Table 3.
Summary of FCS parameters for Y1-arrestin complexes detected by BiFC.
| BiFC complex | τD1 |
τD2 |
D |
N | |
|---|---|---|---|---|---|
| μs | % | ms | (× 10− 9 cm2 s− 1) | ||
| Y1 A1 | |||||
| Control (3) | 246 ± 20 | 52.1 ± 0.8 | 48.4 ± 3.7 | 1.57 ± 0.13 | 21 |
| NPY (4) | 279 ± 29 | 60.3 ± 1.4 | 72.3 ± 5.6 | 1.33 ± 0.10 | 49 |
| Y1 A2 | |||||
| NPY (9) | 278 ± 17 | 63.3 ± 1.4 | 72.6 ± 6.2 | 1.26 ± 0.08 | 64 |
| Y1 A2Δ | |||||
| Control (4) | 259 ± 7 | 60.0 ± 1.6 | 48.8 ± 2.3 | 1.51 ± 0.08 | 35 |
| NPY (4) | 254 ± 11 | 57.8 ± 3.4 | 77.1 ± 7.0 | 1.20 ± 0.11 | 22 |
Fluorescence fluctuations were recorded (2 × 15 s reads) from dual stable HEK293 cells co-expressing Y1 receptor-Gc and β-arrestin-Gn BiFC partners. The development of BiFC complexes was stimulated by 60 min pre-treatment with 100 nM NPY (see Materials and methods, and Fig. 2). Values in parenthesis beside the treatment conditions indicate the number of experiments (no FCS measurements could be taken under control conditions for Y1 A2 cells), whilst n values denote the number of cell recordings. As before, autocorrelation curves were fitted with a two dimensional diffusional model with two dwell time components and a fluorophore blinking component (Zeiss Aim 4.2 software). Pooled data (mean ± s.e.m.) is given for dwell times τD1 and τD2, the percentage contribution of the τD2 component to the autocorrelation curve amplitude, and derived diffusion co-efficient (D). Y1 A2Δ represents the Y1 A2ΔLIEFD cell line.