TABLE 1. Binding characteristics of wild-type and mutant human A_2A-adenosine receptors using the antagonist radioligand [³H]XAC.

Data are presented as mean ± standard deviation of two or three independent experiments, each performed in duplicate. Each sample contained 7–11 μg of membrane protein/tube. Agonist and antagonist binding affinities [K_i values, structures in Fig. 2 and Jacobson et al., (35)] were determined in ³H]XAC (1.0 nM) competition binding studies using membrane homogenates prepared from transiently transfected COS-7 cells, as described in Experimental Procedures. K_i values were calculated from IC₅₀ values by using the KaleidaGraph program. All constructs contain an HA-tag sequence at the amino terminus (15).

	Construct
	Compound	Wild-type	T88A	T88S	188R
Bmax (pmol/mg)	[3H]XAC	14.9 ± 1.2	6.54 ± 1.61b	6.26 ± 070b	5.54 ± 0.47b
Kd (nM)	[3H]XAC	8.83 ± 1.46	10.8 ± 18d	5.76 ± l.82d	4.21 ± 1.17c
Ki (nM)	Agonists
	CADO	118 ± 22	16,400 ± 3,200c	776 ± 313c	9,140 ± 2,590c
	DPMA	65.5 ± 0.3	5,710 ± 2,520c	655 ± 134c	4,090 ± 760c
	NECA	19.1 ± 1.7	1,590 ± 280c	778 ± 224c	2,660 ± 1,390c
	CGS 21680	26.7 ± 6.5	22,100 ± 2,900b	422 ± 180c	4,620 ± 620b
	Antagonists
	CGS 15943	1.20 ± 0.30	7.98 ± 1.71c	6.28 ± 1.68c	5.00 ± 052b
	ZM 241385	0.758 ± 0.135	3.47 ± 2.41d	1.16 ± 0.20d	2.00 ± 0.71d

^ap <0.001.

^bp <0.01.

^cp <0.05.

^dNot significant.