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Chapter 4
General Conclusion
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GPCRs are involved in a wide range of diseases. Growing evidence has suggested the
pluridimensional signal controls of GPCRs by many factors, including ligand-induced unique
structural changes and transient GPCR-GPCR interactions. However, temporally dynamic changes in
signal transductions of GPCRs have not been fully investigated for drug discovery.
In Chapter 2, I established four assay systems to measure the signal transduction efficiency
quantitatively with temporal information. In the conventional drug discovery, the efficacy of signal
transduction has been evaluated mainly by ligand binding and second messengers only at a fixed time
point. The present study added two assay systems to monitor directly the G protein activation and the
β-arrestin recruitment to evaluate ligand bias. All the assays were designed to be performed in the
same cell type and under the same temperature, enabling the unbiased measurements of the efficiency
and the kinetics of signal transduction events. At the equilibrated states, an FPR1 agonist, fMLP,
displayed different dose–response curves among signal transduction events, suggesting an originally
biased signal transduction of FPR1. In addition, I monitored the temporal changes in the dose–
response of β-arrestin recruitment. The effective concentrations of the agonist for β-arrestin
recruitment were decreased over time, suggesting that relative signal strengths among signal pathways
might change dependent on the time point after ligand stimulation. Therefore, the present results
demonstrated the importance of measuring the ligand bias with the temporal information.
In Chapter 3, I visualized oligomer formations of FPR1 in living cells and revealed its roles in signal
transduction. The cluster formation of FPR1 has been widely accepted due to the relatively large size
of the clusters, being visible under conventional microscopy. Recent studies have investigated the 3D
structures of GPCRs using cryo-EM and the monomer–dimer equilibrium of FPR1 using single
molecule microscopy, but the presence and the roles of FPR1 oligomers with intermediate sizes have
not been elucidated. In the present study, by controlling the labeling efficiency of FPR1, both the
clusters and oligomers have been detected in the cells with the same expression levels of FPR1. I
demonstrated that FPR1 on the plasma membrane of HEK293 cells was equilibrated between small
oligomers (2–4 FPR1) and large oligomers (5–11 FPR1) at the physiological expression level of 2
molecules/μm2. The agonist stimulation induced the equilibrium shift toward the large oligomers to
induce G protein binding within 2 min, and induced FPR1 cluster formation (20–40 FPR1) triggered
by β-arrestin recruitment in 5 min. The temporally dynamic complex formation of FPR1 would
provide a robust signal transduction under noisy physiological conditions. The oligomerizationinduced signal transduction may be a novel target for signal specific drug discovery.
I established ligand binding assay, β-arrestin split luciferase assay, bioluminescence cAMP assay, and
G protein FRET assay system for quantitative evaluation of signaling patterns of FPR1 in living
HEK293 cells. Using single molecule and ensemble TIRF imaging, I clarified the size distributions of
FPR1 and their roles in signal transductions under the physiological expression level. The presently
92
established methods enable the detection of signal biases and provide a deep insight into the effects
on dynamic signal control mediated by FPR1 oligomer formation in living systems.
93
Acknowledgments
The present thesis was completed under a supervision of Prof. Takeaki Ozawa. I would like to thank
Prof. Takeaki Ozawa for giving me continuous supports and numerous opportunities to make
presentations in domestic and foreign conferences. I appreciate all staff members in Ozawa Lab.,
especially Dr. Hideaki Yoshimura, Dr. Miho Tanaka for heartful instructions to complete the researches,
Dr. Rintaro Shimada, Dr. Natsumi Noda for exciting scientific talks. My thanks as well go to Dr. Maki
Komiya, Dr. Osamu Takenouchi, Mr. Hiroaki Toyota for daily discussions and helpful suggestions. I
am deeply grateful to Dr. Ayari Takamura, Ms. Aya Yanamisawa, Ms. Qi Dong for sharing their passion
for science. Special thanks to Dr. Genki Kawamura for constant encouragements.
I would like to express my gratitude to Prof. Hirotatsu Kojima, Prof. Takayoshi Okabe, Dr. Naoki
Suto for technical help in developing assay systems. I am truly grateful to Dr. Rinshi S. Kasai, Dr.
Takahiro K. Fujiwara for cheerful discussion and technical help in performing single molecule imaging.
My deep appreciation goes to Prof. Carsten Hoffmann, Prof. Martin J. Lohse for providing me precious
opportunity to join in their laboratory for three months in Würzburg. I owe an important debt to Prof.
Riichiro Abe for generous support to complete the present thesis.
Without their technical and heartful supports, I would not complete the present thesis.
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