A2a adenosine receptors: guanine nucleotide derivative regulation in porcine striatal membranes and digitonin soluble fraction.
We report the characterization of A2a adenosine receptors (A2aARs) in porcine striatal membranes and their solubilization (25%) by the detergent digitonin. After solubilization, the drug specificity and equilibrium [3H]CGS-21680 ([3H]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 [3H]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 Kd1 = 7.4+/-0.5 and Kd2 = 53.1+/-3.6 nM, a Bmax1 = 186+/-15 fmol/mg protein and a Bmax2 = 285+/-20 fmol/mg protein, respectively. In the presence of guanosine-5'-O-(3-thiotriphosphate) (GTPgamma[S]) a shift from two affinity states to a single one was evidenced (Kd = 28.5+/-5.9 nM) and a Bmax 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 Bmax = 285+/-20 fmol/mg protein) and, in the presence of GTPgamma[S]), a two site model likewise provided a significantly (P < 0.01) better fit (Kd1 = 13.9+/-1.2 nM and Kd2 = 72.1+/-6.9 nM, Bmax1 = 125+/-10 fmol/mg protein and Bmax2 = 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.
Dipartimento di Psichiatria, Farmacologia e Biotecnologie dell 'Università degli Studi di Pisa, Italy.
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Costa
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Lucacchini
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Martini
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D003342:Corpus Striatum / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology*; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008970:Molecular Weight; D010627:Phenethylamines / Q000378:metabolism; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D012995:Solubility; D013552:Swine
D006150:Guanine Nucleotides; D010627:Phenethylamines; D018047:Receptors, Purinergic P1; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000241:Adenosine; D004072:Digitonin