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. Fixed receptors were trapped in a high affinity form (attributed to LRG) that could not be rapidly converted to low affinity by the addition of GTP[S]. Adding saturating nucleotide prior to fixation trapped receptors in a low affinity form (attributed to LR). The low affinity receptors retained the sensitivity of the native receptors to the presence of NA+. The distribution between high and low affinity receptors was modulated by GTP[S] in a dose dependent manner. The ability to redistribute low and high affinity receptor forms prior to fixation was unique to GTP[S], as compared to other non-activating nucleotides, suggesting that GTP[S] can regulate the distribution between R and RG. We suggest that precoupled receptors that give rise to high affinity ligand binding are likely to exist in native membranes in human neutrophils.

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. The antibody binding was estimated from the ratio of radioactivity at the origin compared to the total applied. Separations are completed in about 10 min. This method does not use centrifugation or wash steps, and provides an inexpensive and self-contained system to evaluate radioligand binding. Cell binding assay results using this method are approximately the same as those obtained using bead- or cell-type assays.

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. Moreover, acidification in intracellular compartments is a general mechanism occurring in cells during processing of ligand-receptor complexes. Because the protonated form of SNAFL is excited at 488 nm with emission at 530 nm and the unprotonated form is excited at 568 nm with emission at 650 nm, the ratio of protonated and unprotonated forms can be examined by multiparameter flow cytometry. We found that the receptor-bound ligand is sensitive to both the extracellular and intracellular pH. There is a small increase in the pKa of the ligand upon binding to the receptor consistent with protonation in the binding pocket. Once internalized, spectral changes in the probe consistent with acidification and ligand dissociation from the receptor are observed.

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. 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.

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. Excitability was measured by using flow cytometry and the anionic voltage-sensitive dye oxonol. Preincubation with the Na+ channel antagonist tetrodotoxin or removal of the extracellular Na+ blocked the response. Progesterone may rapidly change membrane potential, and eventually function, by acting on putative NK plasma membrane receptors coupled to Na+ conductances.

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. While the degree of quenching of the bound tetrapeptide was largely unchanged, the quenching of the bound pentapeptide decreased with increasing pH over the range of pH 6.5-9.0. Ligand binding studies have shown that the mole fraction of tetrapeptide or pentapeptide bound in kinetic analysis markedly decreased with increasing pH as a consequence of increasing ligand dissociation rate constant. The dependence of the binding parameters for the hexapeptide on pH was much less pronounced. Over a pH range from pH 7.3 to 9.0, the hexapeptide showed little change in binding affinity, while the tetrapeptide and pentapeptide increased in Kd approximately 2.0- and 2.5-fold, respectively. These results indicate that the formyl peptide receptor binding pocket contains at least two microenvironments. The pH sensitivity of the pentapeptide quenching is consistent with a protonating environment, while the pH-independent quenching of the tetrapeptide may reflect aromatic stacking or a hydrophobic microenvironment. The pH-dependent ligand dissociation also suggests that the protonation in the pocket stabilizes ligand binding, which may indicate an alteration in the binding pocket structure. Protonation or hydrogen bonding of the pentapeptide may lead to even further stabilization of that ligand.