Posner R G, Fay S P, Domalewski M D, Sklar L A
National Flow Cytometry Resource M888, Los Alamos National Laboratory, New Mexico 87545.
Mol Pharmacol. 1994 Jan;45(1):65-73.
Fluorescent formyl peptides have made it possible to study ligand-receptor-G protein (ternary complex) dynamics in real-time, but limitations to sample mixing and delivery in flow cytometry have interfered with continuous observation. We have taken advantage of the quenching of a fluoresceinated N-formyl pentapeptide upon binding to its receptor on permeabilized neutrophils to extend the analysis of the ternary complex dynamics to the second time scale. The association and dissociation of ligand in the presence and absence of saturating concentrations of GTP[S] were examined continuously and the results were found to be in agreement with results predicted previously from flow cytometry. We observe comparable initial rates for the formation of ligand-receptor (LR) binary complexes and ligand-receptor guanine nucleotide binding protein (LRG) ternary complexes, dissociation rates differing by two orders of magnitude, and slow interconversions between LR and LRG in the absence of guanine nucleotide. When fit by the ternary complex model, at least three sides of the model are required and the fit is improved if a significant fraction of receptors (RG) are allowed to be precoupled to G protein. One of the limitations of the analysis is that data fits are insensitive to additional parameters in the calculation which would permit analysis of all four sides of the ternary complex model. Experiments performed with subsaturating GTP[S] identified coexisting classes of LR and LRG and allowed analysis of the altered distribution between coupled and uncoupled receptors. At saturating nucleotide levels, the binding of GTP[S] and the breakup of the ternary complex occur on a subsecond time frame. This result is consistent with the idea that inside a neutrophil where GTP levels are several hundred microM, once ternary complex forms, ternary complex decomposition is rapid. Taken together, the observed rapid assembly and disassembly of ternary complex account for subsecond cell responses to ligand.
荧光甲酰肽使得实时研究配体 - 受体 - G蛋白（三元复合物）动力学成为可能，但流式细胞术中样品混合和输送的局限性干扰了连续观察。我们利用了一种荧光标记的N - 甲酰五肽与通透化中性粒细胞上的受体结合时的淬灭现象，将三元复合物动力学的分析扩展到了秒级时间尺度。在有和没有饱和浓度的GTP[S]存在的情况下，连续检测配体的结合和解离，结果发现与先前通过流式细胞术预测的结果一致。我们观察到配体 - 受体（LR）二元复合物和配体 - 受体鸟嘌呤核苷酸结合蛋白（LRG）三元复合物形成的初始速率相当，解离速率相差两个数量级，并且在没有鸟嘌呤核苷酸的情况下，LR和LRG之间存在缓慢的相互转化。当用三元复合物模型拟合时，至少需要模型的三个方面，如果允许相当一部分受体（RG）预先与G蛋白偶联，则拟合效果会更好。该分析的一个局限性是数据拟合对计算中的其他参数不敏感，而这些参数将允许对三元复合物模型的所有四个方面进行分析。用亚饱和GTP[S]进行的实验确定了共存的LR和LRG类别，并允许分析偶联和未偶联受体之间改变的分布。在饱和核苷酸水平下，GTP[S]的结合和三元复合物的分解发生在亚秒级时间框架内。这一结果与中性粒细胞内GTP水平为数百微摩尔的观点一致，即一旦三元复合物形成，三元复合物的分解就很快。综上所述，观察到的三元复合物的快速组装和解组装解释了细胞对配体的亚秒级反应。
