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Supplementary Fig. 1 | Comparison of SDS-PAGE migration profiles of WT Gpr176
and mutants containing a single Asn (N) to Gln (Q) substitution of the potential Nterminal glycosylation sites. The WT Gpr176 and respective N4Q, N11Q, N17Q, and
N26Q mutants were separately expressed in Flp-In TREx293 cells and subjected to SDSPAGE/immunoblot analysis with anti-Gpr176 antibody. A longer exposure of the same
immunoblot is shown in lower panel. A dashed white horizontal line is overlaid on the
high exposure images to emphasize the differences in size between WT and mutants. The
WT Gpr176 was recognized as a broad band with a molecular weight slightly higher than
75 kDa. The broadness of the band implies N-linked glycosylation heterogeneity. All
tested mutants exhibited a small but noticeable change in mobility, with N4Q and N26Q
evoking larger mobility changes than N11Q and N17Q. These results suggest that all four
N-terminal consensus sites under N-glycosylation.
30
Supplementary Fig. 2 (related to Fig. 3) | N-glycosylation deficiency leads to
decreased expression of GFP-fused Gpr176. Flp-In TREx293 cells expressing either
WT or N-ter mut Gpr176-GFP fusion protein were immunoblotted for Gpr176 (upper)
and β-actin (lower). The protein extracts from N-ter mut cells were loaded five-fold over
WT cells to increase the sensitivity of protein detection.
31
Supplementary Figure 3 (related to Fig. 3) | Partially overlapped localization of Nter mut Gpr176 and calnexin in Flp-In TREx293 cells. Cells expressing N-ter mut
Gpr176-GFP (green) were immunolabeled for calnexin (red). Calnexin is an ER-resident
lectin chaperone that binds with N-linked oligosaccharides containing terminal glucose
residues. Merged image is a combined image of GFP and calnexin with DAPI (blue).
Cells are representative of a population with independent experiments. Scale bars, 10 μm.
Note that in the ER, calnexin (red) and N-ter mut Gpr176 (green) were not completely
overlapped, likely consistent with the idea that N-ter mutation of Gpr176 results in the
loss of N-glycans to which calnexin binds.
32
Supplementary Fig. 4 (related to Fig. 3) | Increased level of glycosylation-deficient
Gpr176 by treatment with MG132 but not with bafilomycin A1. In this assay, I
analyzed non-tagged Gpr176 (i.e., no C-terminal GFP). Flp-In TREx293 cells that
express N-glycosylation deficient Gpr176 (i.e., N-ter mut) were treated with either
MG132 or bafilomycin A1 at the indicated concentrations for 6 hours and immunoblotted
for Gpr176 (upper) and β-Actin (lower). Arrowhead indicates the position of nonglycosylated Gpr176. Asterisk, a non-specific band (see Fig. 2B).
33
Supplementary Fig. 5 (related to Fig. 4) | Comparable basal GloSensor levels
between Dox-treated (+) and untreated (−) cells. The GloSensor system relies on the
biosensor expression. To be sure that the intracellular functional GloSensor level does not
differ between Dox-treated and non-treated cells, I verified whether their cell lysates are
able to show similar GloSensor activities when incubated in vitro with fixed amounts of
cAMP. The cells were lysed with or without (w/o) standard cAMP (final conc.: 1 or 4
µM) in buffer containing D-luciferin, Mg2+, and ATP. Luminometry showed that both
cell lysates exhibit equivalent dose-dependent GloSensor activities, providing evidence
that Dox treatment does not affect GloSensor expression in the cell. Data shown are
biological replicates (n = 2, for each condition).
34
Supplementary Fig. 6 (related to Fig. 4) | Correlation between Gpr176 protein
expression level and its cellular cAMP-repressing activity. (A) Immunoblot of Flp-In
TREx293-Gpr176 (tet-on) cells. Cells were treated with increasing doses of Dox (0. 1, 5,
10, 102, and 103 ng ml−1) and immunoblotted for Gpr176 (upper) and β-actin (lower).
Asterisk, nonspecific bands. (B) GloSensor activity traces of Flp-In TREx293-Gpr176
(tet-on) cells. Cells were treated with increasing doses of Dox (0. 1, 5, 10, 102, and 103
ng/mL). For comparison, GloSensor traces of Dox untreated cells (i.e., 0 ng ml−1) are
displayed in parallel. Data represent three independent biological replicates per condition.
RLU, relative light units. (C) Relative protein levels of Gpr176 dependent on Dox doses.
