Palmitoylation of the recombinant human A1 adenosine receptor (A1AR) expressed in HEK-293 cells is demonstrated by showing that hexahistidine (His6)/Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG) (H/F) A1ARs, purified to homogeneity from cells metabolically labelled with [3H]palmitate, incorporate tritium into a 38-42 kDa receptor glycoprotein. The amount of palmitoylation is not affected by incubation of cells with the A1AR-selective agonist N6-cyclopentyladenosine (CPA). A1AR palmitoylation is abolished by treatment with neutral hydroxylamine or by mutation of Cys-309 to Ala (C309 → A). Based on Western blotting and pulse-chase experiments with [35S]methionine, at least 90% of wild-type receptors are palmitoylated and turn over with a t½ of 6.4 h. Of the C309 → A mutated receptors, 40% appear to turn over like wild-type receptors, with a t½ of 7.1 h, and 60% appear to be rapidly cleaved to form a 25 kDa receptor fragment that turns over with a t½ of 0.8 h. In HEK-293 cell lines expressing similar numbers of wild-type or C309 → A mutant A1Rs, there is little difference in the kinetics of CPA-induced receptor internalization (1 h), down-regulation (24 h), inhibition of forskolin-stimulated cAMP accumulation, or activation of co-transfected G-protein-activated inward rectifier K+/cardiac inward rectifying K+ (GIRK1/CIR K+) channels. Also unaffected by palmitoylation is guanosine 5′-[γ-thio]-triphosphate ([S]GTP)-sensitive binding to membranes by the agonist 125I-labelled aminobenzyladenosine. The results suggest that palmitoylation has little effect on receptor-effector coupling, agonist-induced internalization or down-regulation. We speculate that palmitoylation may divert newly synthesized A1ARs from a pathway leading to rapid degradation.
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September 1999
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Research Article|
August 24 1999
Palmitoylation of the recombinant human A1 adenosine receptor: enhanced proteolysis of palmitoylation-deficient mutant receptors
Zhenhai GAO;
Zhenhai GAO
*Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A.
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Yajun NI;
Yajun NI
*Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A.
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Gabor SZABO;
Gabor SZABO
*Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A.
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Joel LINDEN
Joel LINDEN
1
*Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A.
†Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908, U.S.A.
1To whom correspondence should be addressed (e-mail jlinden!virginia.edu).
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Publisher: Portland Press Ltd
Received:
April 01 1999
Revision Received:
May 21 1999
Accepted:
June 16 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 342 (2): 387–395.
Article history
Received:
April 01 1999
Revision Received:
May 21 1999
Accepted:
June 16 1999
Citation
Zhenhai GAO, Yajun NI, Gabor SZABO, Joel LINDEN; Palmitoylation of the recombinant human A1 adenosine receptor: enhanced proteolysis of palmitoylation-deficient mutant receptors. Biochem J 1 September 1999; 342 (2): 387–395. doi: https://doi.org/10.1042/bj3420387
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