title: Effects of urea pretreatment on the binding properties of adenosine A1 receptors.
abstract: The effect of denaturation and/or extraction of nonintegral membrane proteins by 7 M urea on the binding of the antagonist [3H]cyclopentyl-1,3-dipropylxanthine 8 dipropyl-2,3 ([3H]DPCPX), and the agonists adenosine, (-)-N6-(2-phenylisopropyl)-adenosine (R-PIA) and N6-cyclohexyladenosine (CHA), was investigated at human A1 adenosine receptors stably expressed in CHO cells. Pretreatment with urea caused a 56% reduction in membrane proteins. Compared to controls, the use of adenosine deaminase (ADA), 100 microM 5'-guanylylimidodiphosphate (Gpp(NH)p) or urea each caused equivalent increases in specific [3H]DPCPX binding. Neither the binding kinetics nor the affinity of [3H]DPCPX were significantly different in urea-pretreated compared to ADA-pretreated membranes. At 25 degrees C in ADA-pretreated membranes, the competition isotherms for R-PIA and CHA were characterized by two affinity states. Gpp(NH)p (100 microM) reduced, but did not abolish, the value of the high-affinity dissociation constant. Similar results were obtained after treatment with urea for R-PIA, whereas the high-affinity state for CHA was abolished. At 37 degrees C, urea pretreatment, but not 100 microM Gpp(NH)p, abolished high-affinity agonist competition binding. There was no significant effect of any of the treatments on the low-affinity agonist binding state. In urea-pretreated membranes, exogenously added adenosine competed according to a simple mass-action model with a pK(L) of 5.66+/-0.05 (n=3). Compared to the more common approaches of ADA treatment and/or use of guanine nucleotides, our findings suggest that urea pretreatment represents an inexpensive and useful approach for investigating the binding properties of adenosine A1 ligands (including adenosine) to the G protein-uncoupled form of the receptor.
authors.affiliation: Molecular Pharmacology Laboratory, Department of Pharmacology, The University of Melbourne, Parkville, 3010 Victoria, Australia.
authors.forename: Lauren T
authors.initials: LT
authors.lastname: May
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Sexton
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000243:Adenosine Deaminase / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011489:Protein Denaturation; D043682:Receptor, Adenosine A1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014162:Transfection; D014508:Urea / Q000494:pharmacology*; D014970:Xanthines / Q000378:metabolism
chemical_list: D008024:Ligands; D043682:Receptor, Adenosine A1; D014970:Xanthines; C047235:N-(1-methyl-2-phenylethyl)adenosine; D006165:Guanylyl Imidodiphosphate; C027513:N(6)-cyclohexyladenosine; D014508:Urea; C051360:1,3-dipropyl-8-cyclopentylxanthine; D000243:Adenosine Deaminase; D000241:Adenosine