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. 1999 Mar 15;338(Pt 3):729–736.

Purification of A1 adenosine receptor-G-protein complexes: effects of receptor down-regulation and phosphorylation on coupling.

Z Gao 1, A S Robeva 1, J Linden 1
PMCID: PMC1220110  PMID: 10051446

Abstract

We examined the effects of exposing A1 adenosine receptors (A1ARs) to an agonist on the stability and phosphorylation state of receptor-guanine nucleotide-binding regulatory protein (R-G-protein) complexes. Non-denatured recombinant human A1ARs extended on the N-terminus with hexahistidine (His6) and the FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) epitope (H/F) were purified to near homogeneity from stably transfected Chinese-hamster ovary (CHO)-K1 cells. Purified receptors have pharmacological properties similar to receptors in membranes. G-proteins were co-purified with 15+/-2% of H/F-A1AR unless receptor-G-protein (R-G) complexes were uncoupled by pre-treating cell membranes with GTP. By silver staining, purified A1AR-G-protein complexes contain receptors, G-protein alpha and beta subunits and an unidentified 97 kDa protein. Pretreating intact cells with N6-cyclopentyladenosine (CPA) for 24 h decreased both the total number of receptors measured in membranes and the number of purified A1ARs by about 50%. In contrast, pretreating cells with CPA decreased the number of R-G complexes measured in membranes (54+/-6%) significantly less than it decreased the number of purified R-G complexes (78+/-3%) as detected by 125I-N6-(4-aminobenzyl)adenosine binding or by Western blotting Gialpha2. The effect of CPA to decrease the fraction of receptors purified as R-G complexes was not associated with any change in low-level A1AR phosphorylation (found on serine), or low-level phosphorylation of G-protein alpha or beta subunits or the 97 kDa protein. These experiments reveal a novel aspect of agonist-induced down-regulation, namely a diminished stability of receptor-G-protein complexes that is manifested as uncoupling during receptor purification.

