Ligand binding profiles of WT β2-AR and various chimeras
| β2-WT | EX2 | EX3 | EX2+3 | EX1+2+3 | |
|---|---|---|---|---|---|
| β-AR Agonists | |||||
| Epinephrine | 6.85 ± 0.1 (.6) | 6.79 ± 0.23 (.6) | 7.88 ± 0.06 (.3) | 7.75 ± 0.17 (.4) | 7.94 ± 0.04 (.4) |
| Isoproterenol | 7.48 ± 0.1 (.6) | 7.79 ± 0.1 (.6) | 8.53 ± 0.03 (.4) | 8.65 ± 0.13 (.35) | 8.61 ± 0.19 (.35) |
| Albuterol | 6.39 ± 0.1 (.5) | 7.22 ± 0.09 (.37) | 7.62 ± 0.37 (.4) | ||
| β-AR Antagonists | |||||
| ICI 118551 | 8.55 ± 0.19 (.7) | 8.27 ± 0.27 (.6) | 8.34 ± 0.2 (.6) | ||
| Alprenolol | 9.15 ± 0.51 (.6) | 9.30 ± 0.13 (.6) | 9.05 ± 0.25 (.6) | ||
| B max (pmol/mg) | 0.140 ± 0.02 | 0.100 ± 0.01 | 0.050 ± 0.01 | 0.040 ± 0.01 | 0.050 ± 0.004 |
| K D(pm) [125I]CYP | 40.1 ± 4.9 | 64.7 ± 6.5 | 51.9 ± 6.7 | 36.9 ± 5.4 | 60.2 ± 8.1 |
Competition binding studies were used to determine the IC50(−Log) values (mean ± standard error) of adrenergic ligands, as described in Experimental Procedures, using [125I]CYP as the radioligand and membranes prepared from COS-1 cells expressing WT β2-AR or chimeras. Receptor densities (B max) were determined on the same membranes from equilibrium saturation studies. Bold values indicate significant increases (p < 0.05–0.001) in affinity from the corresponding WT β2-AR (one-way ANOVA followed by Student-Newman-Keuls multiple comparison test). Numbers in parentheses represent Hill coefficients. n ≥ 3/each group. EX2, EX3, and EX2+3 are β2-ARs with the corresponding EX replaced with the α1a-AR sequence. EX1+2+3 is the β2-AR with all three EX replacements.