Functional integration of luciferase into the M₃R

To generate a biosensor in which enzyme activity is allosterically controlled by a binding domain of a GPCR, we replaced a 195-amino acid segment of the third ICL (ICL3) of M₃R (amino acid positions 274–469) with the sequence of luciferase from Photinus pyralis (referred to as M₃R-luci, Figures 1 and 2). Previous studies in mammalian expression systems have shown that in M₃R, the removal of the central part of ICL3 has no significant effect on receptor function [29–31]. As shown in Figure 3 and summarized in Table 1, M₃R-luci expressed in COS-7 cells was delivered to the cell surface and displayed both luciferase activity (Figure 3A) and carbachol (CCh)-induced inositol phosphate (IP) formation (Figure 3B). However, enzyme activity was not influenced by application of the agonist CCh but we noted a significant reduction (15%, p < 0.001) in luciferase activity in the presence of the inverse agonist atropine (Figure 3A, Table 1). The effect of atropine was not found when luciferase was inserted into the second intracellular loop (ICL2) or fused to a truncated M₃R (constructs #21 and #22, Figure 2, Table 1) and in several other controls (luciferase alone, V₂ vasopressin receptor (V₂R)) [see Additional file 1] [see Additional file 2] [see Additional file 3].

	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

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 G_q/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.

Significant reduction of the luciferase activity (*p < 0.01; **p < 0.001)

Optimization of the M₃R-luciferase fusion protein

Next, we systematically deleted portions of ICL3 flanking the enzyme (Figure 2) to improve coupling of atropine-induced conformational changes to enzyme activity. Successive N-terminal shortening of ICL3 (constructs #1-#5) progressively reduced cell surface expression, ligand-induced IP formation and luciferase activity but atropine failed to modulate luciferase activity. Likewise, the C-terminal part of ICL3 was shortened (constructs #6-#8). Constructs #6 and #8 were properly delivered to the cell surface and displayed high luciferase activity. Strikingly, both chimeras showed a significant reduction in enzyme activity up to ~40% (construct #8, Table 1) upon atropine binding. G-protein signalling was retained in construct #6 but abolished in construct #8, presumably because the deleted C-terminal part of ICL3 is involved in G-protein coupling [32]. The combination of N- and C-terminal shortening (construct #9) was not advantageous.

We then tested whether N- or C-terminal truncation of the luciferase insert improved coupling to conformational changes of the receptor. Only deletion of the very N-terminal two amino acid residues (construct #10) had a significant effect on atropine-induced reduction of the enzyme activity (Table 1). Combination with ICL3 shortening (construct #11) did not further improve allosteric modulation of the enzyme activity by atropine. In agreement with previous studies [33], further removal of the N terminus abolished enzyme activity completely (Table 1, constructs #12-#18) probably because of destruction of the functionally relevant N-terminal domain. The crystal structure of Photinys pyralis luciferase demonstrates that N-terminal and C-terminal domains form the active site [34]. C-terminal truncation of the enzyme did not result in significantly reduced enzyme activity after atropine application, neither alone (construct #19) nor in combination with ICL3 truncation (construct #20). Preliminary studies with fusion proteins of other GPCR and reporter proteins indicate that integration and optimization is required for every individual biosensor (data not shown).

Allosteric modulation of luciferase activity by M₃R blockers

Finally, we investigated the specificity and potency of other M₃R blockers in M₃R-luci constructs. Scopolamine and butylscopolamine, both are inverse agonists at M₃R, were most efficient in reducing luciferase activity in construct #8 [see Additional file 4]. IC₅₀ values for atropine and scopolamine were 6.6 ± 2.3 nM and 2.1 ± 0.4 nM, respectively (Figure 4). In agreement with functional studies at the wild-type M₃R [35], butylscopolamine was less potent in luciferase inhibition (IC₅₀ value: 1.7 ± 0.5 μM, Figure 4). This indicates an unchanged pharmacology of the ligand-binding domain within the M₃R-luciferase fusion protein.

From the constructs tested, construct #8 met best the criteria of a biosensor, including nanomolar ligand sensitivity, high cell surface expression, and reduced G protein-coupling (Figures 3 and 4, Table 1). In this construct, structural optimization was successfully used to enhance coupling of ligand-induced rigid body movement of the TMH to structural changes within the luciferase domain. As this leads to a reduction in luciferase activity, one can speculate that the underlying conformational change rearranges the orientation of the N- and C-terminal domains within the luciferase molecule that form its active centre. In addition, our data support previous findings [18, 19] that structural changes upon inverse agonist binding are different from those induced by agonists. Following CCh stimulation, fusion protein #6 still activated the G_q/phospholipase C pathway but CCh had no influence on luciferase activity (Table 1). However, atropine reduced luciferase activity by 25% in this construct. It is interesting to note that insertion of luciferase, which itself is bigger than the receptor protein, into the cytoplasmic surface of the GPCR still allows for efficient G-protein coupling. This implicates a rather small interaction site between the receptor and the G protein.

