Recovery of homogeneous and functional 2-adrenergic receptors from extracellular baculovirus particles

Expression in haculovirus-infected insect cells allows sufficient production of G-protein coupled receptor for structural studies. An important drawback of this expression system comes from the presence of unprocessed and biologically inactive receptors that have to be eliminated during receptor purification steps. We show that vire particles released from Sf9 cells infected with a recombinant baculovirus coding for the human 2-adrenergic receptor (2AR) cDNA contain glycosylated and biologically active 2AR. In addition, post-translational modifications known to modulate receptor activity were found to occur in these particles.

1. Strader, C.D., Fong, T.M., Tota, M.R., and Underwood, D. 1994. Structure and function of G protein-coupled receptors. Annu. Rev. Biochem. 63: 101−132. | Article | PubMed  | ISI | ChemPort |
2. Ostrowski, J., Kjelsberg, M.A., Caron, M.G., and Lefkowitz, R.J. 1992. Mutagenesis of the 2-adrenergic receptor: how structure elucidates function. Annu. Rev. Pharmacol. Toxicol. 32: 167−183. | PubMed  | ISI | ChemPort |
3. George, S.T., Arbabian, M.A., Ruoho, A.E., Kiely, J., and Malbon, C.C. 1989. High-efficiency expression of a mammalian -adrenergic receptors in baculovirus-infected insect cells. Biochem. Biophys. Res. Commun. 163: 1265−1269. | PubMed  | ISI | ChemPort |
4. Kobilka, B.K. 1995. Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal. Biochem. 231: 269−271. | Article | PubMed  | ISI | ChemPort |
5. Parker, E.M., Kameyama, K., Higashijima, T., and Ross, E.M. 1991. Reconstitutively active G protein-coupled receptors purified from baculovirus-infected insect cells. J. Biol. Chem. 266: 519−527. | PubMed  | ISI | ChemPort |
6. Mouillac, B., Caron, M., Bonin, H., Dennis, M., and Bouvier, M. 1992. Agonist-modulated palmitoylation of 2-adrenergic receptor in Sf9 cells. J. Biol. Chem. 267: 21733−21737. | PubMed  | ISI | ChemPort |
7. Ravet, V., Blin, N., Guillaume, J.-L., Petitjean, F., Cabanie, L., and Strosberg, A.D. 1993. High level functional expression of human 1-adrenergic receptor in bacutovirus-infected cells screened by a rapid in situ procedure. J. Receptor Res. 13: 541−558. | ISI | ChemPort |
8. Quehenberger, O., Prossnitz, E.R., Cochrane, C.G., and Ye, R.D. 1992. Absence of Gi proteins in the Sf9 insect cell. J. Biol. Chem. 267: 19757−19760. | PubMed  | ISI | ChemPort |
9. Royer, M., Hong, S.-S., Gay, B., Cerutti, M., and Boulanger, P. 1992. Expression and extracellular release of human immunodeficiency virus type 1 Gag precursors by recombinant baculovirus-infected cells. J. Virol. 66: 3230−3235. | PubMed  | ISI | ChemPort |
10. Roy, P. and Jones, I. 1996. Assembly of macromolecular complexes in bacterial and baculovirus expression systems. Curr. Opin. Struct. Biol. 6: 157−161. | Article | PubMed  | ISI | ChemPort |
11. Gheysen, D., Jacobs, E., De Foresta, F., Thiriart, C., Francotta, M., Thines, D., et al. 1989. Assembly and release of HIV-1 precursor Pr55Gag virus-like particles from recombinant baculovirus-infected insect cells. Cell 59: 103−112. | Article | PubMed  | ISI | ChemPort |
12. Delchambre, M., Gheysen, D., Thines, D., Thiriart, C., Jacobs, E., Verdin, E., et al. 1989. The GAG precursor of simian immunodeficiency virus assembles into virus-like particles. EMBO J. 8: 2653−2660. | PubMed  | ISI | ChemPort |
13. Thomsen, D.R., Meyer, A.L., and Post, L.E. 1992. Expression of feline leukaemia virus gp85 and Gag proteins and assembly into virus-like particles using the baculovirus expression system. J. of Gen. Virol. 73: 1819−1824. | ISI | ChemPort |
14. Gamier, L., Ravallec, M., Blanchard, P., Chaabihi, H., Bossy, J.-P., Devauchelle, G., et al. 1995. Incorporation of pseudorabies virus gD into human immunodeficiency virus type 1 Gag particles produced in baculovirus-infected cells. J. Virol. 69: 4060−4068. | PubMed  |
15. Nguyen, C.T., Suzuki, T., Nantel, F., Bonin, H., Valiquette, M., Frielle, T., et al. 1992. Distinct regulation of 1-and 2-adrenergic receptors in Chinese hamster fibroblasts. Mol. Pharmacol. 41: 542−548. | PubMed  | ISI |
16. Hunter, E. 1994. Macromolecular interactions in the assembly of HIV and other retroviruses. Seminars in Virology 5: 71−83. | Article | ISI | ChemPort |
17. Chaabihi, H., Fournier, D., Fedon, Y., Bossy, J.-P., Ravallec, M., Devauchelle, G., et al. 1994. Biochemical characterization of Drosophila malanogaster acetylcholinesterase expressed by recombinant baculoviruses. Biochem. Biophys. Res. Commun. 203: 734−742. | Article | PubMed  | ISI | ChemPort |
18. Miller, L.K. 1996. pp. 533−556. in Insect viruses, Vol 1.Lippincott-Raven, Philadelphia, PA.
