G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma

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

Abstract

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.

Additional Information

Copyright © 1992 by the American Society for Biochemistry and Molecular Biology. (Received for publication, June 25, 1992) We thank Dr. Randall Reed (Johns Hopkins University) and Dr. Boning Gao (University of Texas Southwestern) for β1 and β2 cDNAs, respectively, Dr. N. Gautam (California Institute of Technology) for γ1 and γ2 constructs and NG-1 antiserum, Dr. Susanne Mumby (University of Texas Southwestern) for the γ2C68S construct and for purified transducin βγ, Dr. Wei-Jen Tang (University of Texas Southwestern) for purified rG[], recombinant Drosophila calmodulin, and adenylylcyclase baculoviruses, Dr. Maurine Linder (University of Texas Southwestern) for rG[], and Ethan Lee (University of Texas Southwestern) for bovine brain G[]. We also thank Dr. Paul Sternweis for many helpful suggestions and Vivian Kolman for excellent technical assistance. This work was supported in part by National Institutes of Health Grants GM34497, GM34236, and GM39867, American Cancer Society Grant BE30N, the Perot Family Foundation, the Lucille P. Markey Charitable Trust, and the Raymond and Ellen Willie Chair of Molecular Neuropharmacology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. [J.A.I.-L. was the] [r]ecipient of a Howard Hughes Medical Institute predoctoral training award. [J.D.R. was an] Established Investigator of the American Heart Association.

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