title: Subunit interactions of GTP-binding proteins.
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 G0 with each other and these subunits or trimeric G0 with the beta 1-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, alpha 0- and beta gamma-subunits and trimeric G0 purified from bovine brain, the beta gamma-subunits from bovine rod outer segment membranes and the beta 1-adrenoceptor from the turkey erythrocyte were all labelled with either tetramethylrhodamine maleimide or fluorescein isothiocyanate under conditions which leave the labelled proteins functionally intact. In the case of alpha 0- and beta gamma-interactions, specific high-affinity binding interactions (Kd approximately 10 nM) and nonspecific low-affinity binding interactions (Kd approximately 1 microM) could be readily distinguished by comparing fluorescence energy transfer before and after dissociation with 10 microM guanosine 5'-O-[gamma-thio]triphosphate and 10 mM MgCl2 where only low-affinity binding interactions remained. Interactions between alpha 0- and beta gamma-subunits from bovine brain or from bovine retinal transducin did not differ much. The beta gamma-subunits from bovine brain were found to bind with high transfer efficiency and comparable affinities to the hormone-activated and the nonactivated beta 1-receptor reconstituted in lipid vesicles: Kd = 100 +/- 20 and 120 +/- 20 nM, respectively; however, beta gamma-subunits from transducin appeared to bind more weakly to the beta 1-adrenoceptor than beta gamma-subunits from bovine brain. Separated purified homologous alpha 0- and beta gamma-subunits from bovine brain interfered mutually with each other in binding to the beta 1-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 beta gamma-subunits can bind to the nonactivated beta 1-adrenoceptor.
authors.affiliation: Department of Physiological Chemistry, University of Würzburg Medical School, Federal Republic of Germany.
authors.forename: H
authors.initials: H
authors.lastname: Heithier
authors.forename: M
authors.initials: M
authors.lastname: Fröhlich
authors.forename: C
authors.initials: C
authors.lastname: Dees
authors.forename: M
authors.initials: M
authors.lastname: Baumann
authors.forename: M
authors.initials: M
authors.lastname: Häring
authors.forename: P
authors.initials: P
authors.lastname: Gierschik
authors.forename: E
authors.initials: E
authors.lastname: Schiltz
authors.forename: W L
authors.initials: WL
authors.lastname: Vaz
authors.forename: M
authors.initials: M
authors.lastname: Hekman
authors.forename: E J
authors.initials: EJ
authors.lastname: Helmreich
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D015636:Magnesium Chloride / Q000494:pharmacology; D008567:Membranes, Artificial; D008823:Micelles; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014422:Turkeys
chemical_list: D005456:Fluorescent Dyes; D008567:Membranes, Artificial; D008823:Micelles; D011943:Receptors, Adrenergic, beta; D015636:Magnesium Chloride; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins