Effects of urea pretreatment on the binding properties of adenosine A1 receptors

1 The effect of denaturation and/or extraction of nonintegral membrane proteins by 7M urea on the binding of the antagonist [H-3] cyclopentyl-1,3-dipropylxanthine 8 dipropyl-2,3 ([ 3H] DPCPX), and the agonists adenosine, (-)-N-6-(2-phenylisopropyl)- adenosine (R-PIA) and N-6-cyclohexyladenosine (CHA), was investigated at human A(1) adenosine receptors stably expressed in CHO cells. 2 Pretreatment with urea caused a 56% reduction in membrane proteins. Compared to controls, the use of adenosine deaminase (ADA), 100 mu M 50-guanylylimidodiphosphate (Gpp(NH) p) or urea each caused equivalent increases in specific [H-3] DPCPX binding. 3 Neither the binding kinetics nor the affinity of [3H] DPCPX were significantly different in urea-pretreated compared to ADA-pretreated membranes. 4 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 mM) 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. 5 At 37 degrees C, urea pretreatment, but not 100 mu M 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. 6 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). 7 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 A(1) ligands (including adenosine) to the G protein-uncoupled form of the receptor.