Strategies for positioning fluorescent probes and crosslinkers on formyl peptide ligands.

Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known.

Fixation traps formyl peptide receptors in high and low affinity forms that can be regulated by GTP[S] in the absence of ligand.

The formyl peptide receptor on human neutrophils recognizes bacterial, N-formylated peptides and initiates a cascade of intracellular signals via a pertussis toxin sensitive Gi protein. We used fluorescence techniques to investigate the interactions of ligand (L), receptor (R), and G proteins (G), the ternary complex, in both live and fixed human neutrophils. By lightly fixing permeabilized neutrophils with a procedure that retained ligand binding, we were able to "capture' R and G in different configurations in the absence of ligand.

Quantitation of radiolabeled antibody binding to cells by thin-layer chromatography.

Thin layer chromatography (TLC) was used to monitor binding of radiolabeled antibodies to cells. Labeled antibodies were reacted with cells and aliquots chromatographed on serum-blocked, ITLC strips. The cell-antibody complexes remain at the origin and unbound antibody migrates with the solvent front.

Multiparameter flow cytometric analysis of a pH sensitive formyl peptide with application to receptor structure and processing kinetics.

Environmentally sensitive molecules have many potential cellular applications. We have investigated the utility of a pH sensitive ligand for the formyl peptide receptor, CHO-Met-Leu-Phe-Phe-Lys (SNAFL)-OH (SNAFL-seminaphtho-fluorescein), because in previous studies (Fay et al.: Biochemistry 30:5066-5075, 1991) protonation has been used to explain the quenching when the fluoresceinated formyl pentapeptide ligand binds to this receptor.

Continuous spectrofluorometric analysis of formyl peptide receptor ternary complex interactions.

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.

Progesterone but not estrogen depolarizes natural killer cells.

Natural killer (NK) cell tumoricidal and antimicrobial activities can be rapidly modulated by molecules that interact with membrane receptors. The discovery of NK cell depolarization induced by steroid-like Na+ channel agonists prompted a study of purified human NK cell excitability to a variety of steroids. Progesterone, but not estrogen, depolarized NK cells with concentration and time dependency.

Evidence for protonation in the human neutrophil formyl peptide receptor binding pocket.

We have studied the interaction of a family of fluorescent formyl peptides with their receptor using spectrofluorometric and flow cytometric methods. The peptides contained four (CHO-Met-Leu-Phe-Lys-fluorescein), five (CHO-Met-Leu-Phe-Phe-Lys-fluorescein), or six (CHO-Nle-Leu-Phe-Nle-Tyr-Lys- fluorescein) amino acids. As observed in earlier studies, the fluorescent peptides containing four and five amino acids were quenched upon binding to the receptor, while the hexapeptide was not.