Values are the means ± s.d. (n = 3 for each) of the relative band intensities in (A). *P <
0.05, ***P < 0.005, versus Dox-untreated cells, one-way ANOVA with Bonferroni post
hoc test. (D) Relative area under the curve (AUC) of luminescence values in (B). Light
emissions were integrated and normalized with those of the untreated control. **P <
0.001, ****P < 0.0001, versus Dox-untreated cells, one-way ANOVA with
Bonferroni post hoc test. (n = 3 for each condition). Error bars indicate s.d. (E)
Relationship between protein expression level (x axis) and inhibition activity (y axis) of
Gpr176 in cells treated with different doses of Dox. Values are the means ± s.d. (n = 3 for
each). Correlation coefficient (r) and P value were calculated by Pearson product moment
correlation coefficient analysis (r = 0.9920, P<0.01). The linear regression equation is
shown at the top of figure.
35
Chapter 2
Identification of regulation of Gpr176 activity via
phosphorylation by T-207219 and other non-orphan GPCRs
Gpr176 is an SCN-enriched orphan receptor that is able to set the pace of circadian
behavioural activity rhythm. Therefore, this orphan receptor may be a promising therapeutic
target for circadian disorders such as insomnia and lifestyle-related diseases. However,
biochemical features that control the activity of Gpr176 are still largely unknown. In
Chapter 1, I showed that Gpr176 undergoes N-glycosylation, which is required for proper
cell-surface expression of Gpr176. However, N-glycosylation itself is dispensable for
Gpr176 to keep its basal (constitutive) activity. In this chapter, using a high-throughput
chemical library screening, I identified T-207219 as a modulator for Gpr176 basal activity.
Phosphorylation is a key regulatory post-translational modification described for GPCRs.
However, much less is known about phosphorylation and its potential function(s) in orphan
GPCRs. I found that T-207219 can induce phosphorylation at two separate conserved serine
residues in the C-terminal region of Gpr176, causing receptor internalization. Moreover,
using a receptor co-expression system, I obtained evidence that ligand-mediated activation of
non-orphan GPCRs leads to phosphorylation of the same residues in Gpr176. Induced
phosphorylation, importantly, caused reduced cell-surface expression and second messenger
signaling of Gpr176. My data therefore identified a previously uncharacterised inter-GPCRs
mechanism in which the orphan receptor is regulated by non-orphan receptors through
phosphorylation.
36
ACKNOWLEDGEMENTS
First and foremost, I would like to express my sincere gratitude to Prof. Masao Doi and Prof.
Hitoshi Okamura for accepting me as a member of their laboratory and giving me the
opportunity to meet many wonderful people in this world of chronobiology.
In particular, I am deeply grateful to my direct supervisor, Prof. Masao Doi, for his patient
guidance, warm encouragement, valuable discussions, and all kinds of advice in research and
life. Under Prof. Doi 's instruction, I have learned how to work independently, think logically
and solve difficulties in my research. I would not have been able to complete my Ph.D. course
without his continuous efforts.
I would like to extend my sincere thanks to Associate prof. Akira Hirasawa from the
Department of Genomic Drug Discovery Science for his insightful comments in cell line
preparation and Associate prof. Naoyuki Sugiyama from Laboratory of Molecular Systems
BioAnalysis for his suggestions and technical assistance in mass spectrometry.
I am grateful to all the members of Doi’s lab for their help in life and experiments, which
are lovely memories that I treasure. I would like to especially thank Dr. Iori Murai, Dr. Yukari
Takahashi, Mr. Genzui Setsu, Mr. Shumpei Nakagawa, Mr. Kaoru Goto and Ms. Kaho Tanaka
for their technical contributions in this study.
I would like to thank the Monbukagakusho Honors Scholarship, the AEON Scholarship,
the Kyoto University Doctoral Program (KSPD), the Fujitajinsei Scholarship generously
established by Prof. Emeritus Tetsuro Fujita and his family, and the Japan Society for the
Promotion of Science for financial support during my 10 years of study in Japan. I also thank
the Kyoto International Community for helping me with general advice on living in Japan.
To my dearest friends, Dianhui Wei, Shuheng Yan, Yalin Zhang, Chunmei Zou, Jiachen
Wang, and Yue Song, your companies made me alive.
Last but not least, I would like to express my gratitude to my parents for their unselfish
love and continuous support during my doctoral study and my life in general. Writing this
paper is the hardest thing I have ever encountered, and I cannot overcome this without them.
Thank for raising me and giving me the strength to go forward fearlessly.
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