Solubilisation and immunoprecipitation of rat striatal adenosine A(2A) receptors.
In the present study, we have sought to solubilise adenosine A(2A) receptors from rat striatal membranes using a variety of different detergents. Of the detergents tested, 1% CHAPS (3-[(3-deoxycholic acid (cholamidopropyl) dimethylammonio]-1-propanesulfonate) yielded optimal conditions for solubilisation (in the presence of 3 mg/ml protein, 44% of receptor was solubilised, 50% of total protein was solubilised). An antipeptide antibody was raised against a 15 amino-acid sequence within the predicted third intracellular loop region of the human and rat adenosine A(2A) receptor. The antibody was coupled to protein A immobilised on sepharose CL-4B and used to immunoprecipitate adenosine A(2A) receptors from solubilised rat striatal preparations. Radioligand-binding studies were performed using the selective adenosine A(2) antagonist [(3)H]ZM 241385 (4-(2-[7-amino-2-(2-fury1)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol). Using [(3)H]ZM 241385, the pharmacology of immunoprecipitated adenosine A(2A) receptors was composed to striatal membrane bound adenosine A(2A) receptors and detergent solubilised adenosine A(2A) receptors. [(3)H]ZM 241385 labelled a single saturable binding site with high affinity in all three preparations (membrane bound K(d) 2.7 nM+/-1.0; solubilised K(d) 1.9 nM+/-0.3; immunoprecipitated K(d) 2.2 nM+/-0.7). Additionally, all three assays confirmed a rank order of potency for displacers consistent with adenosine A(2A) receptor pharmacology: ZM 241385>KW 6002 ((E)-8-[2-(3,4-dimethoxyphenyl)ethynyl]-1-3-diethyl-3,7-dihydro-7-methyl-1-purine 2,6 dione)>CGS 21680, (2-(4-(2 carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoadenosine)>DPCPX (8-cyclopentyl-1,3-dipropylxanthine). We conclude that we have solubilised and immunoprecipitated adenosine A(2A) receptors from rat striatum and that their pharmacology is consistent with native striatal adenosine A(2A) receptors.
Harvey
V
V
Department of Molecular Pharmacology, Vernalis Research Ltd., 613 Reading Road, Winnersh, Wokingham, RG41 5UA, UK.
Jones
J
J
Misra
A
A
Knight
A R
AR
Quirk
K
K
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002793:Cholic Acids; D003342:Corpus Striatum / Q000737:chemistry / Q000378:metabolism*; D003902:Detergents; D010627:Phenethylamines / Q000494:pharmacology; D011233:Precipitin Tests; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D011687:Purines / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D043705:Receptor, Adenosine A2A; D018047:Receptors, Purinergic P1 / Q000737:chemistry*; D012995:Solubility; D014227:Triazines / Q000737:chemistry / Q000494:pharmacology; D014230:Triazoles / Q000737:chemistry / Q000494:pharmacology
D002793:Cholic Acids; D003902:Detergents; D010627:Phenethylamines; D058906:Purinergic P1 Receptor Agonists; D058915:Purinergic P1 Receptor Antagonists; D011687:Purines; D043705:Receptor, Adenosine A2A; D018047:Receptors, Purinergic P1; D014227:Triazines; D014230:Triazoles; C097270:ZM 241385; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; C111599:istradefylline; D000241:Adenosine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate