G protein βγ subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of γ

Jorge A. Iñiguez-Lluhi, Melvin I. Simon, Janet D. Robishaw, Alfred G. Gilman

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254 Scopus citations

Abstract

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a nucleotide-binding α subunit and a high-affinity complex of β and γ subunits. There is molecular heterogeneity of β and γ, but the significance of this diversity is poorly understood. Different G protein β and γ subunits have been expressed both singly and in combinations in Sf9 cells. Although expression of individual subunits is achieved in all cases, βγ subunit activity (support of pertussis toxin-catalyzed ADP-ribosylation of rG(iα1)) is detected only when β and γ are expressed concurrently. Of the six combinations of βγ tested (β1 or β2 with γ1, γ2, or γ3), only one, β2γ1, failed to generate a functional complex. Each of the other five complexes has been purified by subunit exchange chromatography using G(oα)-agarose as the chromatographic matrix. We have detected differences in the abilities of the purified proteins to support ADP- ribosylation of G(iα1); these differences are attributable to the γ component of the complex. When assayed for their ability to inhibit calmodulin-stimulated type-I adenylylcyclase activity or to potentiate G(sα)-stimulated type-II adenylylcylase, recombinant β1γ1 and transducin βγ are approximately 10 and 20 times less potent, respectively, than the other complexes examined. Prenylation and/or further carboxyl-terminal processing of γ are not required for assembly of the βγ subunit complex but are indispensable for high affinity interactions of βγ with either G protein α subunits or adenylylcyclases.

Original languageEnglish (US)
Pages (from-to)23409-23417
Number of pages9
JournalJournal of Biological Chemistry
Volume267
Issue number32
StatePublished - Jan 1 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Iñiguez-Lluhi, J. A., Simon, M. I., Robishaw, J. D., & Gilman, A. G. (1992). G protein βγ subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of γ. Journal of Biological Chemistry, 267(32), 23409-23417.