19. Braunagel, S.C. and Summers, M.D. 1994. Autographa califomica nuclear poly-hedrosis virus, PDV, and ECV viral envelopes and nucleocapsids: structural proteins, antigens, lipidsand fatty acid profiles. Virology 202: 315−328. | Article | PubMed  | ISI | ChemPort |
20. Puddington, L., Lively, M.O., and Lyles, D.S. 1985. Role of the nuclear envelope in synthesis, processing, and transportof membrane glycoproteins. J. Biol. Chem. 260: 5641−5647. | PubMed  | ISI | ChemPort |
21. Belcheva, M.M., Gucker, S., Chuang, D.-M., Clark, W.G., Jefcoat, L.B., McHale, R.J., et al. 1995. Modulation of opioid binding associated with nuclear matrix and nuclear membranes of NG108-15 cells. J. Pharmacol. Exp. Ther. 274: 1513−1523. | PubMed  | ISI | ChemPort |
22. Knudson, D.L. and Tinsley, T.W. 1974. Replication of a nuclear polyhedrosis virus in a continuous cell culture of Spodoptera frugiperda: purification, assay of infec-tivity, and growth characteristics of the virus. J. Virol. 14: 934−944. | PubMed  | ISI | ChemPort |
23. Carriere, C., Gay, B., Chazal, N., Morin, N., and Boulanger, P. 1995. Sequence requirements for encapsidation of deletion mutants and chimeras of human immunodeficiency virus type 1 Gag precursor into retrovirus-like particles. J. Virol. 69: 2366−2377. | PubMed  | ISI | ChemPort |
24. Ng, G.Y., Mouillac, B., George, S., Caron, M., Dennis, M., Bouivier, M., et al. 1994. Desensitization, phosphorylation and palmitoylation of the human D1 dopamin receptor. Eur. J. Pharmacol. Mol. Pharmacol. Sec. 267: 7−19. | Article | ISI | ChemPort |
25. Kusui, T., Hellmich, M.R., Wang, L.-H., Evans, R.L., Benya, R.V., Battey, J.F., et al. 1995. Characterization of gastrin-releasing peptide receptor expressed in Sf9 insect cells by baculovirus. Biochemistry 34: 8061−8075. | PubMed  | ISI | ChemPort |
26. Gimpl, G., Anders, J., Thiele, C., and Fahrenholtz, F. 1996. Photoaffinity labeling of the human brain cholecystokinin receptor overexpressed in insect cells. Eur. J. Biochem. 237: 768−777. | PubMed  | ISI | ChemPort |
27. Loisel, T.P., Adam, L., Hebert, T., and Bouvier, M. 1996. Agonist stimulation increases the turnover rate of aAR-bound palmitate and promotes receptor depalmitoylatipn. Biochemistry 35: 15923−15932. | Article | PubMed  | ISI | ChemPort |
28. Sibley, D.R., Benovic, J.L., Caron, M.G., and Lefkowitz, R.J. 1987. Regulation of transmembrane signaling by receptor phosphorylation. Cell 48: 913−922. | Article | PubMed  | ISI | ChemPort |
29. Richardson, R.M., and Hosey, M.M. 1992. Agonist-induced phosphorylation and desensitization of human M2 muscarinic cholinergic receptors in Sf9 insect cells. J. Biol. Chem. 267: 22249−22255. | PubMed  | ISI | ChemPort |
30. Nebigil, C.G., Garnovskaya, M.N., Casanas, S.J., Mulheron, J.G., Parker, E.M., Gettys, T.W., et al. 1995. Agonist-induced desensitization and phosphorylation of human 5-HT1A receptor expressed in Sf9 insect cells. Biochemistry 34: 11954−11962. | PubMed  | ISI | ChemPort |
31. Dearry, A., Gingrich, J.A., Falardeau, P., Fremeau, R.T. Jr., Bates, M.D., Caron, M.G., et al. 1990. Molecular cloning and expression of the gene for the human D1 dopamine receptor. Nature 347: 72−76. | Article | PubMed  | ISI | ChemPort |
32. Allard, W.J., Sigal, I.S., and Dixon, R.A. 1987. Sequence of the gene encoding the human M1 muscarinic acetylcholine receptor. Nucl. Acids Res. 15: 10604. | PubMed  | ISI | ChemPort |
33. O'Reilly, D.R., Miller, L.K., and Luckow, V.A. 1992. Baculovirus expression vectors, a laboratory manual. W.H. Freeman and Co., New York.
34. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227: 680−686. | PubMed  | ISI | ChemPort |
35. Bouvier, M., Hnatowich, M., Collins, S., Kobilka, B.K., De Blasi, A., Lefkowitz, R.J., et al. 1988. Expression of a human cDNA encoding the 2-adrenergic receptor in Chinese hamster fibroblast (CHW): functionality and regulation of the expressed receptors. Mol. Pharmacol. 33: 133−139. | PubMed  | ISI | ChemPort |
36. Salomon, Y., Londos, C., and Rodbell, M. 1974. A highly sensitive adenylate cyclase assay. Anal. Biochem. 58: 541−548. | PubMed  | ISI | ChemPort |
37. Benovic, J.L., Shorr, R.G.L., Caron, M.G., and Lefkowitz, R.J. 1984. The mammalian 2-adrenergic receptor: purification and characterization. Biochemistry 23: 4510−4518. | PubMed  | ISI | ChemPort |
38. Evan, G.I., Lewis, G.K., Ramsay, G., and Bishop, J.M. 1985. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol. 5: 3610−3616. | PubMed  | ISI | ChemPort |
39. Raposo, G., Dunia, I., Delavier-Klutchko, C., Kaveri, S., Strosberg, A.D., Benedetti, E.L., et al. 1989. Intemalization of -Adrenergic receptor in A431 cells involves non-coatedd vesicles. Eur. J. Cell. Biol. 50: 340−352. | PubMed  | ISI | ChemPort |

	Top

	Top