A2a adenosine receptors:: guanine nucleotide derivative regulation in porcine striatal membranes and digitonin soluble fraction

We report the characterization of A(2a) adenosine receptors (A(2a)ARs) in porcine striatal membranes and their solubilization (25%) by the detergent digitonin. After solubilization, the drug specificity and equilibrium [H-3]CGS-21680 ([H-3]2-(4-(2-carboxyethyl)phenylethylamino)-5'-N-ethyl-carboxamido-adenosine) binding parameters were virtually identical to those obtained in intact membranes, indicating a conservation of the binding site after the removal of receptors from their lipid environment. Gel filtration on a calibrated Superdex 200 HR column revealed a main [H-3]CGS-21680 binding peak with an apparent molecular weight of 171,000 +/- 9000 Da. In membranes, Scatchard analysis of saturation data carried out in a wide range of radioligand concentration (1-100 nM) resulted in a biphasic curve and, in accordance with the two binding sites model, yielded a Kd(1) = 7.4 +/- 0.5 and Kd(2) = 53.1 +/- 3.6 nM, a B-max 1 = 186 +/- 15 fmol/mg protein and a B-max 2 = 285 +/- 20 fmol/mg protein, respectively. In the presence of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma[S]) a shift from two affinity states to a single one was evidenced (Kd = 28.5 +/- 5.9 nM) and a B-max value of 504 +/- 10 fmol/mg protein found. In the soluble extract, only one high-affinity state was detected (Kd = 19.3 +/- 1.1 nM and B-max = 285 +/- 20 fmol/mg protein) and, in the presence of (GTP gamma[S]), a two site model likewise provided a significantly (P < 0.01) better fit (Kd(1) = 13.9 +/- 1.2 nM and Kd(2) = 72.1 +/- 6.9 nM, B-max (1) = 125 +/- 10 fmol/mg protein and B-max 2 = 375 +/- 19 fmol/mg protein, respectively). These results suggest a close relation between the receptor and G protein solubilized as a functional unit and open the way to its purification. (C) 1998 Elsevier Science Ltd. All rights reserved.