부산대학교 도서관

Keywords G-protein coupled receptor; GPCR; G-protein; glucagon receptor; receptor activity-modifying protein RAMP; cryo-electron microscopy; cryo-EM; single molecule fluorescence resonance energy transfer; smFRET; hydrogen-deuterium exchange monitored by mass spectrometry; HDX-MS; allostery; protein dynamics; cell signaling Highlights * RAMP2 binds to the glucagon receptor and acts as a negative allosteric modulator * RAMP2 promotes extracellular receptor dynamics resulting in an inactive cytosolic face * Cryo-EM shows an unproductive complex formation with a dynamic extracellular domain * Dynamic allostery is an endogenous GPCR regulatory mechanism Summary Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6.sup.th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-G.sub.s complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD. Author Affiliation: (1) Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305, USA (2) Department of Molecular and Cell Biology, University of California Berkeley, CA 94720, USA (3) Zealand Pharma A/S, Sydmarken 11, Soborg 2860, Denmark (4) QB3 Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley CA 94720, USA (5) Department of Chemistry, University of California, Berkeley, Berkeley CA 94720, USA (6) Department of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, Marburg 35037, Germany (7) Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley CA 94720, USA * Corresponding author Article History: Received 4 October 2022; Revised 23 January 2023; Accepted 17 February 2023 (miscellaneous) Published: March 30, 2023 (footnote)8 These authors contributed equally (footnote)9 Lead contact Byline: Kaavya Krishna Kumar (1,8), Evan S. O'Brien (1,8), Chris H. Habrian (1), Naomi R. Latorraca (2), Haoqing Wang (1), Inga Tuneew (3), Elizabeth Montabana (1), Susan Marqusee (2,4,5), Daniel Hilger (6), Ehud Y. Isacoff (2,7), Jesper Mosolff Mathiesen (3), Brian K. Kobilka [kobilka@stanford.edu] (1,9,*)