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Selected References

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

  1. Aramori I., Ferguson S. S., Bieniasz P. D., Zhang J., Cullen B., Cullen M. G. Molecular mechanism of desensitization of the chemokine receptor CCR-5: receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor. EMBO J. 1997 Aug 1;16(15):4606–4616. doi: 10.1093/emboj/16.15.4606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asano T., Morishita R., Ueda H., Asano M., Kato K. GTP-binding protein gamma12 subunit phosphorylation by protein kinase C--identification of the phosphorylation site and factors involved in cultured cells and rat tissues in vivo. Eur J Biochem. 1998 Jan 15;251(1-2):314–319. doi: 10.1046/j.1432-1327.1998.2510314.x. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bushfield M., Griffiths S. L., Murphy G. J., Pyne N. J., Knowler J. T., Milligan G., Parker P. J., Mollner S., Houslay M. D. Diabetes-induced alterations in the expression, functioning and phosphorylation state of the inhibitory guanine nucleotide regulatory protein Gi-2 in hepatocytes. Biochem J. 1990 Oct 15;271(2):365–372. doi: 10.1042/bj2710365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bushfield M., Lavan B. E., Houslay M. D. Okadaic acid identifies a phosphorylation/dephosphorylation cycle controlling the inhibitory guanine-nucleotide-binding regulatory protein Gi2. Biochem J. 1991 Mar 1;274(Pt 2):317–321. doi: 10.1042/bj2740317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bushfield M., Murphy G. J., Lavan B. E., Parker P. J., Hruby V. J., Milligan G., Houslay M. D. Hormonal regulation of Gi2 alpha-subunit phosphorylation in intact hepatocytes. Biochem J. 1990 Jun 1;268(2):449–457. doi: 10.1042/bj2680449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ciruela F., Saura C., Canela E. I., Mallol J., Lluís C., Franco R. Ligand-induced phosphorylation, clustering, and desensitization of A1 adenosine receptors. Mol Pharmacol. 1997 Nov;52(5):788–797. doi: 10.1124/mol.52.5.788. [DOI] [PubMed] [Google Scholar]
  8. Cole G. M., Reed S. I. Pheromone-induced phosphorylation of a G protein beta subunit in S. cerevisiae is associated with an adaptive response to mating pheromone. Cell. 1991 Feb 22;64(4):703–716. doi: 10.1016/0092-8674(91)90500-x. [DOI] [PubMed] [Google Scholar]
  9. Dennis D. M., Shryock J. C., Belardinelli L. Homologous desensitization of the A1-adenosine receptor system in the guinea pig atrioventricular node. J Pharmacol Exp Ther. 1995 Mar;272(3):1024–1035. [PubMed] [Google Scholar]
  10. Dohlman H. G., Thorner J. RGS proteins and signaling by heterotrimeric G proteins. J Biol Chem. 1997 Feb 14;272(7):3871–3874. doi: 10.1074/jbc.272.7.3871. [DOI] [PubMed] [Google Scholar]
  11. Faurobert E., Chen C. K., Hurley J. B., Teng D. H. Drosophila neurocalcin, a fatty acylated, Ca2+-binding protein that associates with membranes and inhibits in vitro phosphorylation of bovine rhodopsin. J Biol Chem. 1996 Apr 26;271(17):10256–10262. doi: 10.1074/jbc.271.17.10256. [DOI] [PubMed] [Google Scholar]
  12. Feng Y., Song L. Y., Kincaid E., Mahanty S. K., Elion E. A. Functional binding between Gbeta and the LIM domain of Ste5 is required to activate the MEKK Ste11. Curr Biol. 1998 Feb 26;8(5):267–278. doi: 10.1016/s0960-9822(98)70108-3. [DOI] [PubMed] [Google Scholar]
  13. Ferguson S. S., Barak L. S., Zhang J., Caron M. G. G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins. Can J Physiol Pharmacol. 1996 Oct;74(10):1095–1110. doi: 10.1139/cjpp-74-10-1095. [DOI] [PubMed] [Google Scholar]
  14. Freedman N. J., Ament A. S., Oppermann M., Stoffel R. H., Exum S. T., Lefkowitz R. J. Phosphorylation and desensitization of human endothelin A and B receptors. Evidence for G protein-coupled receptor kinase specificity. J Biol Chem. 1997 Jul 11;272(28):17734–17743. doi: 10.1074/jbc.272.28.17734. [DOI] [PubMed] [Google Scholar]
  15. Freedman N. J., Lefkowitz R. J. Desensitization of G protein-coupled receptors. Recent Prog Horm Res. 1996;51:319–353. [PubMed] [Google Scholar]
  16. Freedman N. J., Liggett S. B., Drachman D. E., Pei G., Caron M. G., Lefkowitz R. J. Phosphorylation and desensitization of the human beta 1-adrenergic receptor. Involvement of G protein-coupled receptor kinases and cAMP-dependent protein kinase. J Biol Chem. 1995 Jul 28;270(30):17953–17961. doi: 10.1074/jbc.270.30.17953. [DOI] [PubMed] [Google Scholar]
  17. Georgoussi Z., Milligan G., Zioudrou C. Immunoprecipitation of opioid receptor-Go-protein complexes using specific GTP-binding-protein antisera. Biochem J. 1995 Feb 15;306(Pt 1):71–75. doi: 10.1042/bj3060071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hausdorff W. P., Pitcher J. A., Luttrell D. K., Linder M. E., Kurose H., Parsons S. J., Caron M. G., Lefkowitz R. J. Tyrosine phosphorylation of G protein alpha subunits by pp60c-src. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5720–5724. doi: 10.1073/pnas.89.13.5720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jewell-Motz E. A., Liggett S. B. G protein-coupled receptor kinase specificity for phosphorylation and desensitization of alpha2-adrenergic receptor subtypes. J Biol Chem. 1996 Jul 26;271(30):18082–18087. doi: 10.1074/jbc.271.30.18082. [DOI] [PubMed] [Google Scholar]
