TY - JOUR
T1 - G protein βγ subunits synthesized in Sf9 cells
T2 - Functional characterization and the significance of prenylation of γ
AU - Iñiguez-Lluhi, Jorge A.
AU - Simon, Melvin I.
AU - Robishaw, Janet D.
AU - Gilman, Alfred G.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1992/11/15
Y1 - 1992/11/15
N2 - 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 Goα-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 G8α-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.
AB - 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 Goα-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 G8α-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.
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M3 - Article
C2 - 1429682
AN - SCOPUS:0026452219
SN - 0021-9258
VL - 267
SP - 23409
EP - 23417
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 32
ER -