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 article

Abstract

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)
Number of pages1
JournalAnnals of Biomedical Engineering
Volume19
Issue number5
StatePublished - Dec 1 1991
Externally publishedYes
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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