing low levels of RXFP receptor, we cloned CAMYEL into a lentiviral vector and transduced THP‐1 cells, which express low levels of RXFP1. THP‐1 CAMYEL cells demonstrated robust cAMP activation in response to relaxin. However, the CAMYEL assay was unable to detect the Gαi3‐mediated phase of RXFP1 cAMP activation in PTX‐treated THP‐1 cells or HEK293A cells with knockout of Gαs. Our data demonstrate that cytoplasmically‐expressed CAMYEL efficiently detects real‐time cAMP activation by Gαs or inhibition by Gαi/o but may not detect cAMP generated in specific intracellular compartments such as that generated by Gαi3 upon RXFP1 activation.