IDENTIFICATION OF AN A(2) ADENOSINE RECEPTOR DOMAIN SPECIFICALLY RESPONSIBLE FOR MEDIATING SHORT-TERM DESENSITIZATION

In an attempt to delineate the structural requirements necessary for a gonist-induced desensitization, we have stably expressed wild-type and mutant A(2a) adenosine receptors (A(2a)ARs) in Chinese hamster ovary cells and examined the effects of agonist pretreatment on adenylyl cyc lase activity in subsequently isolated membranes. Deletion of 95 amino acids from the carboxyl-terminus of the A(2a)AR, thereby removing 10 potential phosphorylation sites, failed to alter radioligand binding, adenylyl cyclase activation, or functional desensitization parameters as compared with the wild-type receptor. However, simultaneous mutatio n of Thr 298 and Ser 305 to Ala residues attenuated the desensitizatio n observed in response to short-term (30 min) agonist treatment while not blocking the ability to desensitize after long-term (24 h) agonist exposure. Individual mutation of these residues revealed that mutatio n of Thr 298 alone was sufficient to diminish both short-term desensit ization and agonist-stimulated receptor phosphorylation. These data su ggest that while the majority of the A(2a)AR carboxyl-terminus is disp ensable with regard to observing functional desensitization, the integ rity of Thr 298 is essential for observing agonist-stimulated receptor phosphorylation and short-term desensitization but not long-term dese nsitization of A(2a)AR function.