Heithier H, Fröhlich M, Dees C, Baumann M, Häring M, Gierschik P, Schiltz E, Vaz W L, Hekman M, Helmreich E J
Department of Physiological Chemistry, University of Würzburg Medical School, Federal Republic of Germany.
Eur J Biochem. 1992 Mar 15;204(3):1169-81. doi: 10.1111/j.1432-1033.1992.tb16744.x.
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.
荧光能量转移[参见Förster, T. (1948) Ann. Phys. 6, 55 - 75]被测试其是否适合用于研究去污剂微团中或重构到脂质小泡后G0亚基之间以及这些亚基或三聚体G0与β1 - 肾上腺素能受体的定量相互作用[根据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]。为此，从牛脑中纯化的α0 - 和βγ - 亚基以及三聚体G0、从牛视杆外段膜中提取的βγ - 亚基和从火鸡红细胞中提取的β1 - 肾上腺素能受体均在使标记蛋白功能完整的条件下用四甲基罗丹明马来酰亚胺或异硫氰酸荧光素进行标记。在α0 - 和βγ - 相互作用的情况下，通过比较用10 μM鸟苷5'-O - [γ - 硫代]三磷酸和10 mM氯化镁解离前后的荧光能量转移，可容易地区分特异性高亲和力结合相互作用（Kd约为10 nM）和非特异性低亲和力结合相互作用（Kd约为1 μM），此时仅保留低亲和力结合相互作用。来自牛脑或牛视网膜转导蛋白的α0 - 和βγ - 亚基之间的相互作用差异不大。发现来自牛脑的βγ - 亚基以高转移效率和相当的亲和力与重构在脂质小泡中的激素激活型和非激活型β1 - 受体结合：Kd分别为100 ± 20和120 ± 20 nM；然而，转导蛋白的βγ - 亚基似乎比来自牛脑的βγ - 亚基与β1 - 肾上腺素能受体的结合更弱。从牛脑中分离纯化的同源α0 - 和βγ - 亚基在与β1 - 肾上腺素能受体结合时相互干扰，推测是因为它们彼此之间的亲和力大于与受体的亲和力。这些发现证明荧光能量转移适用于研究G蛋白和G蛋白偶联受体的蛋白质 - 蛋白质相互作用。此外，它们支持了先前的发现[Kurstjens, N. P., Fröhlich, M., Dees, C., Cantrill, R. C., Hekman, M. & Helmreich, E. J. M. (1991) Eur. J. Biochem. 197, 167 - 176]，即βγ - 亚基可以与非激活型β1 - 肾上腺素能受体结合。
