Identification of threonine residues controlling the agonist-dependent phosphorylation and desensitization of the rat A(3) adenosine receptor

Activation of the A(3) adenosine receptor (A(3)AR) contributes to the cardi oprotective, bronchoconstrictive, and hypotensive effects of adenosine. Ago nist occupation of the A(3)AR results in a rapid desensitization of recepto r function, which is associated with the phosphorylation of the receptor pr otein by one or more members of the G protein-coupled receptor kinase famil y of protein kinases. Although we demonstrated previously that phosphorylat ion of the C-terminal 14 amino acids of the rat A(3)AR is crucial for rapid desensitization to occur, the identity of the critical phosphorylation sit es has remained unknown. Here, we demonstrate that the simultaneous mutatio n of Thr(307), Thr(318), and Thr(319) to Ala residues dramatically reduces agonist-stimulated phosphorylation and rapid desensitization of the rat A(3 )AR. Individual mutation of each residue demonstrated that Thr(318) and Thr (319) are the major sites of phosphorylation. Phosphorylation at Thr(318) a ppeared to be necessary to observe phosphorylation at Thr(319), but not vic e versa. However, the replacement of Thr(318) with a glutamate residue demo nstrated that the simple addition of negative charge at position 318 was no t sufficient to rescue phosphorylation at position 319. In addition, the mu tation of two predicted palmitoylation-site cysteine residues proximal to t he regulatory domain resulted in the appearance of an agonist-independent b asal phosphorylation. Therefore, G protein-coupled receptor kinase-mediated phosphorylation of the C-terminal tail of the A(3)AR in situ appears to fo llow a sequential mechanism, perhaps involving receptor depalmitoylation, w ith phosphorylation at Thr(318) being particularly important.