Reconstitutively active G protein-coupled receptors purified from baculovirus-infected insect cells

Eric M. Parker, Kimihiko Kameyama, Tsutomu Higashijima, Elliott M. Ross

Research output: Contribution to journalArticle

260 Scopus citations

Abstract

The turkey β-adrenergic receptor (β-AR),the m1 and m2 forms of the human muscarinic cholingeric 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 β-AR was glycosylated; electrophoretic behavior of the two MAChRs also suggested glycosylation. The β-AR stimulated endogenous adenylyl cyclase in response to β-adrenergic agonists. The β-AR and both MAChRs were purified and core constituted with various purified G proteins in phospholipid vesicles. The recombinant β-AR catalyzed the agonist-dependent activation of G(s) by guanosine 5'-O-(thiotriphosphate) (GTPγS) with the same efficiency as did the natural β-AR. The m2 MAChR efficiently catalyzed GTPγS 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 β-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.

Original languageEnglish (US)
Pages (from-to)519-527
Number of pages9
JournalJournal of Biological Chemistry
Volume266
Issue number1
StatePublished - Jan 31 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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