Materials

The agonist carbamylcholine chloride (carbachol, CCh) and the inverse agonists [36] atropine sulfate, scopolamine hydrochloride, and n-butyl scopolamine bromide were obtained from Sigma. Substances were solved in water and stock solutions (100 mM) were aliquoted and stored at -20°C. Aliquots were thawed only once. All restriction enzymes for cloning purposes were purchased from NEB and primers [see Additional file 5] were synthesized by Invitrogen.

Construction of plasmids and mutants of mAChR

All mutations were introduced into the rat M₃R [30]. In this rat M₃R construct the central part of ICL3 was removed and the N- and C terminus contained an HA- and a Flag-tag, respectively. These modifications were previously demonstrated to have no significant effect on receptor function [29–31].

The cDNA from Photinus pyralis luciferase (without start and stop codons) was amplified and introduced into the ICL3 of M₃R by a PCR-based strategy. Luciferase cDNA was 5' and 3' flanked by SpeI sites which allowed for systematic and convenient shortening of ICL3 during optimization experiments. All other mutations were introduced by PCR-based and fragment replacement strategies into M₃R-luci (Figure 1 and 2). For control purposes, several additional constructs were generated. Here, luciferase was integrated into ICL2 (Figure 2, construct #21) or C-terminally fused to M₃R that was truncated in ICL3 (Figure 2, construct #22). Moreover, luciferase was also integrated into V₂R lacking the central portion of ICL3 (positions R²⁴³ – T²⁵³). The identity of all constructs and the correctness of all PCR-derived sequences were confirmed by restriction analysis and DNA sequencing.

COS-7 cell culture and transfection

COS-7 cells were grown in Dulbecco's modified Eagle medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified 7% CO₂ incubator. Rotifect (Roth) was used for transient transfection. For measurement of luciferase activity, 6 × 10⁵ cells were seeded into 6-cm dishes and transfected with 2 μg of plasmid DNA. Twenty-four hours after transfection, cells were split into white 96-well culture plates (PerkinElmer) at 30,000 cells per well. IP formation was determined in 12-well plates (1.5 × 10⁵ cells/well transfected with 0.5 μg of plasmid DNA/well). For the measurement of cell surface expression, cells were seeded into 48-well plates (3.5 × 10⁴ cells/well) and transfected with 0.2 μg of plasmid DNA/well.

Functional assays

To measure IP formation transfected COS-7 cells were incubated with 2 μCi/ml of myo-³H-inositol (18.6 Ci/mmol, PerkinElmer) for 18 h. Thereafter, cells were washed once with serum-free DMEM containing 10 mM LiCl, followed by incubation with ligands for 30 min at 37°C. Intracellular IP levels were determined by anion-exchange chromatography as described previously [37].

The luciferase activity assay was performed with a luciferase activity detection system based on the method described by van Leeuwen et al. [38]. Briefly, after incubation with the ligand in DMEM for 20 min at 37°C, cells were washed with ice-cold PBS. Cell lysis was performed on ice by adding 20 μl of lysis buffer (77 mM K₂HPO₄, 23 mM KH₂PO₄, 0.2% Triton X-100, 1 mM DTT, pH 7.8) per well. The lysate was mixed with 100 μl of luciferase buffer (20 mM tricine, 2.67 mM MgSO₄, 0.1 mM EDTA, 33.3 mM DTT, 270 μM coenzyme A, 530 μM ATP, 470 μM d-luciferin, pH 7.8). After 3 min luminescence was measured for 1 sec with a Victor²-1420 Multilabel counter (PerkinElmer). Prior to data-collecting functional assays, several tests were performed to ascertain the adequate time for cell lysis and incubation with luciferase buffer. Since both buffers did not contain ligands, diffusion from the receptor was possible. The ligand-induced changes in luciferase activity were stable for at least 10 min after cell lysis [see Additional file 6].

To estimate receptor surface expression, we used an indirect cellular ELISA [39]. Three days after transfection cells were fixed with 4% formaldehyde in PBS for 30 min at room temperature. After washing with PBS and blocking with 10% FBS in DMEM at 37°C for 1 h cells were incubated with biotinylated anti-HA-antibody (Roche, 1 μg/ml in DMEM with 10% FBS) at room temperature for 2 h. Plates were washed and incubated with streptavidin-horse-radish peroxidase conjugate (Roche, 1:5000 dilution in DMEM with 10% FBS) at room temperature for 1 h, followed by extensive washing. Enzymatic reactions were carried out at room temperature in the presence of H₂O₂ and o-phenylenediamine. The reaction was stopped by adding 50 μl of 50 mM Na₂SO₃ in 1 M HCl. Colour development was measured bichromatically at 492 nm and 620 nm using a Sunrise™ plate reader (Tecan).

A paired, two-tailed Student's t-test was used to detect significant differences in luciferase activity after ligand binding. The number of replicates is given in the respective legends of the figures and table.