  20. Limbird L. E., Gill D. M., Lefkowitz R. J. Agonist-promoted coupling of the beta-adrenergic receptor with the guanine nucleotide regulatory protein of the adenylate cyclase system. Proc Natl Acad Sci U S A. 1980 Feb;77(2):775–779. doi: 10.1073/pnas.77.2.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Liu W. W., Mattingly R. R., Garrison J. C. Transformation of Rat-1 fibroblasts with the v-src oncogene increases the tyrosine phosphorylation state and activity of the alpha subunit of Gq/G11. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8258–8263. doi: 10.1073/pnas.93.16.8258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Luthin D. R., Eppler C. M., Linden J. Identification and quantification of Gi-type GTP-binding proteins that copurify with a pituitary somatostatin receptor. J Biol Chem. 1993 Mar 15;268(8):5990–5996. [PubMed] [Google Scholar]
  24. Luthin D. R., Eppler C. M., Linden J. Identification and quantification of Gi-type GTP-binding proteins that copurify with a pituitary somatostatin receptor. J Biol Chem. 1993 Mar 15;268(8):5990–5996. [PubMed] [Google Scholar]
  25. Matesic D. F., Manning D. R., Wolfe B. B., Luthin G. R. Pharmacological and biochemical characterization of complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein. J Biol Chem. 1989 Dec 25;264(36):21638–21645. [PubMed] [Google Scholar]
  26. McGraw D. W., Donnelly E. T., Eason M. G., Green S. A., Liggett S. B. Role of beta ARK in long-term agonist-promoted desensitisation of the beta 2-adrenergic receptor. Cell Signal. 1998 Mar;10(3):197–204. doi: 10.1016/s0898-6568(97)00112-5. [DOI] [PubMed] [Google Scholar]
  27. Morris N. J., Bushfield M., Lavan B. E., Houslay M. D. Multi-site phosphorylation of the inhibitory guanine nucleotide regulatory protein Gi-2 occurs in intact rat hepatocytes. Biochem J. 1994 Aug 1;301(Pt 3):693–702. doi: 10.1042/bj3010693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  29. Motulsky H. J., Ransnas L. A. Fitting curves to data using nonlinear regression: a practical and nonmathematical review. FASEB J. 1987 Nov;1(5):365–374. [PubMed] [Google Scholar]
  30. Munshi R., Linden J. Co-purification of A1 adenosine receptors and guanine nucleotide-binding proteins from bovine brain. J Biol Chem. 1989 Sep 5;264(25):14853–14859. [PubMed] [Google Scholar]
  31. Munshi R., Pang I. H., Sternweis P. C., Linden J. A1 adenosine receptors of bovine brain couple to guanine nucleotide-binding proteins Gi1, Gi2, and Go. J Biol Chem. 1991 Nov 25;266(33):22285–22289. [PubMed] [Google Scholar]
  32. Nanoff C., Mitterauer T., Roka F., Hohenegger M., Freissmuth M. Species differences in A1 adenosine receptor/G protein coupling: identification of a membrane protein that stabilizes the association of the receptor/G protein complex. Mol Pharmacol. 1995 Nov;48(5):806–817. [PubMed] [Google Scholar]
  33. Nanoff C., Waldhoer M., Roka F., Freissmuth M. G protein coupling of the rat A1-adenosine receptor--partial purification of a protein which stabilizes the receptor-G protein association. Neuropharmacology. 1997 Sep;36(9):1211–1219. doi: 10.1016/s0028-3908(97)00135-4. [DOI] [PubMed] [Google Scholar]
  34. Palmer T. M., Benovic J. L., Stiles G. L. Molecular basis for subtype-specific desensitization of inhibitory adenosine receptors. Analysis of a chimeric A1-A3 adenosine receptor. J Biol Chem. 1996 Jun 21;271(25):15272–15278. doi: 10.1074/jbc.271.25.15272. [DOI] [PubMed] [Google Scholar]
  35. Pals-Rylaarsdam R., Xu Y., Witt-Enderby P., Benovic J. L., Hosey M. M. Desensitization and internalization of the m2 muscarinic acetylcholine receptor are directed by independent mechanisms. J Biol Chem. 1995 Dec 1;270(48):29004–29011. doi: 10.1074/jbc.270.48.29004. [DOI] [PubMed] [Google Scholar]
  36. Parsons W. J., Stiles G. L. Heterologous desensitization of the inhibitory A1 adenosine receptor-adenylate cyclase system in rat adipocytes. Regulation of both Ns and Ni. J Biol Chem. 1987 Jan 15;262(2):841–847. [PubMed] [Google Scholar]
  37. Polakis P. G., Uhing R. J., Snyderman R. The formylpeptide chemoattractant receptor copurifies with a GTP-binding protein containing a distinct 40-kDa pertussis toxin substrate. J Biol Chem. 1988 Apr 5;263(10):4969–4976. [PubMed] [Google Scholar]
  38. Ramkumar V., Olah M. E., Jacobson K. A., Stiles G. L. Distinct pathways of desensitization of A1- and A2-adenosine receptors in DDT1 MF-2 cells. Mol Pharmacol. 1991 Nov;40(5):639–647. [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Shryock J., Patel A., Belardinelli L., Linden J. Downregulation and desensitization of A1-adenosine receptors in embryonic chicken heart. Am J Physiol. 1989 Feb;256(2 Pt 2):H321–H327. doi: 10.1152/ajpheart.1989.256.2.H321. [DOI] [PubMed] [Google Scholar]
  41. Strassheim D., Malbon C. C. Phosphorylation of Gi alpha 2 attenuates inhibitory adenylyl cyclase in neuroblastoma/glioma hybrid (NG-108-15) cells. J Biol Chem. 1994 May 13;269(19):14307–14313. [PubMed] [Google Scholar]
  42. Wennogle L. P., Conder L., Winter C., Braunwalder A., Vlattas S., Kramer R., Cioffi C., Hu S. I. Stabilization of C5a receptor--G-protein interactions through ligand binding. J Cell Biochem. 1994 Jul;55(3):380–388. doi: 10.1002/jcb.240550316. [DOI] [PubMed] [Google Scholar]
  43. Yang J., Logsdon C. D., Johansen T. E., Williams J. A. Human m3 muscarinic acetylcholine receptor carboxyl-terminal threonine resides are required for agonist-induced receptor down-regulation. Mol Pharmacol. 1993 Dec;44(6):1158–1164. [PubMed] [Google Scholar]
  44. Yasuda H., Lindorfer M. A., Myung C. S., Garrison J. C. Phosphorylation of the G protein gamma12 subunit regulates effector specificity. J Biol Chem. 1998 Aug 21;273(34):21958–21965. doi: 10.1074/jbc.273.34.21958. [DOI] [PubMed] [Google Scholar]
  45. Zhang J., Pratt R. E. The AT2 receptor selectively associates with Gialpha2 and Gialpha3 in the rat fetus. J Biol Chem. 1996 Jun 21;271(25):15026–15033. [PubMed] [Google Scholar]

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