Biology subjects

Oshima, H. S.

Publications and source records attributed to Oshima, H. S..

4 recordsLinked to original sources

bioRxiv2024

Cryo-EM structure of the bicarbonate receptor GPR30

G-protein-coupled receptor 30 (GPR30) is a bicarbonate receptor that plays a vital role in cellular responses to extracellular pH and ion homeostasis. Despite its significance, the mechanisms by which GPR30 interacts with bicarbonate ions remain elusive. There is no consensus on a drug that targets GPR30, and difficulties in pharmacological analyses have limited biological and drug discovery research on GPR30. Here, we present the cryo-electron microscopy structure of human GPR30 in the presence of bicarbonate ions, at 3.15 [A] resolution. Our structure reveals unique extracellular pockets and critical residues for bicarbonate binding and activation. Functional assays demonstrate that mutations in these residues impair bicarbonate-induced GPR30 activation, underscoring their importance in receptor function. This study also provides insights into G-protein coupling, highlighting the structural divergence between GPR30 and other GPCRs. Our findings not only advance the understanding of the role of GPR30 in pH homeostasis but also pave the way for the development of high-affinity drugs targeting GPR30 for therapeutic interventions in diseases associated with acid-base imbalance.

biochemistry↗

bioRxiv2024

Structural insights into the agonist selectivity and structure-based engineering of the adenosine A3 receptor

Adenosine receptors, expressed across various tissues, play pivotal roles in physiological processes and are implicated in diverse diseases, including neurological disorders and inflammation, highlighting the therapeutic potential of receptor-selective agents. The Adenosine A3 receptor (A3R), the last identified adenosine receptor, is also activated by breakdown products of post-transcriptionally modified tRNA and exhibits dual roles in neuron, heart, and immune cells, and is often overexpressed in tumors, making it a target for anticancer therapy. Despite extensive studies on the other adenosine receptors, the structure and activation mechanism of A3R, especially by selective agonists like N6-methyladenosine (m6A) and namodenoson, remained elusive. Here, we identified N6-isopentenyl adenosine (i6A), a novel A3R-selective ligand, via comprehensive modified adenosine library screening. Cryo-EM analyses of A3R-Gi signaling complexes with two nonselective and three selective agonists revealed the structural basis for A3R activation. We further conducted structure-guided engineering of m6A-insensitive A3R, which would greatly facilitate future discoveries of the physiological functions of the selective activation of A3R by modified adenosines. Our results clarify the selective activation of adenosine receptors, providing the basis for future drug discovery.

biophysics↗

bioRxiv2023

Structure and dynamics of the RF-amide QRFP receptor GPR103

Pyroglutamylated RF amide peptide (QRFP) is a type of peptide hormone with a C-terminal RF-amide motif. QRFP selectively activates class-A categorized GPCR, GPR103 to exert various physiological functions such as energy metabolism and appetite regulation. Here, we report the cryo-electron microscopy structure of the QRFP-GPR103-Gq complex at 3.3 [A] resolution. Unlike class-A GPCR, QRFP adopts an extended structure baring no secondary structure, with its N-terminal and C-terminal sides recognized by extracellular and transmembrane domains, respectively, of GPR103. The C-terminal heptapeptide of QRFP penetrates into the orthosteric pocket to act in receptor activation. Particularly, the residues that recognize the RF-amide are highly conserved in the RF-amide receptors. Notably, the unique N-terminal helix-loop-helix of the receptor traps the N-terminal side of QRFP with the pendulum-like motion to guide QRFP into the ligand-binding pocket. This movement, reminiscent of class B1 GPCRs except for orientation and structure of the ligand, is critical for the high affinity binding and receptor specificity of QRFP. Structural comparisons with closely related receptors, including RY-amide peptide-recognizing GPCRs, revealed conserved and diversified peptide recognition mechanisms, providing profound insights into the biological significance of RF-amide peptides. This study not only advances our understanding of GPCR-ligand interactions, but also paves the way for the development of novel therapeutics targeting metabolic and appetite disorders and emergency medical care.

biochemistry↗

bioRxiv2023

Optimizing Cryo-EM Structural Analysis of Gi-coupling Receptors via Engineered Gt and Nb35 Application

Cryo-EM single particle analysis has recently facilitated the high-resolution structural determination of numerous GPCR-G complexes. Diverse methodologies have been devised with this trend, and in the case of GPCR-Gi complexes, scFv16, an antibody that recognizes the intricate interface of the complex, has been mainly implemented to stabilize the complex. However, owing to their flexibility and heterogeneity, structural determinations of GPCR-Gi complexes remain both challenging and resource-intensive. By employing eGt, which exhibits binding affinity to modified nanobody Nb35, the cryo-EM structure of Rhodopsin-eGt complex was previously reported. Using this modified G protein, we determined the structure of the ETB-eGt complex bound to the modified Nb35. The determined structure of ETB receptor was the same as the previously reported ETB-Gi complex, and the resulting dataset demonstrated significantly improved anisotropy. This modified G protein will be utilized for the structural determination of other GPCR-Gi complexes. HighlightsO_LIThe study introduces the engineered G protein subunit eGT, which enhances the resolution of GPCR-G protein structures by suppressing G protein conformational fluctuations and is particularly beneficial for Gi-coupled receptors. C_LIO_LIThe cryo-EM structure of the ETB receptor complexed with eGt-Nb35 reveals improved map quality, reduced anisotropy, and isotropic density distribution, increasing the accuracy of structural analysis. C_LIO_LIStructural comparison between ETB-Gi and ETB-eGt reveals similar receptor-G protein interactions, demonstrating the utility of eGt-Nb35 for studying GPCR-Gi complexes and the potential for broader applications within the Gi family. C_LI

bioengineering↗