G protein-coupled receptors (GPCRs) play an important role in cellular signaling and serve as important therapeutic targets for a variety of diseases. Upon binding to extracellular agonists, GPCRs stimulate distinct signaling pathways by recruiting different G proteins (Gs, Gi, Gq, etc.,) to mediate a variety of physiological functions. Selective conjugation between GPCRs and specific G proteins is essential for the biological role of such receptors.

However, determining the molecular details of how individual GPCRs recognize different G protein isoforms has remained elusive, limiting the understanding of GPCR signaling mechanisms.

In a new study, researchers from the Shanghai Institute of Pharmaceutical Sciences, Chinese Academy of Sciences, Fudan University and the University of Science and Technology Shanghai, among others, used cryo-electron microscopy (cryo-EM) techniques to resolve the 3D structure of the human glucagon receptor (GCGR) when bound to its cognate agonists and different types of G proteins (Gs or Gi). The results of the study was published in the March 20, 2020 issue of Science in a paper titled "Structural basis of Gs and Gi recognition by the human glucagon receptor".

These structures provide the first detailed molecular maps of the interaction patterns between GPCRs and different G-protein isoforms, and unexpectedly reveal many molecular features that regulate G-protein specificity, greatly advancing the understanding of GPCR signaling mechanisms.

GCGR is a member of the class B GPCR family and is essential for glucose homeostasis by triggering glucose release in the liver, making it a potential drug target for type 2 diabetes and obesity.

Although GCGR normally exerts its physiological effects through Gs signaling, it can also couple to other G proteins (e.g., Gi and Gq), leading to multiple cellular responses. In 2017 and 2018, scientists at the Shanghai Institute of Pharmaceutical Sciences, Chinese Academy of Sciences, solved the crystal structure of full-length GCGR when bound to a negative conformation regulator or a partial peptide agonist, providing new insights into signal recognition and regulation of class B GPCRs.

In this new study, these researchers made further progress by resolving the complex structure of GCGR when bound to two transduction proteins with opposing biological activities. This study provides valuable insights into GPCR-G protein coupling and G protein specificity. In particular, it shows that the sixth transmembrane helix of GCGR (helix VI) adopts a similar outward shift in the structure of GCGR bound by the two G proteins, resulting in a common binding cavity to accommodate Gs and Gi. This contradicts the hypothesis, based on the previously determined structure of the GPCR-G protein complex, that the difference in position of helix VI is the main distinguishing factor for Gs and Gi binding specificity.

This common G protein binding pocket observed in these GCGR-G protein complex structures is consistent with the signaling pleiotropy of GCGR and is maximally efficient in activating different signaling pathways. Although GCGR couples to both G proteins through this common pocket, it does so through different modes of interaction, explaining the specificity of the G proteins. The measured interaction interface between GCGR and Gs is much larger than that between GCGR and Gi, resulting in a higher binding affinity of Gs to this receptor. This provides a structural basis for the preferential coupling of GCGR to Gs.

Based on the structure of the GCGR-Gs and GCGR-Gi complexes, these researchers performed extensive functional studies using techniques such as mutagenesis, G protein activation, and cellular signaling to investigate the role of key residues in the GCGR-G protein binding interface in Gs and Gi activation.

These results suggest that conformational differences in the intracellular loop and side chains of residues in GCGR are sufficient to control G protein selectivity. The second intracellular loop of GCGR (ICL2) and the interaction facilitated by the helix VII/VIII junction play a key role in Gs coupling, while the other two intracellular loops of GCGR, ICL1 and ICL3, and its hydrophobic intracellular binding lumen are more important for Gi recognition.

These findings expand the knowledge of GPCR activation, pleiotropic coupling and G protein specificity. They also offer new opportunities for drug discovery: designing biased ligands to selectively block a specific signaling pathway, thereby reducing side effects.

RESOURCE: https://www.creative-biolabs.com/gpcr-preparation-as-immunogen.html

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