TABLE 8

Log EC50 values and % maximum response to isoprenaline for CRE-SPAP production for the agonists cimaterol and CGP12177 at β1-WT, the chimeric β1-TM4 receptor (containing the full TM4 mutations), and chimeric β1/β2-adrenoceptors with single point mutations and then combinations of these mutations

Log KB values for several antagonists for inhibition of the cimaterol and CGP12177 responses are given. These data were obtained from between 4 and 24 stable mixed populations of cells for each receptor, and n in the table refers to the number of separate experiments. This table shows that the mutations L195Q and W199Y, both alone and in combination, have a very significant effect on the responses to CGP12177 and affinity of antagonist measured in the presence of CGP12177 (i.e., the secondary conformation of the β1-receptor). Every time the mutation V189T is present, the affinity of ICI118551 is increased, both at the catecholamine conformation (in the presence of cimaterol) and at the secondary conformation (presence of CGP12177).

Cimaterol Log EC50% IsoprenalinenLog KB CGP20712AnLog KB ICI118551nLog KB propranololnLog KB CGP12177n
Cimaterol as agonist: stable mixed populations of cells
 β1-WT−8.29 ± 0.0481.2 ± 2.124−9.36 ± 0.0735−7.13 ± 0.0631−8.69 ± 0.0830−10.01 ± 0.0633
 β1-A187G−8.22 ± 0.0878.9 ± 2.38−9.23 ± 0.0814−7.02 ± 0.078−8.72 ± 0.098−9.99 ± 0.0617
 β1-V189T−8.20 ± 0.0480.6 ± 1.812−9.26 ± 0.0920−7.73 ± 0.07*15−8.61 ± 0.0514−9.81 ± 0.0827
 β1-L195Q−8.20 ± 0.0580.6 ± 2.712−8.97 ± 0.1019−7.12 ± 0.0715−8.86 ± 0.1114−9.85 ± 0.0725
 β1-W199Y−8.46 ± 0.0677.3 ± 2.412−9.66 ± 0.0819−7.39 ± 0.0716−9.01 ± 0.1215−10.06 ± 0.0526
 β1-W199A−8.63 ± 0.07*85.4 ± 3.28−9.51 ± 0.1413−7.26 ± 0.0811−9.12 ± 0.1113−10.07 ± 0.164
 β1-W199D−8.91 ± 0.07*87.6 ± 3.58−9.88 ± 0.0812−7.48 ± 0.1110−9.17 ± 0.1012−9.85 ± 0.128
 β1-W199F−8.71 ± 0.09*80.0 ± 2.68−10.14 ± 0.10*14−7.19 ± 0.1212−9.09 ± 0.1314−9.95 ± 0.1611
 β1-W199K−7.64 ± 0.05*86.4 ± 3.28−9.46 ± 0.0814−6.41 ± 0.06*9−8.00 ± 0.12*10a
 β1-W199L−7.60 ± 0.06*75.2 ± 2.98−10.06 ± 0.11*12−6.90 ± 0.1111−8.72 ± 0.0612a
 β1-W199N−8.88 ± 0.06*84.6 ± 5.48−9.93 ± 0.0911−7.21 ± 0.0812−9.22 ± 0.1013−10.12 ± 0.1212
 β1-V189T-L195Q−8.22 ± 0.0487.0 ± 3.48−9.49 ± 0.0913−7.67 ± 0.10*11−8.81 ± 0.1114−9.58 ± 0.0912
