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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)