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File S1: Supporting Information on line PLOS ONE
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Characterisation of AmphiAmR11, an amphioxus (Branchiostoma floridae) D2-dopamine-
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like G protein-coupled receptor
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By
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Asha L. Bayliss and Peter D. Evans
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Peter.evans@babraham.ac.uk
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Supplementary Figures S1-S6
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Supplementary Figure Legends Bayliss and Evans
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Figure S1. Effect of biogenic amines (A) and synthetic agonists (B) on forskolin-
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stimulated cAMP levels in AmphiAmR11-expressing CHO-K1 cells. Cells were pre-
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incubated with 100 µM IBMX for 20 min, followed by incubation with 10 µM forskolin and
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100 µM IBMX in the presence of 1 µM biogenic amines (A) or synthetic agonists (B) for a
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further 20 min. Data are expressed as the mean + SEM. n ≥ 3.
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Figure S2. Effect of yohimbine and WB4101 on tyramine-induced decreases in
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forskolin-stimulated cAMP levels in AmphiAmR11-expressing CHO-K1 cells. Cells were
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pre-incubated with 100 µM IBMX in the presence or absence of varying concentrations of
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the antagonists, yohimbine (A) or WB4101 (B), for 20 minutes, followed by incubation with
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either varying concentrations of antagonist (black bars) or 30 nM tyramine plus varying
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concentrations of antagonist (striped bars) in the presence of 10 µM forskolin and 100 µM
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IBMX for a further 20 minutes. The basal value in the absence of agonist and antagonist
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(grey bar) and the tyramine-only value (open bar) are shown. Data are expressed as the mean
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+ SEM. n = 3. Yoh, yohimbine; TA, tyramine; WB, WB4101.
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Figure S3. Time courses for the effect of dopamine and tyramine on ERK1/2
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phosphorylation in AmphiAmR11-expressing CHO-K1 cells. (A and B) AmphiAmR11-
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expressing CHO-K1 cells were serum-starved for 2 hours, prior to stimulation with tyramine
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or dopamine at the specified concentration for the specified length of time. Equal quantities
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of cell lysates were separated by SDS-PAGE and analysed for pERK or tERK by Western
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blotting. (C and D): AmphiAmR11-expressing CHO-K1 cells were incubated with pertussis
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toxin (200 ng/mL) for 16 hours followed by stimulation as described for Figures A and B. (A
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to D) Representative blot for each condition from three to four independent experiments. (E)
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Summary of the quantified blots. Data are expressed as the mean ± SEM. n = 3-4. PTX,
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pertussis toxin; Min, minutes.
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Figure S4. The relative effectiveness of the biogenic amines and synthetic agonists on
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ERK1/2 activation in AmphiAmR11-expressing CHO-K1 cells. Cells were serum-starved
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for 2 h, prior to stimulation with 10 nM biogenic amines (A and B) or 100 nM synthetic
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agonists (C and D) for 5 min. Equal quantities of cell lysates were separated by SDS-PAGE
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and analysed for p-ERK or t-ERK by Western blotting. (A and C) Representative blots from
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three to four independent experiments. (Band D) Summary of the quantified blots. Data are
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expressed as the mean + SEM.
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Phenylethylamine, NA, noradrenaline; Adr, adrenaline; OA, octopamine; Syn, synephrine;
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His, histamine; Nap, naphazoline; Phe, phenylephrine; UK, UK14,304; Clo, clonidine; Iso,
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isoproterenol; SKF, SKF38393; 6-C, 6-Chloro-APB; Qui, quinpirole.
n = 3-4. DA, Dopamine; TA, tyramine; Phen,
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Figure S5. Calcium mobilisation in AmphiAmR11-expressing and wild type CHO-K1
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cells. (A) Representative traces for agonist-induced calcium mobilisation in AmphiAmR11-
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expressing cells. During agonist stimulation, some cells in a field of view coupled to calcium
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mobilisation (represented by Cells 1-3), while others did not (represented by Cell 4). (B)
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Wild type CHO-K1 cells were stimulated with various biogenic amines at 10 µM for 8
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minutes followed by stimulation with adenosine trisphosphate (ATP) at 10 µM for 5 minutes.
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n ≥ 3.
