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

Research output: Contribution to journalArticle

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Abstract

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a nucleotide-binding a 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 rGiα1) 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-agarose as the chromatographic matrix. We have detected differences in the abilities of the purified proteins to support ADP-ribosylation of Giα1, these differences are attributable to the 7 component of the complex. When assayed for their ability to inhibit calmodulin-stimulated type-I adenylylcyclase activity or to potentiate G-stimulated type-II adenylylcyclase, recombinant β1γ1 and transducin βγ are approximately 10 and 20 times less potent, respectively, than the other complexes examined. Prenylation and/or further carboxylterminal 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 - Nov 15 1992

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Prenylation
Sf9 Cells
Guanine Nucleotides
Protein Subunits
GTP-Binding Proteins
Carrier Proteins
Adenosine Diphosphate
Transducin
Pertussis Toxin
Calmodulin
Chromatography
Sepharose
Proteins
Nucleotides
Processing

ASJC Scopus subject areas

  • Biochemistry

Cite this

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.

G protein βγ subunits synthesized in Sf9 cells : Functional characterization and the significance of prenylation of γ. / Iñiguez-Lluhi, Jorge A.; Simon, Melvin I.; Robishaw, Janet D.; Gilman, Alfred G.

In: Journal of Biological Chemistry, Vol. 267, No. 32, 15.11.1992, p. 23409-23417.

Research output: Contribution to journalArticle

Iñiguez-Lluhi, JA, Simon, MI, Robishaw, JD & Gilman, AG 1992, 'G protein βγ subunits synthesized in Sf9 cells: Functional characterization and the significance of prenylation of γ', Journal of Biological Chemistry, vol. 267, no. 32, pp. 23409-23417.
Iñiguez-Lluhi, Jorge A. ; Simon, Melvin I. ; Robishaw, Janet D. ; Gilman, Alfred G. / G protein βγ subunits synthesized in Sf9 cells : Functional characterization and the significance of prenylation of γ. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 32. pp. 23409-23417.
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