Gα protein | [35S]GTPγS Binding | |||
---|---|---|---|---|
(−)-Epinephrine | Clonidine | |||
KD | Bmax | KD | Bmax | |
nM | pmol/mg protein | nM | pmol/mg protein | |
Gαo/DEINLL6-150 | 11.68 ± 1.56 | 5.80 ± 0.25 | weak stimulation (not determined) | |
Gαo/DEIGLL | 4.49 ± 1.026-a | 5.11 ± 0.79 | 6.59 ± 1.28 | 6.24 ± 1.04 |
[3H]RX 821002 | [3H]Clonidine | [3H]UK 14304 | ||||
---|---|---|---|---|---|---|
KD | Bmax | KD | Bmax | KD | Bmax | |
nM | pmol/mg protein | nM | pmol/mg protein | nM | pmol/mg protein | |
Gαo/DEINLL6-150 | 0.85 ± 0.03 | 34.41 ± 6.71 | 64.26 ± 7.12 | 4.87 ± 1.10 | 11.24 ± 0.53 | 2.63 ± 0.47 |
Gαo/DEIGLL | 0.83 ± 0.05 | 32.77 ± 5.35 | 1.25 ± 0.066-a | 2.24 ± 0.046-a | 0.24 ± 0.046-a | 1.96 ± 0.30 |
Co-expression of α2A-AR and mutant Gαo 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 α2A-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. KD (nM) andBmax (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 (KD, nM) and maximal radioligand binding capacity (Bmax, 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 KD andBmax values between Gαo/DEINLL and Gαo/DEIGLL proteins.