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Figure S6. Localization of AmphiAmR11 in CHO-K1 cells. AmphiAmR11-expressing
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CHO-K1 cells were fixed with 4% paraformaldehyde and stained with an anti-V5-FITC
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antibody (green). Nuclei were stained with DAPI (blue). All cells from the clonal cell line
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used in the present study showed strong staining in the periphery of the cells consistent with
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its function as a biogenic amine receptor since biogenic amines are cell membrane
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impermeable. Additional staining was observed particularly in the perinuclear region of the
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cells. Wild type cells showed no staining.
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TA
(
(1 100
00
n
nM M)
)+
W
TA
W
B
B
(1
(1
0
0
n
nM M
)
)
+
TA
0
B
96
20
B
95
40
W
94
60
)
93
80
)+
92
100
W
91
120
M
90
A
M
89
Biogenic Amine (1 M)
(1
82
TA
88
% Forskolin-stimulated Basal cAMP
0
(1
86
20
B
81
40
W
80
60
B
78
80
W
77
ap
Ph haz
en oli
n
yl
ep e
hr
in
U
e
K
14
,3
40
C
l
Is oni
d
op
ro ine
te
re
SK n o
F3 l
683
C
hl
or 93
oQ AP
B
ui
np
iro
le
76
100
B
87
A
N
75
% Forskolin-stimulated Basal cAMP
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Y
TA
Yo oh
(
h
(1 1 
M M)
)+
Y
Yo oh
TA
(
h
(1 100
00
n
nM M)
)+
Y
Yo oh
TA
h
(1
(1
0
0
n
nM M)
)
+
TA
73
B
72
% Forskolin-stimulated Basal cAMP
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op
N
a
or
ad min
re
e
na
li
A
dr ne
en
al
Ty ine
ra
O
ct min
op
e
am
in
Ph Sy
en nep e
yl
hr
et
hy ine
la
m
i
H
is ne
ta
m
in
e
71
D
70
% Forskolin-stimulated Basal cAMP
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Bayliss and Evans Figure S1
B
120
100
80
60
40
20
0
Synthetic Agonist (1 M)
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Bayliss and Evans Figure S2
B
120
100
80
60
40
20
0
102
103
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Bayliss and Evans Figure S3
A
C
Tyramine 1 µM
Tyramine 1 µM + PTX
Time (Min):
Time (Min):
0
1
2
5
10
15
20
0
1
2
5
10
15
20
30
30
106
107
p-ERK
p-ERK
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t-ERK
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t-ERK
B
D
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Dopamine 30 nM
Time (Min):
0
1
2
5
10
15
Dopamine 30 nM + PTX
20
30
Time (Min):
p-ERK
p-ERK
t-ERK
t-ERK
0
1
2
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E
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22
119
Tyramine 1 M
Tyramine 1 M + PTX
Dopamine 30 nM
Dopamine 30 nM + PTX
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120
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125
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ERK1/2 Phosphorylation (Fold Increase)
121
16
14
12
10
8
6
4
131
2
132
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0
0
5
10
15
20
Time (Min)
25
30
5
10
15
20
30
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Bayliss and Evans Figure S4
A
C
Amine:
B
DA
TA
Phen NA Adr
OA Syn His
Agonist:
B DA Nap Phe UK Clo
Iso SKF 6-C Qui
p-ERK
p-ERK
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t-ERK
t-ERK
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154
% Dopamine Response
60
40
Biogenic Amine (10 nM)
is
H
Sy
n
A
D
-20
A
0
152
153
80
20
O
151
20
dr
150
40
A
149
60
A
148
80
N
147
100
Ph
en
146
120
100
% Dopamine Response
145
D
120
0
D
A
N
ap
Ph
e
U
K
C
lo
Is
o
SK
F
6C
Q
ui
B
143
TA
142
Synthetic Agonist (100 nM)
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Bayliss and Evans Figure S5
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A
200
% Change in Ratio (F340/F380)
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Agonist
Buffer
Cell 1
Cell 2
Cell 3
Cell 4
150
100
50
0
1
2
3
Time (Mins)
4
5
-50
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169
170
B
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350
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175
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177
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180
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183
300
% Change in Ratio (F340/F380)
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Tyramine
Dopamine
Noradrenaline
Octopamine
Adrenaline
250
200
150
100
50
0
2
184
185
186
187
4
-50
Buffer
Amine (10 M)
6
8
10
Time (Mins)
12
ATP (10 M)
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16
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Bayliss and Evans Figure S6