Many drugs that target G-protein-coupled receptors (GPCRs) induce or inhibit their signal transduction with different strengths, which affect their therapeutic properties. However, the mechanism underlying the differences in the signalling levels is still not clear, although several structures of GPCRs complexed with ligands determined by X-ray crystallography are available. Here we utilized NMR to monitor the signals from the methionine residue at position 82 in neutral antagonist- and partial agonist-bound states of β₂-adrenergic receptor (β₂AR), which are correlated with the conformational changes of the transmembrane regions upon activation. We show that this residue exists in a conformational equilibrium between the inverse agonist-bound states and the full agonist-bound state, and the population of the latter reflects the signal transduction level in each ligand-bound state. These findings provide insights into the multi-level signalling of β₂AR and other GPCRs, including the basal activity, and the mechanism of signal transduction mediated by GPCRs.

G‐protein‐coupled receptor (GPCR) ligands impart differing degrees of signaling in the G‐protein and arrestin pathways, in phenomena called “biased signaling”. However, the mechanism underlying the biased signaling of GPCRs is still unclear, although crystal structures of GPCRs bound to the G protein or arrestin are available. In this study, we observed the NMR signals from methionine residues of the μ‐opioid receptor (μOR) in the balanced‐ and biased‐ligand‐bound states. We found that the intracellular cavity of μOR exists in an equilibrium between closed and multiple open conformations with coupled conformational changes on the transmembrane helices 3, 5, 6, and 7, and that the population of each open conformation determines the G‐protein‐ and arrestin‐mediated signaling levels in each ligand‐bound state. These findings provide insight into the biased signaling of GPCRs and will be helpful for development of analgesics that stimulate μOR with reduced tolerance and dependence.

The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 β-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the “fly-casting” mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging.

Discoidin domain receptor (DDR) is a cell‐surface receptor tyrosine kinase activated by the binding of its discoidin (DS) domain to fibrillar collagen. Here, we have determined the NMR structure of the DS domain in DDR2 (DDR2‐DS domain), and identified the binding site to fibrillar collagen by transferred cross‐saturation experiments. The DDR2‐DS domain structure adopts a distorted jellyroll fold, consisting of eight β‐strands. The collagen‐binding site is formed at the interloop trench, consisting of charged residues surrounded by hydrophobic residues. The surface profile of the collagen‐binding site suggests that the DDR2‐DS domain recognizes specific sites on fibrillar collagen. This study provides a molecular basis for the collagen‐binding mode of the DDR2‐DS domain.