Table I. Binding characteristics of wild type and mutant human A2a adenosine receptors using an agonist radioligand.
Data are presented as means ± S.E. of two or three independent experiments, each performed in duplicate. Each sample contained 7–11 µg of membrane protein/tube.
| [3H]CGS 21680 bindinga | Competitor | |||||||
|---|---|---|---|---|---|---|---|---|
| Mutantb | Kd | Bmax | Agonists | Antagonists | ||||
| CADO | DPMA | NECA | R-PIA | CGS15943 | XAC | |||
| nm | pmol/mg | nm | nm | |||||
| wt (Tag3) | 21.7 ± 2.6 | 34.5 ± 20.0 | 152 ± 10 | 244 ± 43 | 19.9 ± 8.3 | 318 ± 8.0 | 10.1 ± 2.0 | 63.2 ± 5.5 |
| F180A | 15.1 ± 1.3 | 9.6 ± 0.8 | NDc | 104 ± 19 | 46.0 ± 11.1 | ND | 13.7 ± 3.2 | 90.3 ± 7.2 |
| N181S | 24.8 ± 5.6 | 21.9 ± 0.1 | 1340 ± 200 | 981 ± 52 | 16.4 ± 2.5 | 4500 ± 1200 | 23.7 ± 2.0 | 130 ± 10.0 |
| F182Y | 57 ± 15 | 8.9 ± 1.0 | 1890 ± 170 | 1720 ± 98 | 170 ± 11 | 2800 ± 1100 | 19.2 ± 4.2 | 140 ± 20.1 |
| F182w | 66 ± 6 | 7.4 ± 3.0 | 776 ± 120 | 756 ± 102 | 144 ± 12 | 1600 ± 460 | 10.3 ± 3.3 | 66.8 ± 1.0 |
| H250F | 14.4 ± 4.0 | 3.8 ± 1.8 | 2130 ± 100 | 1650 ± 107 | 41.9 ± 12 | 7950 ± 1750 | 25.7 ± 5.0 | 60.3 ± 3.0 |
| H250Y | 14.9 ± 1.0 | 5.2 ± 0.5 | 8250 ± 1400 | 3000 ± 80 | 43.8 ± 16 | 9580 ± 280 | 8.4 ± 1.0 | 52.7 ± 11 |
| C254A | 23.7 ± 2.4 | 11.8 ± 0.6 | ND | 99.6 ± 14.0 | 18.2 ± 10.3 | ND | 19.4 ± 3.4 | 54.1 ± 8.1 |
| S277N | 25.0 ± 5.0 | 23.4 ± 3.0 | 221 ± 21 | 311 ± 32 | 43.0 ± 17.1 | 631 ± 329 | 12.8 ± 2.8 | 60.0 ± 17.4 |
| S277T | 23.6 ± 6.3 | 27.3 ± 3.2 | 201 ± 5.0 | 305 ± 20 | 39.8 ± 6.8 | 534 ± 152 | 12.2 ± 1.0 | 65.9 ± 2.2 |
| S281T | 17.1 ± 2.0 | 10.2 ± 1.0 | 13.7 ± 1.3 | 80.5 ± 22 | 6.6 ± 0.8 | 30.5 ± 8.6 | 15.6 ± 3.4 | 17.3 ± 1.6 |
Agonist and antagonist binding affinities (Ki values, structures in Jacobson et al. (1992)) were determined in [3H]CGS 21680 (15 nm) competition binding studies using membrane homogenates prepared from transiently transfected COS-7 cells, as described under “Experimental Procedures.” Ki values were calculated from IC50 values by using the GraphPad program. All constructs contain the Tag3 sequence at the NH2 terminus (Fig. 3A).
Constructs that showed that <2% of specific binding of [3H]CGS21680 (15 nm) found for wild type Tag3 receptors were: F182A, H250A, N253A, N253S, N253Q, F257A, Y271A, I274A, S277A, H278A, H278Q, H278F, H278Y, S281A.
ND, not determined.