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| RECONSTITUTIVELY ACTIVE G-PROTEIN-COUPLED RECEPTORS PURIFIED FROM BACULOVIRUS-INFECTED INSECT CELLS |
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| Author(s): PARKER EM, KAMEYAMA K, HIGASHIJIMA T, ROSS EM |
| Source: JOURNAL OF BIOLOGICAL CHEMISTRY Volume: 266 Issue: 1 Pages: 519-527 Published: JAN 5 1991 |
| Times Cited: 288 References: 71 |
| Abstract: The turkey beta-adrenergic receptor (beta-AR), the m1 and m2 forms of the human muscarinic cholinergic receptor (MAChR) and several other mutant and wild-type G protein-coupled receptors were produced in insect Sf9 cells by infection with recombinant baculoviruses. Maximal expression for most receptors was 5-30 pmol receptor/mg protein (2-15 nmol/liter culture). The receptors displayed typical ligand binding characteristics. The beta-AR was glycosylated; electrophoretic behavior of the two MAChRs also suggested glycosylation. The beta-AR stimulated endogenous adenylyl cyclase in response to beta-adrenergic agonists. The beta-AR and both MAChRs were purified and coreconstituted with various purified G proteins in phospholipid vesicles. The recombinant beta-AR catalyzed the agonist-dependent activation of G(s) by guanosine5'-O-(thiotriphosphate) (GTP-gamma-S) with the same efficiency as did the natural beta-AR. The m2 MAChR efficiently catalyzed GTP-gamma-S binding to G(o) and to the recently identified G protein G(z) (G(x)). The m2 MAChR also catalyzed the activation of G(i),1 and G(i),3 weakly. Activation of these same G proteins by the ml MAChR was much less efficient, consistent with its known selectivity for pertussis toxin-insensitive G proteins ("G(p)") that have not yet been isolated. The beta-AR and m2 MAChR were characteristically stimulated by reduction of disulfides. These results demonstrate the general utility of the baculovirus system for production of large quantities of native G protein-coupled receptors. |
| Document Type: Article |
| Language: English |
| Reprint Address: PARKER, EM (reprint author), UNIV TEXAS, SW MED CTR, SW GRAD SCH BIOMED SCI, DEPT PHARMACOL, DALLAS, TX 75235 USA |
Addresses:
1. UNIV TOKYO, FAC MED, INST BRAIN RES, DEPT BIOCHEM, BUNKYO KU, TOKYO 113, JAPAN |
| Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: EQ339 |
| ISSN: 0021-9258 |
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| | | | | | | | | Record from Web of Science® | | | | | | | | | |