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

Additional file 1:

Description of control experiments. This file summarises all control experiments carried out.

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Additional file 2:

Luciferase can be integrated into other GPCR (e.g. V₂R) without loosing G protein-coupling abilities. COS-7 cells were transfected with wild type V₂R and V₂R-luci. 48 h after transfection cells were incubated with 100 nM arginine-vasopressin (AVP). Cyclic AMP levels were determined using the non-radioactive cAMP-determination kit (AlphaScreening technology, PerkinElmer). The cAMP level (atmol/cell) of two independent experiments performed in triplicate is given (means ± S.E.M.).

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Additional file 3:

M₃R ligands have no effect on soluble luciferase and V₂R-luciferase fusion protein. COS-7 cells were transfected with luciferase (A) and V₂R-luci (B). Increasing concentrations of CCh (square), atropine (open circle), scopolamine (filled circle) and butylscopolamine (diamond) were applied and luciferase activity was determined as described under Methods. The enzyme activities without ligands were 1,905,212 ± 172,463 AU (luciferase), 182,120 ± 18,188 AU (V₂R-luci). Enzyme activity of the individual constructs without ligands was set 100%. All data are given as mean ± S.E.M. of three independent experiments each performed in triplicate.

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Additional file 4:

Modulation of enzyme activity in M₃R-luciferase fusion proteins by different receptor ligands. COS-7 cells transfected with M₃R-luci and construct #8 were stimulated with the indicated ligands (100 μM) and luciferase activity was determined. The luminescence of the fusion constructs were 184,314 ± 75,049 AU (M₃R-luci) and 140,452 ± 55,426 AU (construct #8). Enzyme activity of the individual constructs without ligands were set 100%. All data are given as mean ± S.E.M. of four independent experiments each performed in triplicate.

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Additional file 5:

Primer used in this study. This table listes all primers used to generate the mutants of the M₃R-luciferase fusion protein.

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Additional file 6:

Kinetics of luciferase activity assay. Cells were transfected with M₃R-luci and luciferase and the assay was performed as described in the Method section with the exception that the luciferase buffer was added at different incubation times after lysis. Data are given as mean ± S.D. of one experiment performed in triplicate.

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