DOI: 10.1111/j.1432-1033.1992.tb16744.x. Subunit interactions of GTP‐binding proteins

Subunit interactions of GTP‐binding proteins

10.1111/j.1432-1033.1992.tb16744.x

Crossref journal-article

Wiley

European Journal of Biochemistry (311)

Abstract

Fluorescence energy transfer [cf. Förster, T. (1948) Ann. Phys. 6, 55–75] was tested for its suitability to study quantitative interactions of subunits of Go with each other and these subunits or trimeric Go with the β1‐adrenoceptor in detergent micelles or after reconstitution into lipid vesicles [according to Feder, D., Im, M.‐J., Klein, H. W., Hekman, M., Holzhöfer, A, Dees, C., Levitzki, A., Helmreich, E. J. M. & Pfeuffer, T. (1986) EMBO J. 5, 1509–1514]. For this purpose, αo‐ and βγ‐subunits and trimeric Go purified from bovine brain, the βγ‐subunits from bovine rod outer segment membranes and the β1‐adrenoceptor from the turkey erythrocyte were all labelled with either tetramethylrhodaminmaleimide or fluorescein isothiocyanate under conditions which leave the labelled proteins functionally intact. In the case of αo‐ and βγ‐interactions, specific high‐affinity binding interactions (Kd∼ 10 nM) and nonspecific low‐affinity binding interactions (Kd∼ 1 μM) could be readily distinguished by comparing fluorescence energy transfer before and after dissociation with 10 μM guanosine 5′‐O‐[γ‐thio]triphosphate and 10 mM MgCl2 where only low‐affinity binding interactions remained. Interactions between αo‐ and βγ‐subunits from bovine brain or from bovine retinal transducin did not differ much. The βγ‐subunits from bovine brain were found to bind with high transfer efficiency and comparable affinities to the hormone‐activated and the nonactivated β1‐receptor reconstituted in lipid vesicles: Kd= 100 ± 20 and 120 ± 20 nM, respectively; however, βγ‐subunits from transducin appeared to bind more weakly to the β1‐adrenoceptor than βγ‐subunits from bovine brain. Separated purified homologous αo‐ and βγ‐subunits from bovine brain interfered mutually with each other in binding to the β1‐adrenoceptor presumably because they had a greater affinity for each other than for the receptor. These findings attest to the suitability of fluorescence energy transfer for studying protein – protein interactions of G‐proteins and G‐protein‐linked receptors. Moreover, they supported the previous finding [Kurstjens, N. P., Fröhlich, M., Dees, C., Cantrill, R. C., Hekman, M. & Helmreich, E. J. M. (1991) Eur. J. Biochem. 197, 167–176] that βγ‐subunits can bind to the nonactivated β1‐adrenoceptor.

Bibliography

HEITHIER, H., FRÃHLICH, M., DEES, C., BAUMANN, M., HÃRING, M., GIERSCHIK, P., SCHILTZ, E., VAZ, W. L. C., HEKMAN, M., & HELMREICH, E. J. M. (1992). Subunit interactions of GTPâbinding proteins. European Journal of Biochemistry, 204(3), 1169â1181. Portico.

Authors 10

Helmuth HEITHIER (first)
Margareta FRÖHLICH (additional)
Christian DEES (additional)
Martin BAUMANN (additional)
Martin HÄRING (additional)
Peter GIERSCHIK (additional)
Emile SCHILTZ (additional)
Winchil L. C. VAZ (additional)
Mirko HEKMAN (additional)
Ernst J. M. HELMREICH (additional)

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Dates

Type	When
Created	20 years, 5 months ago (March 4, 2005, 12:46 a.m.)
Deposited	1 year, 9 months ago (Nov. 23, 2023, 12:09 p.m.)
Indexed	1 year, 9 months ago (Nov. 23, 2023, 12:44 p.m.)
Issued	33 years, 5 months ago (March 1, 1992)
Published	33 years, 5 months ago (March 1, 1992)
Published Online	20 years, 5 months ago (March 3, 2005)
Published Print	33 years, 5 months ago (March 1, 1992)

Funders 0

None

BibTeX

@article{HEITHIER_1992, title={Subunit interactions of GTP‐binding proteins}, volume={204}, ISSN={1432-1033}, url={http://dx.doi.org/10.1111/j.1432-1033.1992.tb16744.x}, DOI={10.1111/j.1432-1033.1992.tb16744.x}, number={3}, journal={European Journal of Biochemistry}, publisher={Wiley}, author={HEITHIER, Helmuth and FRÖHLICH, Margareta and DEES, Christian and BAUMANN, Martin and HÄRING, Martin and GIERSCHIK, Peter and SCHILTZ, Emile and VAZ, Winchil L. C. and HEKMAN, Mirko and HELMREICH, Ernst J. M.}, year={1992}, month=mar, pages={1169–1181} }

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