G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma.
Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a nucleotide-binding alpha subunit and a high-affinity complex of beta and gamma subunits. There is molecular heterogeneity of beta and gamma, but the significance of this diversity is poorly understood. Different G protein beta and gamma subunits have been expressed both singly and in combinations in Sf9 cells. Although expression of individual subunits is achieved in all cases, beta gamma subunit activity (support of pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1) is detected only when beta and gamma are expressed concurrently. Of the six combinations of beta gamma tested (beta 1 or beta 2 with gamma 1, gamma 2, or gamma 3), only one, beta 2 gamma 1, failed to generate a functional complex. Each of the other five complexes has been purified by subunit exchange chromatography using Go alpha-agarose as the chromatographic matrix. We have detected differences in the abilities of the purified proteins to support ADP-ribosylation of Gi alpha 1; these differences are attributable to the gamma component of the complex. When assayed for their ability to inhibit calmodulin-stimulated type-I adenylylcyclase activity or to potentiate Gs alpha-stimulated type-II adenylylcyclase, recombinant beta 1 gamma 1 and transducin beta gamma are approximately 10 and 20 times less potent, respectively, than the other complexes examined. Prenylation and/or further carboxyl-terminal processing of gamma are not required for assembly of the beta gamma subunit complex but are indispensable for high affinity interactions of beta gamma with either G protein alpha subunits or adenylylcyclases.
Iñiguez-Lluhi
J A
JA
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Simon
M I
MI
Robishaw
J D
JD
Gilman
A G
AG
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007313:Insecta; D007700:Kinetics; D046911:Macromolecular Substances; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D014162:Transfection
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000246:Adenosine Diphosphate Ribose; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases