Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Sep 1;342(Pt 2):387–395.

Palmitoylation of the recombinant human A1 adenosine receptor: enhanced proteolysis of palmitoylation-deficient mutant receptors.

Z Gao 1, Y Ni 1, G Szabo 1, J Linden 1
PMCID: PMC1220476  PMID: 10455026

Abstract

Palmitoylation of the recombinant human A(1) adenosine receptor (A(1)AR) expressed in HEK-293 cells is demonstrated by showing that hexahistidine (His(6))/Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG) (H/F) A(1)ARs, purified to homogeneity from cells metabolically labelled with [(3)H]palmitate, incorporate tritium into a 38-42 kDa receptor glycoprotein. The amount of palmitoylation is not affected by incubation of cells with the A(1)AR-selective agonist N(6)-cyclopentyladenosine (CPA). A(1)AR palmitoylation is abolished by treatment with neutral hydroxylamine or by mutation of Cys-309 to Ala (C(309)-->A). Based on Western blotting and pulse-chase experiments with [(35)S]methionine, at least 90% of wild-type receptors are palmitoylated and turn over with a t1/2 of 6.4 h. Of the C(309)-->A mutated receptors, 40% appear to turn over like wild-type receptors, with a t1/2 of 7.1 h, and 60% appear to be rapidly cleaved to form a 25 kDa receptor fragment that turns over with a t1/2 of 0.8 h. In HEK-293 cell lines expressing similar numbers of wild-type or C(309)-->A mutant A(1)Rs, 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'-[gamma-thio]-triphosphate ([S]GTP)-sensitive binding to membranes by the agonist (125)I-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 A(1)ARs from a pathway leading to rapid degradation.

Full Text

The Full Text of this article is available as a PDF (240.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alaluf S., Mulvihill E. R., McIlhinney R. A. The metabotropic glutamate receptor mGluR4, but not mGluR1 alpha, is palmitoylated when expressed in BHK cells. J Neurochem. 1995 Apr;64(4):1548–1555. doi: 10.1046/j.1471-4159.1995.64041548.x. [DOI] [PubMed] [Google Scholar]
  2. Bhattacharya S., Linden J. Effects of long-term treatment with the allosteric enhancer, PD81,723, on Chinese hamster ovary cells expressing recombinant human A1 adenosine receptors. Mol Pharmacol. 1996 Jul;50(1):104–111. [PubMed] [Google Scholar]
  3. Bouvier M., Chidiac P., Hebert T. E., Loisel T. P., Moffett S., Mouillac B. Dynamic palmitoylation of G-protein-coupled receptors in eukaryotic cells. Methods Enzymol. 1995;250:300–314. doi: 10.1016/0076-6879(95)50080-4. [DOI] [PubMed] [Google Scholar]
  4. Brooker G., Terasaki W. L., Price M. G. Gammaflow: a completely automated radioimmunoassay system. Science. 1976 Oct 15;194(4262):270–276. doi: 10.1126/science.184530. [DOI] [PubMed] [Google Scholar]
  5. Bünemann M., Pott L. Down-regulation of A1 adenosine receptors coupled to muscarinic K+ current in cultured guinea-pig atrial myocytes. J Physiol. 1995 Jan 1;482(Pt 1):81–92. doi: 10.1113/jphysiol.1995.sp020501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eason M. G., Jacinto M. T., Theiss C. T., Liggett S. B. The palmitoylated cysteine of the cytoplasmic tail of alpha 2A-adrenergic receptors confers subtype-specific agonist-promoted downregulation. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11178–11182. doi: 10.1073/pnas.91.23.11178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Figler R. A., Graber S. G., Lindorfer M. A., Yasuda H., Linden J., Garrison J. C. Reconstitution of recombinant bovine A1 adenosine receptors in Sf9 cell membranes with recombinant G proteins of defined composition. Mol Pharmacol. 1996 Dec;50(6):1587–1595. [PubMed] [Google Scholar]
  8. Gao Z., Robeva A. S., Linden J. Purification of A1 adenosine receptor-G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling. Biochem J. 1999 Mar 15;338(Pt 3):729–736. [PMC free article] [PubMed] [Google Scholar]
  9. Horstmeyer A., Cramer H., Sauer T., Müller-Esterl W., Schroeder C. Palmitoylation of endothelin receptor A. Differential modulation of signal transduction activity by post-translational modification. J Biol Chem. 1996 Aug 23;271(34):20811–20819. doi: 10.1074/jbc.271.34.20811. [DOI] [PubMed] [Google Scholar]
  10. Karnik S. S., Ridge K. D., Bhattacharya S., Khorana H. G. Palmitoylation of bovine opsin and its cysteine mutants in COS cells. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):40–44. doi: 10.1073/pnas.90.1.40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kawate N., Menon K. M. Palmitoylation of luteinizing hormone/human choriogonadotropin receptors in transfected cells. Abolition of palmitoylation by mutation of Cys-621 and Cys-622 residues in the cytoplasmic tail increases ligand-induced internalization of the receptor. J Biol Chem. 1994 Dec 2;269(48):30651–30658. [PubMed] [Google Scholar]
  12. Kennedy M. E., Limbird L. E. Mutations of the alpha 2A-adrenergic receptor that eliminate detectable palmitoylation do not perturb receptor-G-protein coupling. J Biol Chem. 1993 Apr 15;268(11):8003–8011. [PubMed] [Google Scholar]
  13. Kennedy M. E., Limbird L. E. Palmitoylation of the alpha 2A-adrenergic receptor. Analysis of the sequence requirements for and the dynamic properties of alpha 2A-adrenergic receptor palmitoylation. J Biol Chem. 1994 Dec 16;269(50):31915–31922. [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Leid M., Schimerlik M. I., Murray T. F. Agonist radioligand interactions with the solubilized porcine atrial A1 adenosine receptor. Mol Pharmacol. 1989 Apr;35(4):450–457. [PubMed] [Google Scholar]
  16. Linden J. Cloned adenosine A3 receptors: pharmacological properties, species differences and receptor functions. Trends Pharmacol Sci. 1994 Aug;15(8):298–306. doi: 10.1016/0165-6147(94)90011-6. [DOI] [PubMed] [Google Scholar]
  17. Linden J., Patel A., Sadek S. [125I]Aminobenzyladenosine, a new radioligand with improved specific binding to adenosine receptors in heart. Circ Res. 1985 Feb;56(2):279–284. doi: 10.1161/01.res.56.2.279. [DOI] [PubMed] [Google Scholar]
  18. Linden J., Patel A., Sadek S. [125I]Aminobenzyladenosine, a new radioligand with improved specific binding to adenosine receptors in heart. Circ Res. 1985 Feb;56(2):279–284. doi: 10.1161/01.res.56.2.279. [DOI] [PubMed] [Google Scholar]
  19. Loisel T. P., Adam L., Hebert T. E., Bouvier M. Agonist stimulation increases the turnover rate of beta 2AR-bound palmitate and promotes receptor depalmitoylation. Biochemistry. 1996 Dec 10;35(49):15923–15932. doi: 10.1021/bi9611321. [DOI] [PubMed] [Google Scholar]