 β1-V189T-W199Y−8.62 ± 0.07*84.2 ± 2.18−9.93 ± 0.0812−7.85 ± 0.08*10−8.80 ± 0.0812−10.10 ± 0.1511
 β1-L195Q-W199Y−8.41 ± 0.0679.6 ± 2.08−9.68 ± 0.0613−7.24 ± 0.0611−9.04 ± 0.1112−9.82 ± 0.149
 β1-V189T-L195Q-W199Y−8.44 ± 0.0482.7 ± 2.78−9.85 ± 0.0814−7.92 ± 0.06*11−8.99 ± 0.0914−9.79 ± 0.1112
 β1-TM4−8.32 ± 0.0577.3 ± 1.923−9.43 ± 0.0935−7.94 ± 0.06*31−9.08 ± 0.0729−9.98 ± 0.0935
CGP12177 Log EC50% IsoprenalinenLog KB CGP20712AnLog KB ICI118551nLog KB propranololn
CGP12177 as agonist: stable mixed populations of cells
 β1-WT−8.12 ± 0.0753.3 ± 1.521−7.37 ± 0.0837−5.86 ± 0.0822−6.69 ± 0.0827
 β1-A187G−8.21 ± 0.0941.5 ± 3.17−7.25 ± 0.1112−5.81 ± 0.087−6.72 ± 0.1210
 β1-V189T−8.01 ± 0.0744.1 ± 2.311−7.64 ± 0.0821−6.36 ± 0.09*16−6.98 ± 0.0824
 β1-L195Q−9.00 ± 0.06*32.9 ± 1.4*11−8.53 ± 0.09*19−6.64 ± 0.11*13−7.74 ± 0.12*21
 β1-W199Y−9.16 ± 0.05*40.2 ± 2.3*11−8.46 ± 0.07*23−6.69 ± 0.09*17−7.90 ± 0.09*29
 β1-W199A−9.46 ± 0.11*68.6 ± 4.0*8−8.79 ± 0.10*12−6.88 ± 0.11*9−7.96 ± 0.15*9
 β1-W199D−9.48 ± 0.08*45.3 ± 3.38−8.94 ± 0.09*17−6.98 ± 0.16*13−8.87 ± 0.12*20
 β1-W199F−8.95 ± 0.13*58.6 ± 3.48−8.10 ± 0.07*13−6.53 ± 0.10*10−7.61 ± 0.17*12
 β1-W199K−9.40 ± 0.05*85.1 ± 2.5*8−8.13 ± 0.10*18−6.34 ± 0.09*13−7.47 ± 0.08*20
 β1-W199L−9.20 ± 0.06*82.9 ± 3.2*8−8.12 ± 0.09*20−6.45 ± 0.08*13−7.56 ± 0.11*20
 β1-W199N−9.27 ± 0.12*47.7 ± 3.28−8.26 ± 0.11*12−6.67 ± 0.10*10−8.19 ± 0.09*17
 β1-V189T-L195Q−9.21 ± 0.06*35.1 ± 2.6*8−8.64 ± 0.09*15−7.11 ± 0.08*12−8.17 ± 0.10*14
 β1-V189T-W199Y−9.07 ± 0.11*43.2 ± 2.98−8.70 ± 0.08*18−7.42 ± 0.14*13−7.82 ± 0.10*16
 β1-L195Q-W199Y−9.51 ± 0.06*41.3 ± 2.38−9.17 ± 0.12*15−6.94 ± 0.09*12−8.15 ± 0.11*15
 β1-V189T-L195Q-W199Y−9.40 ± 0.07*39.2 ± 2.4*8−9.21 ± 0.12*14−7.49 ± 0.07*12−8.66 ± 0.11*16
 β1-TM4−9.43 ± 0.06*33.9 ± 1.5*19−8.98 ± 0.07*39−7.64 ± 0.08*32−8.39 ± 0.06*40

  • ↵a CGP12177 is very efficacious in these mutants, similar to cimaterol, and it is therefore not possible to measure a log KB value for CGP12177 by a rightward shift of the cimaterol concentration response.

  • ↵* P < 0.001 One-way ANOVA with post hoc Newman–Keuls comparing values from the mutant receptors with those obtained from the β1-WT, thus the log KB for ICI118551 at β1-V189T is different from that obtained from the β1-WT with P < 0.001