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Table 1 Functional properties of wild-type M3R and M3R-luciferase fusion proteins.

From: Luciferase activity under direct ligand-dependent control of a muscarinic acetylcholine receptor

 

luciferase activity

IP accumulation (fold over GFP basal)

Cell surface expression

mutant

basal (% of M3R-luci)

100 μM CCh (% of basal activity)

100 μM atropine (% of basal activity)

basal

100 μM CCh

(% of M3R-luci)

M3R

0.08 ± 0.03 (3)

-

-

2.93 ± 0.37

12.1 ± 2.0

229 ± 13

M3R-luci

100 (31)

105 ± 5

84.6 ± 3.5**

1.60 ± 0.15

10.6 ± 0.2

100

#1

71.2 ± 0.7 (3)

103 ± 9

74.8 ± 9.3

1.40 ± 0.12

9.60 ± 0.45

74.7 ± 5.3

#2

42.8 ± 4.8 (3)

116 ± 5

93.0 ± 4.2

1.20 ± 0.12

4.37 ± 0.09

56.1 ± 4.1

#3

29.8 ± 3.0 (4)

91.0 ± 11.0

95.2 ± 10.9

0.93 ± 0.03

1.57 ± 0.12

32.8 ± 5.3

#4

16.6 ± 3.0 (3)

110 ± 3.8

104 ± 6.8

0.97 ± 0.03

1.10 ± 0.06

13.5 ± 0.7

#5

11.1 ± 1.5 (3)

104 ± 10

96.0 ± 21.2

0.97 ± 0.09

1.10 ± 0.06

3.9 ± 0.9

#6

95.7 ± 5.5 (20)

94.0 ± 7.1

75.7 ± 5.2**

1.27 ± 0.09

9.50 ± 0.61

105 ± 3.2

#7

42.0 ± 3.7 (11)

95.9 ± 7.5

93.8 ± 5.9

1.00 ± 0.10

3.27 ± 0.17

35.8 ± 4.2

#8

62.6 ± 5.2 (22)

98.5 ± 4.7

60.2 ± 2.8**

0.87 ± 0.03

1.37 ± 0.09

114 ± 7

#9

25.9 ± 3.7 (5)

98.4 ± 8.7

87.3 ± 8.6

1.03 ± 0.09

1.43 ± 0.12

27.3 ± 3.5

#10

37.0 ± 4.0 (5)

95.2 ± 7.3

76.4 ± 4.1*

1.53 ± 0.17

9.67 ± 0.78

31.6 ± 4.4

#11

30.3 ± 3.9 (5)

83.4 ± 7.3

73.8 ± 3.1*

1.40 ± 0.06

8.33 ± 0.54

28.9 ± 4.8

#12

37.2 ± 6.8 (4)

111 ± 17

78.6 ± 10.1

1.43 ± 0.17

9.90 ± 0.70

56.6 ± 7.8

#13

27.8 ± 6.7 (3)

80.2 ± 8.3

76.3 ± 8.5

1.13 ± 0.09

8.87 ± 1.01

36.7 ± 11.6

#14

42.0 ± 11.3 (4)

106 ± 10

85.5 ± 6.4

1.50 ± 0.12

9.57 ± 0.95

39.5 ± 4.3

#15

31.8 ± 6.5 (4)

102 ± 5

84.3 ± 7.4

1.10 ± 0.10

8.80 ± 0.95

25.5 ± 2.5

#16

0.4 ± 0.1 (3)

-

-

1.07 ± 0.09

5.23 ± 0.41

14.6 ± 3.8

#17

0.4 ± 0.1 (3)

-

-

1.00 ± 0.06

4.40 ± 0.45

12.3 ± 5.7

#18

0.5 ± 0.3 (3)

-

-

0.87 ± 0.07

1.80 ± 0.15

5.9 ± 2.5

#19

69.3 ± 7.6 (4)

92.3 ± 16.1

66.4 ± 9.4

1.47 ± 0.19

10.9 ± 1.5

76.6 ± 12.6

#20

19.5 ± 1.3 (4)

99.6 ± 4.5

91.8 ± 9.0

0.93 ± 0.09

4.13 ± 0.12

35.2 ± 2.9

#21

17.8 ± 2.4 (7)

99.5 ± 3.3

96.1 ± 4.1

1.07 ± 0.19

1.03 ± 0.07

6.6 ± 1.6

#22

24.3 ± 10.9 (5)

97.4 ± 4.2

97.2 ± 9.1

1.03 ± 0.07

0.93 ± 0.09

0.6 ± 0.5

  1. To evaluate the functional properties of M3R-enzyme chimeras, luciferase activities, measured in the absence (basal activity) and presence of the indicated ligands, were determined (see Methods). Basal activity is given as percentage of M3R-luci (411,043 ± 95,142 AU). Activity in the presence of ligands is given as percentage of the activity of the individual constructs. Data are presented as means ± S.E.M. of the indicated number of experiments, each carried out in triplicate. For measurement of Gq/phospholipase C activation IP accumulation assays were performed as described under Methods. Basal IP values were determined in control (GFP)-transfected COS-7 cells (716 ± 81 cpm/well). Data (three independent assays performed in triplicate) are given as means ± S.E.M. fold over GFP-transfected IP values. Cell surface expression was assayed with indirect cell surface ELISA. Specific optical density (OD) readings (OD value of HA-tagged construct minus OD value of GFP-transfected cells) are given as a percentage of M3R-luci. The nonspecific OD (GFP) was 0.156 ± 0.02 (set as 0%) and the OD value of M3R-luci was 0.66 ± 0.06 (set as 100%). ELISA data are given as means ± S.E.M. of three independent experiments, each carried out in triplicate.
  2. Significant reduction of the luciferase activity (*p < 0.01; **p < 0.001)
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BMC Biotechnology

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