SUBUNIT INTERACTIONS OF GTP-BINDING PROTEINS

被引:39
作者
HEITHIER, H
FROHLICH, M
DEES, C
BAUMANN, M
HARING, M
GIERSCHIK, P
SCHILTZ, E
VAZ, WLC
HEKMAN, M
HELMREICH, EJM
机构
[1] UNIV WURZBURG,INST PHYSIOL CHEM,DEPT PHYSIOL CHEM,KOELLIKERSTR 2,W-8700 WURZBURG,GERMANY
[2] UNIV HEIDELBERG,SCH MED,DEPT PHARMACOL,W-6900 HEIDELBERG,GERMANY
[3] UNIV FREIBURG,INST BIOCHEM & ORGAN CHEM,W-7800 FREIBURG,GERMANY
[4] MAX PLANCK INST BIOPHYS CHEM,KARL FRIEDRICH BONHOEFFER INST,DEPT MOLEC BIOL,W-3400 GOTTINGEN,GERMANY
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1992年 / 204卷 / 03期
关键词
D O I
10.1111/j.1432-1033.1992.tb16744.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Fluorescence energy transfer [cf. Forster, 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., Holzhofer, 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 tetramethylrhodaminmaleimide 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 (K(d) almost-equal-to 10 nM) and nonspecific low-affinity binding interactions (K(d) almost-equal-to 1-mu-M) could be readily distinguished by comparing fluorescence energy transfer before and after dissociation with 10-mu-M 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: K(d) = 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., Frohlich, 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.
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收藏
页码:1169 / 1181
页数:13
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