TABLE 4

Affinity (log KD values) of β-adrenoceptor ligands for the wild-type β-AR and receptors containing single and double point mutations in the extracellular loops in transiently transfected populations of cells

The KD value of [3H]CGP 12177 and the receptor expression level in these transient populations are also given. The values are mean ± S.E.M. for n separate experiments, and each separate n number has been obtained in a separate transiently transfected population of cells.

KD [3H]CGP 121777 from SaturationProteinLog KD SalmeterolnLog KD SalbutamolnLog KD FormoterolnLog KD FenoterolnLog KD AdrenalinenLog KD Clenbuteroln
fmol/mg
β2-WT0.18 ± 0.01165 ± 1330−8.67 ± 0.0367−5.76 ± 0.0338−7.92 ± 0.0352−6.56 ± 0.0246−5.64 ± 0.0517−7.44 ± 0.0420
Point mutations in β2 resulting in a change to β1 amino acids
  β2-EL20.17 ± 0.02188 ± 336−8.07 ± 0.07*19−5.59 ± 0.049−7.51 ± 0.034−6.19 ± 0.033−5.20 ± 0.105−7.26 ± 0.045
  β2-F194V0.24 ± 0.03207 ± 477−7.92 ± 0.05*21−5.50 ± 0.0614−7.57 ± 0.05*12−6.18 ± 0.03*12−5.37 ± 0.108−7.13 ± 0.048
  β2-EL30.22 ± 0.04126 ± 216−6.56 ± 0.04*,#25−5.54 ± 0.059−7.61 ± 0.05*9−6.33 ± 0.038−4.97 ± 0.08*5−7.10 ± 0.144
  β2-Q299H0.25 ± 0.03216 ± 319−8.30 ± 0.06*10−5.76 ± 0.037−7.84 ± 0.0513−6.54 ± 0.0412NDND
  β2-D300R0.27 ± 0.03271 ± 479−8.27 ± 0.10*10−5.74 ± 0.057−7.99 ± 0.0612−6.59 ± 0.0612NDND
  β2-N301E0.23 ± 0.03176 ± 308−8.44 ± 0.0910−5.69 ± 0.046−7.83 ± 0.0412−6.46 ± 0.0412NDND
  β2-I303V0.25 ± 0.04224 ± 389−8.34 ± 0.05*10−5.66 ± 0.057−7.78 ± 0.0513−6.40 ± 0.0413NDND
  β2-R304P0.22 ± 0.04267 ± 599−8.10 ± 0.04*16−5.65 ± 0.0314−7.71 ± 0.0419−6.29 ± 0.03*18−5.59 ± 0.0711−7.26 ± 0.0310
  β2-K305D0.21 ± 0.03159 ± 268−7.18 ± 0.04*15−5.61 ± 0.0312−7.56 ± 0.04*17−6.43 ± 0.0317−5.30 ± 0.108−7.33 ± 0.068
  β2-F194V-K305D0.19 ± 0.03128 ± 219−6.88 ± 0.03*17−5.54 ± 0.0412−7.33 ± 0.05*20−6.31 ± 0.04*19−5.16 ± 0.136−7.25 ± 0.108
  β2-R304P-K305D0.22 ± 0.06109 ± 127−6.85 ± 0.06*15−5.50 ± 0.0713−7.41 ± 0.04*13−6.28 ± 0.04*14−5.19 ± 0.155−7.22 ± 0.075
Point mutations in β2 resulting in a change to non-β1 amino acids
  β2-F194A0.22 ± 0.03170 ± 239−7.36 ± 0.05*10−5.43 ± 0.0411−7.60 ± 0.06*9−6.17 ± 0.05*9−5.24 ± 0.108−6.98 ± 0.03*8
  β2-K305A0.16 ± 0.02151 ± 458−8.05 ± 0.09*9−5.52 ± 0.099−7.74 ± 0.048−6.44 ± 0.087−5.54 ± 0.088−7.30 ± 0.078
  β2-K305E0.22 ± 0.03114 ± 169−7.62 ± 0.07*10−5.28 ± 0.10*11−7.46 ± 0.04*9−6.34 ± 0.049NDND
