Table 4

Agonist efficacies and potencies at hH₂R-A271D-G_sαS, NgpChH₂R-G_sαS, and NhCgpH₂R-G_sαSexpressed in Sf9 cell membranes

Compound	hH₂R-A271D-G_sαS		NgpChH₂R-G_sαS		NhCgpH₂R-G_sαS
Compound	Efficacy	EC₅₀	Rel. Pot.	Efficacy	EC₅₀	Rel. Pot.	Efficacy	EC₅₀	Rel. Pot.
		μM			μM			μM
1(HIS)	1.00	0.35 ± 0.05	100	1.00	1.98 ± 0.67	100	1.00	0.71 ± 0.26	100
5 (IMP)	0.85 ± 0.12	0.03 ± 0.01	1300	0.78 ± 0.10	0.19 ± 0.03	1040	0.99 ± 0.09	0.04 ± 0.01	1780
6 (BU-E-42)	0.95 ± 0.08	0.08 ± 0.01	438	0.87 ± 0.02	0.25 ± 0.04	792	0.91 ± 0.02	0.11 ± 0.03	645
7 (BU-E-43)	0.76 ± 0.09	0.28 ± 0.04	125	0.70 ± 0.03	0.21 ± 0.03	943	0.79 ± 0.01	0.46 ± 0.03	154
8 (ARP)	0.91 ± 0.04	0.06 ± 0.01	583	0.95 ± 0.06	0.27 ± 0.04	733	0.93 ± 0.06	0.11 ± 0.05	645
10 (BU-E-48)	0.83 ± 0.04	0.03 ± 0.00	1170	0.85 ± 0.03	0.12 ± 0.02	1650	0.85 ± 0.03	0.08 ± 0.02	888
11 (BU-E-75)	0.80 ± 0.10	0.04 ± 0.01	875	0.91 ± 0.06	0.19 ± 0.01	1040	0.97 ± 0.05	0.09 ± 0.01	789
13 (D281)	0.62 ± 0.11	0.11 ± 0.02	318	0.45 ± 0.06	0.16 ± 0.05	1240	0.52 ± 0.14	0.13 ± 0.04	546

Potencies and efficacies of ligands at hH₂R-A271D-G_sαS, NgpChH₂R-G_sαS, and NhCgpH₂R-G_sαS were determined in the GTPase assay. GTP hydrolysis was determined as described under Experimental Procedures. Reaction mixtures contained Sf9 membranes expressing fusion proteins and agonists at concentrations from 1 nM to 1 mM as appropriate to generate saturated concentration/response curves. Curves were analyzed by nonlinear regression. Typical basal GTPase activities ranged between ∼1 and 2 pmol/mg/min, and typical GTPase activities stimulated by HIS (100 μM) ranged between ∼4 and 8 pmol/mg/min. To calculate agonist efficacies, the maximum stimulatory effect of HIS was set at 1.00, and the stimulatory effects of other agonists were referred to this value. Data shown are the means ± S.D. of three to six experiments performed in duplicates. The relative potency (Rel. Pot.) of HIS was set at 100, and the potencies of other agonists were referred to this value to facilitate comparison of agonist potencies with hH₂R-G_sαS and gpH₂R-G_sαS(Table 2).