Establishment of a stable cell clone co-expressing the hY 4 receptor, the chimeric G protein qi5

CHO-K1 cells were transfected with the pcDNA3-hY4 vector. Isolated cell clones resistant to 400 µg/ml G418 were tested for binding of cy5-[K⁴]-hPP. The cell clone CHO-hY4-K13b was found to bind the fluorescent ligand in a concentration dependent manner (Fig. 90).

The bound ligand was displaced by 1 µM hPP as shown in Fig. 90b. However, the cells did not equally express the receptor, which became obvious after incubation with 10 and 20 nM of 10 (Fig. 90a). The CHO-hY4-K13b cells consisted of at least two subpopulations binding the fluorescent ligand to different extents.

Nevertheless, the cells were further transfected with the pcDNA3.1/hygro-qi5 vector.

Selected cell clones were tested for an increase in intracellular calcium upon stimulation with hPP. Injection of 20 nM of hPP led to a weak but distinct increase in fluorescence of fluo-4 loaded CHO-hY4-K13b-qi5-K8 cells (Fig. 91).

Fig. 90: Binding of 10 to CHO-hY4-K13b cells. a: Total binding. b: Unspecific binding in presence of 1 µM hPP.

fluorescence FL-4

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Fig. 91: Calcium response of CHO-hY4 -K13b-qi5-K8 cells after stimulation with 20 nM hPP. Raw data were smoothed with WinMDI and SigmaPlot software.

The cells were further transfected with the pcDNA3.1/zeo-mtAEQ vector. After selection with G418, hygromycin and zeocin, cell clones were isolated and screened for a luminescence signal following receptor activation with 50 nM hPP. The total luminescence light emitted was divided by the intensity of DAPI fluorescence representing the different cell numbers. The result is shown in Fig. 92.

E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12

Fig. 92: Screening of CHO-hY4-K13b-qi5-K8-mtAEQ cell clones.

The cell clone E11 showed the strongest luminescence signal in relation to the cell number. CHO-hY4-K13b-qi5-K8-mtAEQ-E11 cells were further analyzed for their response to peptide agonists in the aequorin assay. As shown in Fig. 93, the courses of the luminescence signals were different from the luminescence signals measured with the CHO-hY2-K9-qi5-K9-mtAEQ-A7 cells (see Fig. 56a). Injection of the cells to the full agonist hPP led to an initial increase of luminescence which was followed by a delayed rise of luminescence (Fig. 93a, red).

Fig. 93: Aequorin assay with CHO-hY4-K13b-qi5-K8-mtAEQ-E11 cells. a: Luminescence signal of hPP (red) and GW1229 (green). b: Dose-response curves of peptide agonists (mean values ± SEM, n=3).

This effect was less pronounced with the partial agonist GW1229 (Fig. 93a, green).

Nevertheless, concentration-response curves for various peptide agonists were constructed with the expected pharmacological rank order concerning the full agonists hPP, rPP and [K⁴]-hPP. The partial agonist GW1229 reached 57 % of the maximal signal elicited by full agonists. The EC50 was with 9.4 nM about 4-fold lower compared to hPP (37.3 nM).

Because the course of the aequorin assay indicated the existence of at least two cell populations with different kinetic responses upon agonist stimulation, the hY4

receptors were labeled by binding to 10 and the receptor distribution was analyzed by flow cytometry.

In fact, as shown in Fig. 94a, the CHO-hY4-K13b-qi5-K8-mtAEQ-E11 cells bound the fluorescent ligand inhomogeneously indicated by the two maxima in the histogram presentation (Fig. 94a). Both maxima were shifted to the right with increasing concentrations of 10, indicating the existence of two cell populations with different receptor expression. Therefore, new subclones were isolated and tested for their binding of 10. The cell clone CHO-hY4-K13b-qi5-K8-mtAEQ-E11-K11 exhibited high and homogeneous binding of the fluorescent ligand (Fig. 94b). The labeled ligands cy5-[K⁴]-hPP and S0586-[K⁴]-hPP could be displaced by 1 µM hPP and Kd values were determined after saturation analysis.

