Abstract
Despite coupling to the same class of inhibitory G proteins and binding the same physiological ligand, the human A1 and rat A3 adenosine receptors (ARs) desensitize at different rates in response to sustained agonist exposure. This is due to the ability of the A3AR, but not the A1AR, to serve as a substrate for rapid phosphorylation and desensitization by members of the G protein-coupled receptor kinase (GRK) family. The aim of this study was to investigate whether these differences were also manifested in their abilities to undergo agonist-dependent receptor internalization. For the first time, we report that A3ARs internalize profoundly in response to short-term exposure to agonist but not activators of second messenger-regulated kinases. The A3AR-selective antagonist MRS1523 blocked both A3AR phosphorylation and internalization. Moreover, in contrast to the A1AR, which internalized quite slowly (t 1/2 = 90 min), A3ARs internalized rapidly (t 1/2 = 10 min) over a time frame that followed the onset of receptor phosphorylation. A nonphosphorylated A3AR mutant failed to internalize over a 60-min time course, suggesting that receptor phosphorylation was essential for rapid A3AR internalization to occur. In addition, fusion onto the A1AR of the A3AR C-terminal domain containing the sites for phosphorylation by GRKs conferred rapid agonist-induced internalization kinetics (t 1/2 = 10 min) on the resulting chimeric AR. In conclusion, these data suggest that GRK-stimulated phosphorylation of threonine residues within the C-terminal domain of the A3AR is obligatory to observe rapid agonist-mediated internalization of the receptor.
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 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. K.R.W. was supported by a studentship from the UK Biotechnology and Biological Sciences Research Council.
- The American Society for Pharmacology and Experimental Therapeutics
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