Table 1.

Binding Characteristics of Wild Type and Mutant Human A_2a Adenosine Receptors Using the Agonist Radioligand [³H]CGS21680^a

	constructb
	compound	WT	V84L	H250N
Bmax (pmol/mg)	[3H]CGS21680	5.69 ± 0.51	3.16 ± 0.15**	1.67 ± 0.11**
Kd (nM)	[3H]CGS21680	36.4 ± 2.7	27.3 ± 3.2**	7.60 ± 1.49**
Ki (nM)	agonists
	DPMA	38.1 ± 0.8	18.9 ± 8.1**	35.9 ± 4.4
	NECA	21.6 ± 4.5	21.5 ± 9.5	5.24 ± 4.69**
	IB-MECA	370 ± 91	143 ± 28*	32.0 ± 13.7**
	antagonists
	CGS15943	0.142 ± 0.047	0.842 ± 0.433	0.457 ± 0.225
	XAC	7.89 ± 1.17	45.6 ± 12.6**	15.6 ± 6.4**
	galangin	16700 ± 900	18100 ± 5000	33000 ± 5800
	nifedipine	24400 ± 6300	22200 ± 1700	22200 ± 1900
	CPX	226 ± 28	788 ± 93**	291 ± 104
	BTH4	106000c	72600 ± 44000	54300 ± 4400
	amiloride	12000 ± 4100	11600 ± 2400	3280 ± 1330*

^aAgonist and antagonist binding affinities (K_i values, structures in Figure 2 and Jacobson et al., 1992) were determined in [3H]CGS21680 (15 nM) competition binding studies at pH 6.8 using membrane homogenates prepared from transiently transfected COS-7 cells, as described in the Experimental Section. Data are presented as means ± SD of three independent experiments, unless indicated, each performed in duplicate. Each sample contained 7–11 μg of membrane protein/tube. K_i values were calculated from IC₅₀ values using the KaleidaGraph program. All constructs contain an HA tag sequence at the N-terminus (Kim et al., 1995).

^**P ≤ 0.01,

^*P ≤ 0.05 vs wild type receptors.

^bConstructs that showed <3% of specific binding of [³H]CGS21680 (15 nM) found for HA-tagged wild type receptors were V84A, V84D, H278N, and H278K mutant receptors. The expression levels at these four mutants showed levels comparable to HA-tagged wild type receptors. K_d was determined in saturation experiments using [³H]CGS21680 at the V84L and H250N mutant receptors transfected in COS-7 cells.

^cn = 2 (105 and 107 μM).