Selective reduction in A(2) adenosine receptor desensitization following antisense-induced suppression of G protein-coupled receptor kinase 2 expression

Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRK s) is considered to play a critical role in the desensitization of response s mediated by these receptors. To explore the role of GRK2 in A(2) adenosin e receptor desensitization, we attempted to reduce specifically GRK2 expres sion in NG108-15 cells by stable transfection with an antisense rat GRK2 cD NA sequence. This yielded up to a 69% loss of GRK2 when compared with plasm id-transfected control cells, which correlated with a reduction in kinase a ctivity when measured by the ability of cell lysates to promote light-depen dent phosphorylation of rhodopsin, Levels of GRK3 were the same in antisens e and plasmid-transfected controls. On addition of the A(2) adenosine recep tor agonist 5'-(N-ethylcarboxamido)adenosine, cyclic AMP accumulation was g reater in GRK2 antisense cells as compared with plasmid control cells. In c ontrast, cyclic AMP accumulation via agonist stimulation of either IP-prost anoid or secretin receptors or by addition of forskolin was not significant ly different among all clones examined. The increase in A(2) adenosine rece ptor response could not be explained by changes in A(2A) adenosine receptor expression, as assessed by ligand binding experiments with the radioligand 2-H-3-labelled 4-[2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triaz in-5-ylamino]ethyl]phenol ([H-3]ZM241385). These data show for the first ti me a direct correlation between expression of GRK2 and desensitization of n atively expressed A(2) adenosine receptors in intact cells, suggesting that GRK2 plays a major role in the regulation of these receptors.