  20. Moffett S., Adam L., Bonin H., Loisel T. P., Bouvier M., Mouillac B. Palmitoylated cysteine 341 modulates phosphorylation of the beta2-adrenergic receptor by the cAMP-dependent protein kinase. J Biol Chem. 1996 Aug 30;271(35):21490–21497. doi: 10.1074/jbc.271.35.21490. [DOI] [PubMed] [Google Scholar]
  21. Moffett S., Mouillac B., Bonin H., Bouvier M. Altered phosphorylation and desensitization patterns of a human beta 2-adrenergic receptor lacking the palmitoylated Cys341. EMBO J. 1993 Jan;12(1):349–356. doi: 10.1002/j.1460-2075.1993.tb05663.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Morello J. P., Bouvier M. Palmitoylation: a post-translational modification that regulates signalling from G-protein coupled receptors. Biochem Cell Biol. 1996;74(4):449–457. doi: 10.1139/o96-049. [DOI] [PubMed] [Google Scholar]
  23. Mouillac B., Caron M., Bonin H., Dennis M., Bouvier M. Agonist-modulated palmitoylation of beta 2-adrenergic receptor in Sf9 cells. J Biol Chem. 1992 Oct 25;267(30):21733–21737. [PubMed] [Google Scholar]
  24. Ng G. Y., George S. R., Zastawny R. L., Caron M., Bouvier M., Dennis M., O'Dowd B. F. Human serotonin1B receptor expression in Sf9 cells: phosphorylation, palmitoylation, and adenylyl cyclase inhibition. Biochemistry. 1993 Nov 2;32(43):11727–11733. doi: 10.1021/bi00094a032. [DOI] [PubMed] [Google Scholar]
  25. Ng G. Y., Mouillac B., George S. R., Caron M., Dennis M., Bouvier M., O'Dowd B. F. Desensitization, phosphorylation and palmitoylation of the human dopamine D1 receptor. Eur J Pharmacol. 1994 Mar 15;267(1):7–19. doi: 10.1016/0922-4106(94)90219-4. [DOI] [PubMed] [Google Scholar]
  26. Ng G. Y., O'Dowd B. F., Caron M., Dennis M., Brann M. R., George S. R. Phosphorylation and palmitoylation of the human D2L dopamine receptor in Sf9 cells. J Neurochem. 1994 Nov;63(5):1589–1595. doi: 10.1046/j.1471-4159.1994.63051589.x. [DOI] [PubMed] [Google Scholar]
  27. O'Dowd B. F., Hnatowich M., Caron M. G., Lefkowitz R. J., Bouvier M. Palmitoylation of the human beta 2-adrenergic receptor. Mutation of Cys341 in the carboxyl tail leads to an uncoupled nonpalmitoylated form of the receptor. J Biol Chem. 1989 May 5;264(13):7564–7569. [PubMed] [Google Scholar]
  28. Okamoto Y., Ninomiya H., Tanioka M., Sakamoto A., Miwa S., Masaki T. Palmitoylation of human endothelinB. Its critical role in G protein coupling and a differential requirement for the cytoplasmic tail by G protein subtypes. J Biol Chem. 1997 Aug 22;272(34):21589–21596. doi: 10.1074/jbc.272.34.21589. [DOI] [PubMed] [Google Scholar]
  29. Papac D. I., Thornburg K. R., Büllesbach E. E., Crouch R. K., Knapp D. R. Palmitylation of a G-protein coupled receptor. Direct analysis by tandem mass spectrometry. J Biol Chem. 1992 Aug 25;267(24):16889–16894. [PubMed] [Google Scholar]
  30. Patel A., Craig R. H., Daluge S. M., Linden J. 125I-BW-A844U, an antagonist radioligand with high affinity and selectivity for adenosine A1 receptors, and 125I-azido-BW-A844U, a photoaffinity label. Mol Pharmacol. 1988 Jun;33(6):585–591. [PubMed] [Google Scholar]
  31. Pepperberg D. R., Morrison D. F., O'Brien P. J. Depalmitoylation of rhodopsin with hydroxylamine. Methods Enzymol. 1995;250:348–361. doi: 10.1016/0076-6879(95)50084-7. [DOI] [PubMed] [Google Scholar]
  32. Prater M. R., Taylor H., Munshi R., Linden J. Indirect effect of guanine nucleotides on antagonist binding to A1 adenosine receptors: occupation of cryptic binding sites by endogenous vesicular adenosine. Mol Pharmacol. 1992 Nov;42(5):765–772. [PubMed] [Google Scholar]
  33. Robeva A. S., Woodard R., Luthin D. R., Taylor H. E., Linden J. Double tagging recombinant A1- and A2A-adenosine receptors with hexahistidine and the FLAG epitope. Development of an efficient generic protein purification procedure. Biochem Pharmacol. 1996 Feb 23;51(4):545–555. doi: 10.1016/0006-2952(95)02235-x. [DOI] [PubMed] [Google Scholar]
  34. Sadeghi H. M., Innamorati G., Dagarag M., Birnbaumer M. Palmitoylation of the V2 vasopressin receptor. Mol Pharmacol. 1997 Jul;52(1):21–29. doi: 10.1124/mol.52.1.21. [DOI] [PubMed] [Google Scholar]
  35. Trussell L. O., Jackson M. B. Adenosine-activated potassium conductance in cultured striatal neurons. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4857–4861. doi: 10.1073/pnas.82.14.4857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wang J., Tu Y., Woodson J., Song X., Ross E. M. A GTPase-activating protein for the G protein Galphaz. Identification, purification, and mechanism of action. J Biol Chem. 1997 Feb 28;272(9):5732–5740. doi: 10.1074/jbc.272.9.5732. [DOI] [PubMed] [Google Scholar]
  37. Zhu H., Wang H., Ascoli M. The lutropin/choriogonadotropin receptor is palmitoylated at intracellular cysteine residues. Mol Endocrinol. 1995 Feb;9(2):141–150. doi: 10.1210/mend.9.2.7776964. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES