Analysis of ligand, receptor, and G-protein interaction in the n-formyl peptide receptor of the human neutrophil

S. P. Fay, R. G. Posner, M. D. Domalewski, L. A. Sklar

Research output: Contribution to journalConference articlepeer-review


We have used a combination of spectrofluorometric and flow cytometric methods to characterize the interaction of fluorescent formyl peptide ligands with cell surface receptors. Using commercially available fluorescent microbeads as calibration standards, a family of fluoresceinated formyl peptides (N-formyl-met-leu-(phe)n-lys-fluorescein, n = 1-3), and digitoninpermeabilized human neutrophils, we were able to examine both equilibrium and kinetic aspects of ligand binding. Equilibrium studies showed that GTP[S] caused a loss of binding affinity of approximately two orders of magnitude, from approximately 0.04 nM (LRG) to -3 nM (LR), resp. Kinetic studies revealed that this change in affinity was due to principally an increase in the dissociation rate constant from -1 × 10-3 sec-1 (LRG) to approximately 1 × 10-1 sec-1 (LR). In contrast the association rate constants in the presence and absence of guanine nuclcotide (-3 × 107 sec-1 M-1) were statistically indistinguishable, and close to the diffusion limit. In the presence of guanine nuclocotide (LR), the kinetic data were adequately fit by a single step reversible model. However, in the absence of guanine nucleotide, while a large fraction of the receptors has essentially instantaneous access to G proteins, a substantial fraction is initially uncoupled from G proteins and only has access to them over a period of minutes. The binding data are consistent with the idea that those receptors with rapid access to the G proteins may be physically pre-coupled to the receptors in permeabilized neutrophil preparations even in the absence of the peptide ligand. Quenching of the fluorescein of the shorter peptides (n = 1-2) upon binding suggests that the pocket is large enough to contain at least five, but no more than six amino acids, while pH-dependent intensity measurements suggest that the mechanism of quenching is dependent upon the position of fluorescein within the pocket.

Original languageEnglish (US)
Pages (from-to)602
Number of pages1
JournalAnnals of Biomedical Engineering
Issue number5
StatePublished - 1991
Event1991 Annual Fall Meeting of the Biomedical Engineering Society - Charlottesville, VA, USA
Duration: Oct 12 1991Oct 14 1991

ASJC Scopus subject areas

  • Biomedical Engineering


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