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

Br J Pharmacol. 2005 Dec;146(8):1119-29. doi: 10.1038/sj.bjp.0706419.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / analogs & derivatives
  • Adenosine / metabolism
  • Adenosine Deaminase / pharmacology
  • Animals
  • Binding, Competitive
  • CHO Cells
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism*
  • Cricetinae
  • Cricetulus
  • Guanylyl Imidodiphosphate / pharmacology
  • Humans
  • Kinetics
  • Ligands
  • Protein Denaturation
  • Receptor, Adenosine A1 / drug effects
  • Receptor, Adenosine A1 / genetics
  • Receptor, Adenosine A1 / metabolism*
  • Temperature
  • Transfection
  • Urea / pharmacology*
  • Xanthines / metabolism


  • Ligands
  • Receptor, Adenosine A1
  • Xanthines
  • N-(1-methyl-2-phenylethyl)adenosine
  • Guanylyl Imidodiphosphate
  • N(6)-cyclohexyladenosine
  • Urea
  • 1,3-dipropyl-8-cyclopentylxanthine
  • Adenosine Deaminase
  • Adenosine