The A2 adenosine receptor: guanine nucleotide modulation of agonist binding is enhanced by proteolysis.

Agonist binding to the A2 adenosine receptor (A2AR) and its regulation by guanine nucleotides was studied using the newly developed radioligand 125I-2-[4-(2-[2-[(4-aminophenyl)methylcarbonylamino] ethylaminnocarbonyl]ethyl)phenyl]ethylamino-5'-N- ethylcarboxamidoadenosine (125I-PAPA-APEC) and its photoaffinity analog 125I-azido-PAPA-APEC. A single protein of Mr 45,000, displaying the appropriate A2AR pharmacology, is labeled in membranes from bovine striatum, PC12 cells, and frog erythrocytes. In DDT1 MF2 cells the labeled protein has a slightly lower molecular weight. Incorporation of 125I-azido-PAPA-APEC into membranes from rabbit striatum, however, reveals two specifically labeled peptides (Mr approximately 47,000 and 38,000), both of which display A2AR pharmacology. Inhibition of protease activity leads to a decrease in the amount of the Mr 38,000 protein, with only the Mr 47,000 protein remaining. This suggests that the Mr 38,000 peptide is a proteolytic product of the Mr 47,000 A2AR protein. In membranes containing the intact undigested A2AR protein, guanine nucleotides induce a small to insignificant decrease in agonist binding, which is atypical of stimulatory GS-coupled receptors. This minimal effect is observed in rabbit striatal membranes prepared in the presence of protease inhibitors, as well as in the other tissues studied. Binding to rabbit striatal membranes that possess the partially digested receptor protein, however, reveals a 50% reduction in maximal specific agonist binding upon addition of guanine nucleotides. Inhibition of proteolysis in rabbit striatum, on the other hand, results in a diminished ability of guanine nucleotides to regulate agonist binding. Thus, the enhanced effectiveness of guanine nucleotides in rabbit striatal membranes is associated with the generation of the Mr 38,000 peptide fragment. Guanosine 5'-(beta,gamma-imido)triphosphate reduces photoaffinity labeling by 55% in the Mr 38,000 protein, whereas the labeling is decreased by only 28% in the Mr 47,000 receptor protein. Our data, therefore, suggest that, unless proteolysis occurs, the A2AR in all tissues studied is tightly associated with the GS protein and displays minimal guanine nucleotide modulation of agonist binding, which makes the A2AR an atypical stimulatory receptor.