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RG Posner, SP Fay, MD Domalewski and LA Sklar
National Flow Cytometry Resource M888, Los Alamos National Laboratory, New Mexico 87545.
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.
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