Fig. 94: Binding of cy5-[K⁴]-hPP to CHO-hY4-K13b-qi5-K8-mtAEQ-E11 (panel a) and CHO-hY₄-K13b-qi5-K8-mtAEQ-E11-K11 (panel b) cells.

fluorescence FL-4

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a b

The determined Kd values of 5.62 ± 1.08 nM for 10 and 9.24 ± 1.34 nM for 11 are in good agreement with the Kd values determined with the transduced P388 cells (see section 5.2.3.2). The ligand 10 (10 nM) was used in flow cytometric competition assays and Ki values of peptide ligands were determined. According to the literature, hPP (Ki = 239 ± 36 pM) and GW1229 (Ki = 217 ± 44 pM) showed the highest affinity to the hY4 receptor. Rat PP bound with slightly lower affinity but still in the picomolar range (Ki = 443 ± 85 pM). The peptides [L³¹,P³⁴]-pNPY (Ki = 7.08 ± 1.49 nM), BW1911U90 (Ki = 10.77 ± 1.84 nM) and [hPP^19-23,P³⁴]-pNPY (Ki = 20.07 ± 3.03 nM) bound in the nanomolar range and pNPY showed only low affinity with a Ki value of 365 ± 79 nM.

Fig. 95: Saturation assay with CHO-hY₄-K13b-qi5-K8-mtAEQ-E11-K11 cells and cy5-[K⁴]-hPP (left panel; mean values ± SEM, n=3) and S0586-[K⁴]-hPP (right panel; mean values ± SEM, n=2).

Fig. 96: Flow cytometric binding assay with CHO-hY4 -K13b-qi5-K8-mtAEQ-E11-K11 cells. Displace-ment of 10 nM cy5-[K⁴ ]-hPP by various peptide ligands (mean values ± SEM,n=3-4).

The same isolated cell clone CHO-hY4-K13b-qi5-K8-mtAEQ-E11-K11 was used in an aequorin assay. In order to prevent adsorption of the peptides to the well-plate material, 1 % BSA and 0.1 mg/ml bacitracin were added to the peptide solutions.

5.3.3.2 Aequorin assay with two injectors

After the upgrading of the Tecan Genios Pro™ plate reader with a second injector an additional injection of a triton-X-100 solution was included into the assay design by analogy with the assay described for the hY2 receptor (see 4.2.3.5). The course of the measurement is shown in Fig. 98. The aequorin signal was elicited with increasing concentrations of agonist (hPP) at time point ‘a’. The peak followed by the injection of triton-X-100 (time point ‘b’) decreased with increasing concentrations of hPP as more active aequorin was discharged by the previous aequorin signal.

time [s]

Fig. 97: Aequorin assay with CHO-hY4-K13b-qi5-K8-mtAEQ-E11-K11

Fig. 98: Aequorin assay with CHO-hY4 -K13b-qi5-K8-mtAEQ-E11-K11 cells using 2 injectors.

The cell suspension is injected to the agonist (hPP) solution at time point ‘a’ and the aequorin signal is recorded for 43 s. Injection of 0.1 % triton-X-100 at time point ‘b’ leads to cell lysis and causes consumption of residual active aequorin.

The influence of the postincubation step was analyzed in the next experiment. The cells were loaded with coelenterazine h for 2 h and postincubated for various times before injection to the full agonist hPP or to the partial agonist GW1229.

As shown in Fig. 100, the signals varied broadly when the postincubation step was

omitted. The fractional luminescence elicited by the agonists increased during the measurement tempering the concentration-response curves. Although the EC50

values were constant during the whole experiment (EC50 (hPP) = 6.6 – 8.5 nM and EC50 (GW1229) = 7.3 – 9.0 nM), the maxima of the concentration-response curves increased within the first 3 h after the loading procedure for hPP and GW1229. In agreement with the aequorin assay using CHO-hY2-K9-qi5-K9-mtAEQ-A7 cells (see section 4.2.3.5), reproducible concentration-response curves were obtained after 3 h of postincubation allowing a discrimination between full and partial agonists.

c (peptide) [nM] 8.19 ± 0.56 nM) were slightly lower compared to the ones obtained from the aequorin

c (hPP) [nM]

Fig. 100: Dose-response curves of hPP (a) and GW1229 (b) after various postincubation times.

CHO-hY4-K13b-qi5-K8-mtAEQ-E11-K11 cells were incubated with 2 µM coelenterazine h for 2 h at room temperature, diluted to 5 x 10⁵ cells/ml in loading buffer and postincubated for various periods before injection to the ligands (mean values ± SEM, n=3).

Fig. 99: Concentration-response curves of hPP and GW1229. Cells were postincubated for 3-6 h (mean values ± SEM, n=9-13 in 3 independent experiments).

assay performed with one injector and the triton-X-100 sample as external standard.

The maximal signal elicited with GW1229 is 62 % of the maximal signal induced by the full agonist hPP.