Agonist-modulated palmitoylation of beta 2-adrenergic receptor in Sf9 cells.
The palmitoylation of the human beta 2-adrenergic receptor (beta 2-AR) was studied in recombinant baculovirus-infected insect Sf9 cells. At 48 h post-infection, a high level expression of an epitope-tagged beta 2-AR (10-25 pmol/mg protein) was detected by [125I]iodocyanopindolol ([125I]CYP) binding assays. The identity of the receptor was confirmed both by photoaffinity labeling and immunoblotting. The fusion receptor displayed typical beta 2-AR pharmacological properties and conferred a beta-adrenergic sensitive adenylyl cyclase activity to the Sf9 cells. Moreover, exposure of the Sf9 cells to the beta-adrenergic agonist isoproterenol induced a rapid desensitization of the receptor-stimulated adenylyl cyclase activity. Purification of the epitope-tagged beta 2-AR by immunoprecipitation as well as by alprenolol-Sepharose affinity chromatography revealed that the receptor is covalently modified with palmitic acid in the insect cells as is observed in mammalian cells. In addition, short-term incubation of the cells with isoproterenol led to a specific increase in the incorporation of [3H]palmitate in the receptor, consistent with a rapid agonist-modulated turnover of the beta 2-AR-attached palmitic acid. These results suggest that agonist-mediated regulation of beta 2-AR post-translational palmitoylation could represent an other regulatory process for G protein-coupled receptors.
Mouillac
B
B
Department of Biochemistry, University of Montreal, Quebec, Canada.
Caron
M
M
Bonin
H
H
Dennis
M
M
Bouvier
M
M
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D002478:Cells, Cultured; D005822:Genetic Vectors; D007313:Insecta; D020120:Iodocyanopindolol; D007545:Isoproterenol / Q000378:metabolism*; D008969:Molecular Sequence Data; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D010869:Pindolol / Q000031:analogs & derivatives / Q000737:chemistry; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics
D000345:Affinity Labels; D010169:Palmitic Acids; D016271:Proto-Oncogene Proteins c-myc; D011943:Receptors, Adrenergic, beta; D011993:Recombinant Fusion Proteins; D019308:Palmitic Acid; D020120:Iodocyanopindolol; D010869:Pindolol; D007545:Isoproterenol