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Adenosine receptors play pivotal roles in physiological processes. Adenosine A3 receptor (A3R), the most recently identified adenosine receptor, is expressed in various tissues, exhibiting important roles in neuron, heart, and immune cells, and is often overexpressed in tumors, highlighting the therapeutic potential of A3R-selective agents. Recently, we identified RNA-derived N6-methyladenosine (m6A) as an endogenous agonist for A3R, suggesting the relationship between RNA-derived modified adenosine and A3R. Despite extensive studies on the other adenosine receptors, the selectivity mechanism of A3R, especially for A3R-selective agonists such as m6A and namodenoson, remained elusive. Here, we identify tRNA-derived N6-isopentenyl adenosine (i6A) as an A3R-selective ligand via screening of modified nucleosides against the adenosine receptors. Like m6A, i6A is found in the human body and may be an endogenous A3R ligand. Our cryo-EM analyses elucidate the A3R-Gi complexes bound to adenosine, 5'-N-ethylcarboxamidoadenosine (NECA), m6A, i6A, and namodenoson at overall resolutions of 3.27 Å (adenosine), 2.86 Å (NECA), 3.19 Å (m6A), 3.28 Å (i6A), and 3.20 Å (namodenoson), suggesting the selectivity and activation mechanism of A3R. We further conduct structure-guided engineering of m6A-insensitive A3R, which may aid future research targeting m6A and A3R, providing a molecular basis for future drug discovery. Adenosine A3 receptor (A3R) plays important roles in neurons, heart, and immune cells, and is often overexpressed in tumors. Oshima et al. identify tRNA-derived i6A as an A3R-selective ligand and use cryo-EM to reveal A3R's selectivity and activation mechanisms. [ABSTRACT FROM AUTHOR]

β-arrestins (βarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo–electron microscopy structures of βarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the βarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of βarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of βarr2 from a β strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-βarr complexes with direct implications for exploring novel therapeutic avenues. [ABSTRACT FROM AUTHOR]

The endothelin ETB receptor is a promiscuous G-protein coupled receptor that is activated by vasoactive peptide endothelins. ETB signaling induces reactive astrocytes in the brain and vasorelaxation in vascular smooth muscle. Consequently, ETB agonists are expected to be drugs for neuroprotection and improved anti-tumor drug delivery. Here, we report the cryo-electron microscopy structure of the endothelin-1-ETB-Gi complex at 2.8 Å resolution, with complex assembly stabilized by a newly established method. Comparisons with the inactive ETB receptor structures revealed how endothelin-1 activates the ETB receptor. The NPxxY motif, essential for G-protein activation, is not conserved in ETB, resulting in a unique structural change upon G-protein activation. Compared with other GPCR-G-protein complexes, ETB binds Gi in the shallowest position, further expanding the diversity of G-protein binding modes. This structural information will facilitate the elucidation of G-protein activation and the rational design of ETB agonists. [ABSTRACT FROM AUTHOR]

Lysophosphatidic acid receptor 1 (LPA1) is one of the six G protein-coupled receptors activated by the bioactive lipid, lysophosphatidic acid (LPA). LPA1 is a drug target for various diseases, including cancer, inflammation, and neuropathic pain. Notably, LPA1 agonists have potential therapeutic value for obesity and urinary incontinence. Here, we report a cryo-electron microscopy structure of the active human LPA1-Gi complex bound to ONO-0740556, an LPA analog with more potent activity against LPA1. Our structure elucidated the details of the agonist binding mode and receptor activation mechanism mediated by rearrangements of transmembrane segment 7 and the central hydrophobic core. A structural comparison of LPA1 and other phylogenetically-related lipid-sensing GPCRs identified the structural determinants for lipid preference of LPA1. Moreover, we characterized the structural polymorphisms at the receptor-G-protein interface, which potentially reflect the G-protein dissociation process. Our study provides insights into the detailed mechanism of LPA1 binding to agonists and paves the way toward the design of drug-like agonists targeting LPA1. LPA1 is one of the GPCRs that are drug targets for various diseases. Here the authors report a cryo-EM structure of the active human LPA1-Gi complex bound to an LPA analog with more potent activity against LPA1 and clarified the ligand recognition mechanism. [ABSTRACT FROM AUTHOR]