Table 6

K_D and B_max values for the binding of various radioligands to membrane preparations of COS-7 cells expressing the α_2A-AR and mutant G_αo/DEINLL and G_αo/DEIGLL proteins

G_α protein	[³⁵S]GTPγS Binding
	(−)-Epinephrine		Clonidine
	K_D	B_max	K_D	B_max
	nM	pmol/mg protein	nM	pmol/mg protein
G_αo/DEINLL^6-150	11.68 ± 1.56	5.80 ± 0.25	weak stimulation (not determined)
G_αo/DEIGLL	4.49 ± 1.02^6-a	5.11 ± 0.79	6.59 ± 1.28	6.24 ± 1.04

	[³H]RX 821002		[³H]Clonidine		[³H]UK 14304
	K_D	B_max	K_D	B_max	K_D	B_max
	nM	pmol/mg protein	nM	pmol/mg protein	nM	pmol/mg protein
G_αo/DEINLL^6-150	0.85 ± 0.03	34.41 ± 6.71	64.26 ± 7.12	4.87 ± 1.10	11.24 ± 0.53	2.63 ± 0.47
G_αo/DEIGLL	0.83 ± 0.05	32.77 ± 5.35	1.25 ± 0.06^6-a	2.24 ± 0.04^6-a	0.24 ± 0.04^6-a	1.96 ± 0.30

Co-expression of α_2A-AR and mutant G_αo protein was performed as described under Experimental Procedures. All conditions were treated with PTX (20 ng/ml). Saturation [³⁵S]GTPγS binding responses mediated by the α_2A-AR were performed as described. Membranes were incubated with 0.5 nM [³⁵S]GTPγS, 30 μM GDP, and either without or with 0.1 to 300 nM unlabeled GTPγS. K_D (nM) andB_max (pmol/mg of protein) values were deduced from saturation analysis for specific (−)-epinephrine (10 μM) and/or clonidine (10 μM)-stimulated [³⁵S]GTPγS binding. The equilibrium dissociation constant (K_D, nM) and maximal radioligand binding capacity (B_max, pmol/mg of protein) were determined for each condition as described underExperimental Procedures according to a monophasic Scatchard analysis. Data represent mean values ± S.E.M. of four independent transfection experiments, each performed in duplicate. Statistical analysis was performed on ligand's K_D andB_max values between G_αo/DEINLL and G_αo/DEIGLL proteins.

↵6-150 G_αo/15 protein.
↵6-a P < 0.05.