TABLE 6

Log EC₅₀ values and % maximum response to isoprenaline for CRE-SPAP production for the agonists cimaterol and CGP12177 at the β₁-WT, the chimeric β₁-TM4 receptor (containing the full TM4 mutations), and chimeric β₁/β₂-adrenoceptors with several amino acids in TM4 mutated to that of the β₂-WT

Log K_B values for several antagonists for inhibition of the cimaterol and CGP12177 responses are also given. Table 1 lists the detail of each mutation. These data were obtained from between 4 and 24 stable mixed populations of cells for each chimera, and n in the table refers to the number of separate experiments. This table shows that within TM4, it is only the amino acid changes in stage 5 and the full TM4 (i.e., L195Q and W199Y) that are very important for the alteration of the secondary conformation (i.e., responses to CGP12177 and antagonist affinities obtained in the presence of CGP12177). Stage 4 (addition of V189T to the other changes) affects the affinity of ICI18551. There is no change in the pharmacology of either conformation, even when the majority of amino acids have been changed (up to and including stage 3).

	Cimaterol Log EC₅₀	Isoprenaline %	n	Log K_B CGP20712A	n	Log K_B ICI118551	n	Log K_B Propranolol	n	Log K_B CGP12177	n
Cimaterol as agonist: stable mixed populations of cells
β₁-WT	−8.29 ± 0.04	81.2 ± 2.1	24	−9.36 ± 0.07	35	−7.13 ± 0.06	31	−8.69 ± 0.08	30	−10.01 ± 0.06	33
β₁-TM4 stage 1	−8.26 ± 0.11	85.6 ± 3.9	4	−9.10 ± 0.09	4	−7.04 ± 0.09	6	−8.64 ± 0.14	4	−9.96 ± 0.33	3
β₁-TM4 stage 2	−8.20 ± 0.06	77.8 ± 1.5	12	−9.34 ± 0.10	18	−7.14 ± 0.06	14	−8.79 ± 0.11	12	−9.81 ± 0.09	14
β₁-TM4 stage 3	−8.13 ± 0.07	78.2 ± 3.9	11	−8.95 ± 0.09	13	−7.00 ± 0.07	10	−8.63 ± 0.07	11	−9.93 ± 0.06	15
β₁-TM4 stage 4	−8.08 ± 0.05	76.3 ± 1.5	12	−9.14 ± 0.08	18	−7.62 ± 0.05^*	16	−8.59 ± 0.06	12	−9.77 ± 0.06	19
β₁-TM4 stage 5	−8.14 ± 0.06	78.2 ± 3.2	11	−8.97 ± 0.09	16	−7.71 ± 0.07^*	15	−8.81 ± 0.04	11	−9.93 ± 0.08	19
β₁-TM4	−8.32 ± 0.05	77.3 ± 1.9	23	−9.43 ± 0.09	35	−7.94 ± 0.06^*	31	−9.08 ± 0.07	29	−9.98 ± 0.09	35
	CGP12177 Log EC₅₀	Isoprenaline %	n	Log K_B CGP20712A	n	Log K_B ICI118551	n	Log K_B Propranolol	n
CGP12177 as agonist: stable mixed populations of cells
β₁-WT	−8.12 ± 0.07	53.3 ± 1.5	21	−7.37 ± 0.08	37	−5.86 ± 0.08	22	−6.69 ± 0.08	27
β₁-TM4 stage 1	−8.43 ± 0.06	50.4 ± 2.2	4	−7.32 ± 0.25	5	−6.21 ± 0.50	3	−6.17 ± 0.17	4
β₁-TM4 stage 2	−8.46 ± 0.07	46.5 ± 1.9	11	−7.55 ± 0.12	17	−6.08 ± 0.11	10	−6.78 ± 0.09	20
β₁-TM4 stage 3	−8.48 ± 0.08	45.9 ± 3.0	11	−7.13 ± 0.10	18	−5.90 ± 0.11	10	−6.88 ± 0.11	13
β₁-TM4 stage 4	−7.93 ± 0.12	40.0 ± 1.6^*	10	−7.26 ± 0.10	15	−6.57 ± 0.13^*	10	−6.98 ± 0.13	18
β₁-TM4 stage 5	−9.29 ± 0.06^*	30.2 ± 2.3^*	11	−8.74 ± 0.09^*	19	−7.54 ± 0.09^*	10	−8.18 ± 0.08^*	25
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

↵* 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 EC₅₀ for CGP12177 at β₁-TM4 Stage 5 receptor is different from that obtained from the β₁-WT with P < 0.001