  β2-K305G0.24 ± 0.0477 ± 76−7.51 ± 0.11*9−5.70 ± 0.069−7.74 ± 0.095−6.38 ± 0.125−5.47 ± 0.055−7.35 ± 0.075
  β2-K305H0.23 ± 0.03157 ± 208−7.57 ± 0.06*9−5.65 ± 0.1110−7.24 ± 0.07*8−6.49 ± 0.058NDND
  β2-K305R0.24 ± 0.03135 ± 1415−8.37 ± 0.05*12−5.64 ± 0.0516−8.02 ± 0.0614−6.55 ± 0.0511−5.87 ± 0.076−7.26 ± 0.114
  β2-K305S0.19 ± 0.0483 ± 116−7.57 ± 0.06*9−5.58 ± 0.078−7.66 ± 0.115−6.54 ± 0.093−5.38 ± 0.075−7.55 ± 0.105
  β2-R304D0.22 ± 0.0294 ± 146−8.30 ± 0.11*8−5.63 ± 0.106−7.86 ± 0.076−6.70 ± 0.114−5.66 ± 0.086−7.39 ± 0.068
  β2-R304D-K305G0.19 ± 0.0258 ± 36−7.56 ± 0.09*6−5.63 ± 0.156−7.49 ± 0.18*5−6.42 ± 0.073−5.38 ± 0.125−7.50 ± 0.065
β1-WT0.28 ± 0.02731 ± 9627−5.67 ± 0.0144−4.74 ± 0.0228−5.86 ± 0.0242−4.90 ± 0.0244−4.74 ± 0.0411−6.58 ± 0.0611
Point mutations in β1 resulting in a change to β2 amino acids
  β1-EL20.32 ± 0.011135 ± 1796−5.82 ± 0.059−4.79 ± 0.085−5.99 ± 0.104−4.98 ± 0.063−4.80 ± 0.055−6.48 ± 0.025
  β1-V219F0.38 ± 0.041224 ± 2619−5.48 ± 0.02*10−4.76 ± 0.017−5.80 ± 0.0313−5.06 ± 0.0313−4.89 ± 0.108−6.51 ± 0.038
  β1-EL30.33 ± 0.02209 ± 116−6.29 ± 0.07*9−4.90 ± 0.065−6.14 ± 0.06*5−5.26 ± 0.03*3−5.24 ± 0.09*4−6.76 ± 0.055
  β1-P355R0.22 ± 0.02283 ± 726−5.77 ± 0.0511−4.82 ± 0.075−5.98 ± 0.0211−4.98 ± 0.049−4.76 ± 0.075−6.56 ± 0.035
  β1-D356K0.69 ± 0.04945 ± 1379−5.38 ± 0.02*12−4.48 ± 0.03*7−5.59 ± 0.03*15−4.64 ± 0.02*15−4.67 ± 0.068−6.31 ± 0.048
  β1-V219F-D356K0.70 ± 0.06631 ± 746−5.52 ± 0.04*9−4.58 ± 0.025−5.70 ± 0.039−4.81 ± 0.077−4.61 ± 0.025−6.36 ± 0.095
  β1-P355R-D356K0.61 ± 0.03191 ± 295−5.53 ± 0.03*9−4.61 ± 0.075−5.80 ± 0.039−4.91 ± 0.077−4.67 ± 0.105−6.42 ± 0.065

  • ND, not determined.

  • ↵* P < 0.001. One-way analysis of variance with post hoc Newman-Keuls comparing values from the mutant receptors with those obtained from the β2-WT or the β1-WT. Thus, the log KD for salmeterol at the β2-EL2 is different from that obtained from the β2-WT with P < 0.001. Likewise, the log KD for salmeterol at the β1-EL3 is different from that obtained from the β1-WT with P < 0.001; #P < 0.001. One-way analysis of variance with post hoc Newman-Keuls comparing each value with all other values in this set. Thus, the log KD for salmeterol at β2-EL3 is different from that obtained at β2-WT and all other β2-mutant receptors with P < 0.001 in all cases.