Wu Yang, Buranda Tione, Simons Peter C, Lopez Gabriel P, McIntire William E, Garrison James C, Prossnitz Eric R, Sklar Larry A
Department of Pathology and Cancer Research Facility, University of New Mexico Health Sciences Center, University of New Mexico, Albuquerque, NM 87131, USA.
Anal Biochem. 2007 Dec 1;371(1):10-20. doi: 10.1016/j.ab.2007.08.011. Epub 2007 Aug 14.
We have used rapid-mix flow cytometry to analyze the early subsecond dynamics of the disassembly of ternary complexes of G protein-coupled receptors (GPCRs) immobilized on beads to examine individual steps associated with guanine nucleotide activation. Our earlier studies suggested that the slow dissociation of Galpha and Gbetagamma subunits was unlikely to be an essential component of cell activation. However, these studies did not have adequate time resolution to define precisely the disassembly kinetics. Ternary complexes were assembled using three formyl peptide receptor constructs (wild type, formyl peptide receptor-Galpha(i2) fusion, and formyl peptide receptor-green fluorescent protein fusion) and two isotypes of the alpha subunit (alpha(i2) and alpha(i3)) and betagamma dimer (beta(1)gamma(2) and beta(4)gamma(2)). At saturating nucleotide levels, the disassembly of a significant fraction of ternary complexes occurred on a subsecond time frame for alpha(i2) complexes and tau(1/2)< or =4s for alpha(i3) complexes, time scales that are compatible with cell activation. beta(1)gamma(2) isotype complexes were generally more stable than beta(4)gamma(2)-associated complexes. The comparison of the three constructs, however, proved that the fast step was associated with the separation of receptor and G protein and that the dissociation of the ligand or of the alpha and betagamma subunits was slower. These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Galphabetagamma heterotrimer.
我们使用快速混合流式细胞术分析固定在珠子上的G蛋白偶联受体（GPCR）三元复合物解离的早期亚秒级动力学，以研究与鸟嘌呤核苷酸激活相关的各个步骤。我们早期的研究表明，Gα和Gβγ亚基的缓慢解离不太可能是细胞激活的关键组成部分。然而，这些研究没有足够的时间分辨率来精确确定解离动力学。使用三种甲酰肽受体构建体（野生型、甲酰肽受体-Gα（i2）融合体和甲酰肽受体-绿色荧光蛋白融合体）以及α亚基的两种亚型（α（i2）和α（i3））和βγ二聚体（β（1）γ（2）和β（4）γ（2））组装三元复合物。在饱和核苷酸水平下，对于α（i2）复合物，很大一部分三元复合物的解离发生在亚秒时间范围内，对于α（i3）复合物，解离半衰期τ（1/2）≤4秒，这些时间尺度与细胞激活相兼容。β（1）γ（2）亚型复合物通常比β（4）γ（2）相关复合物更稳定。然而，对这三种构建体的比较证明，快速步骤与受体和G蛋白的分离有关，而配体或α和βγ亚基的解离较慢。这些结果与涉及G蛋白构象变化而非Gαβγ异源三聚体解离的细胞激活模型一致。
