Abstract
Activation of the A3 adenosine receptor (A3AR) contributes to the cardioprotective, bronchoconstrictive, and hypotensive effects of adenosine. Agonist occupation of the A3AR results in a rapid desensitization of receptor function, which is associated with the phosphorylation of the receptor protein by one or more members of the G protein-coupled receptor kinase family of protein kinases. Although we demonstrated previously that phosphorylation of the C-terminal 14 amino acids of the rat A3AR is crucial for rapid desensitization to occur, the identity of the critical phosphorylation sites has remained unknown. Here, we demonstrate that the simultaneous mutation of Thr307, Thr318, and Thr319 to Ala residues dramatically reduces agonist-stimulated phosphorylation and rapid desensitization of the rat A3AR. Individual mutation of each residue demonstrated that Thr318 and Thr319 are the major sites of phosphorylation. Phosphorylation at Thr318 appeared to be necessary to observe phosphorylation at Thr319, but not vice versa. However, the replacement of Thr318 with a glutamate residue demonstrated that the simple addition of negative charge at position 318 was not sufficient to rescue phosphorylation at position 319. In addition, the mutation of two predicted palmitoylation-site cysteine residues proximal to the regulatory domain resulted in the appearance of an agonist-independent basal phosphorylation. Therefore, G protein-coupled receptor kinase-mediated phosphorylation of the C-terminal tail of the A3AR in situ appears to follow a sequential mechanism, perhaps involving receptor depalmitoylation, with phosphorylation at Thr318 being particularly important.
Footnotes
- Received July 15, 1999.
- Accepted December 14, 1999.
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Send reprint requests to: Timothy M. Palmer, Ph.D., Room 407, Davidson Building, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. E-mail:T.Palmer{at}bio.gla.ac.uk
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T.M.P. was supported by a Postdoctoral Fellowship from the American Heart Association, North Carolina Affiliate; project grants from the British Heart Foundation and Royal Society; a Medical Research Council Co-operative Group Grant in Cellular Signalling and Molecular Genetics in Metabolic and Cardiovascular Syndromes; and equipment grants from the Wellcome Trust and Tenovus-Scotland. G.L.S. was supported by a National Heart, Lung, and Blood Institute SCOR Grant (5P50-HL54314) in Ischaemic Disease.
- The American Society for Pharmacology and Experimental Therapeutics
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