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

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.