TY - JOUR
T1 - Structural insights into the G protein selectivity revealed by the human EP3-Gi signaling complex
AU - Suno, Ryoji
AU - Sugita, Yukihiko
AU - Morimoto, Kazushi
AU - Takazaki, Hiroko
AU - Tsujimoto, Hirokazu
AU - Hirose, Mika
AU - Suno-Ikeda, Chiyo
AU - Nomura, Norimichi
AU - Hino, Tomoya
AU - Inoue, Asuka
AU - Iwasaki, Kenji
AU - Kato, Takayuki
AU - Iwata, So
AU - Kobayashi, Takuya
N1 - Funding Information:
The plasmids for expressing Gi and Gβ1γ2 were provided by Brian K. Kobilka (Stanford University). This research was supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED (JP21am0101072 [support number 1113] to K.I. and T. Kato, and JP19am0101079 [support number 2365] to S.I.); the Takeda Science Foundation (to R.S. Yuijnolk.S. K.M. and T. Kobayashi); Ono Medical Research Foundation (to K.M.), AMED Core Research for Evolutional Science and Technology (CREST) (JP21gm0910007 to T. Kobayashi); AMED Science and Technology Platform Program for Advanced Biological Medicine under grant number JP21am0401020 (to T. Kobayashi); AMED Research on Development of New Drugs (JP20ak0101103 to T. Kobayashi); The Naito Foundation (to T. Kobayashi); Koyanagi Foundation (to T. Kobayashi); FOREST Program JPMJFR215T (to A.I.); JST Moonshot Research and Development Program JPMJMS2023 (to A.I.) from Japan Science and Technology Agency (JST), JSPS KAKENHI (15K08268 and 19H03428 to R.S.; 20H03434 to K.M.; and 21H04791, 21H05113, and JPJSBP120218801 to A.I.); and a Grant-in-Aid for Transformative Research Areas (21H05112 to R.S.) and for MEXT LEADER Program (to Y.S.). DNA sequencing analysis was performed at the Medical Research Support Center, Graduate School of Medicine at Kyoto University. R.S. H.T. and C.S.-I. carried out protein expression and purification of the receptors, G proteins, and GFP nanobody. N.N. purified scFv16 fragment. T.H. provided the plasmids for expressing GFP nanobody and purified GFP nanobody. R.S. prepared the cryo-EM sample of EP3-Gi-scFv16 complex. K.M. and A.I. carried out the signaling assays. R.S. Y.S. and M.H. carried out the cryo-EM data collection. Y.S. and H.T. carried out cryo-EM data processing and model building of EP3-Gi-scFv16 complex. R.S. designed the project, and K.I. T. Kato. T. Kobayashi, and S.I. supervised the overall project. R.S. Y.S. and K.M. wrote the manuscript. All authors discussed the results and commented on the manuscript. The authors declare no competing financial interests.
Funding Information:
The plasmids for expressing G i and G β1 γ 2 were provided by Brian K. Kobilka (Stanford University). This research was supported by Platform Project for Supporting Drug Discovery and Life Science Research ( Basis for Supporting Innovative Drug Discovery and Life Science Research [ BINDS ]) from AMED ( JP21am0101072 [support number 1113] to K.I. and T. Kato, and JP19am0101079 [support number 2365] to S.I.); the Takeda Science Foundation (to R.S., Yuijnolk.S., K.M., and T. Kobayashi); Ono Medical Research Foundation (to K.M.), AMED Core Research for Evolutional Science and Technology (CREST) ( JP21gm0910007 to T. Kobayashi); AMED Science and Technology Platform Program for Advanced Biological Medicine under grant number JP21am0401020 (to T. Kobayashi); AMED Research on Development of New Drugs ( JP20ak0101103 to T. Kobayashi); The Naito Foundation (to T. Kobayashi); Koyanagi Foundation (to T. Kobayashi); FOREST Program JPMJFR215T (to A.I.); JST Moonshot Research and Development Program JPMJMS2023 (to A.I.) from Japan Science and Technology Agency (JST), JSPS KAKENHI ( 15K08268 and 19H03428 to R.S.; 20H03434 to K.M.; and 21H04791 , 21H05113 , and JPJSBP120218801 to A.I.); and a Grant-in-Aid for Transformative Research Areas ( 21H05112 to R.S.) and for MEXT LEADER Program (to Y.S.). DNA sequencing analysis was performed at the Medical Research Support Center , Graduate School of Medicine at Kyoto University .
Publisher Copyright:
© 2022 The Author(s)
PY - 2022/9/13
Y1 - 2022/9/13
N2 - Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E2. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-Gi signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of Gi to EP3 and the structural changes induced in EP3 by Gi binding. In addition, we compare the structure of the EP3-Gi complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to Gs that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5.
AB - Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E2. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-Gi signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of Gi to EP3 and the structural changes induced in EP3 by Gi binding. In addition, we compare the structure of the EP3-Gi complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to Gs that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5.
UR - http://www.scopus.com/inward/record.url?scp=85137778924&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85137778924&partnerID=8YFLogxK
U2 - 10.1016/j.celrep.2022.111323
DO - 10.1016/j.celrep.2022.111323
M3 - Article
C2 - 36103815
AN - SCOPUS:85137778924
SN - 2211-1247
VL - 40
JO - Cell Reports
JF - Cell Reports
IS - 11
M1 - 111323
ER -
