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.

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.

Real-time analysis of the assembly of ligand, receptor, and G protein by quantitative fluorescence flow cytometry.

We describe a general approach for the quantitative analysis of the interaction among fluorescent peptide ligands (L), receptors (R), and G proteins (G) using fluorescence flow cytometry. The scheme depends upon the use of commercially available fluorescent microbeads as standards to calibrate the concentration of fluorescent peptides in solution and the receptor number on cells in suspension. We have characterized a family of fluoresceinated formyl peptides and analyzed both steady-state and dynamic aspects of ligand formyl peptide-receptor interactions in digitonin-permeabilized human neutrophils. Detailed receptor-binding studies were performed with the pentapeptide N-formyl-Met-Leu-Phe-Phe-Lys-fluorescein. 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 approximately 3 nM (LR), respectively. Kinetic studies revealed that this change in affinity was principally due to an increase in the dissociation rate constants from approximately 1 x 10(-3) s-1 (LRG) to approximately 1 x 10(-1) s-1 (LR). In contrast, the association rate constants in the presence and absence of guanine nucleotide (approximately 3 x 10(7) s-1 M-1) were statistically indistinguishable and close to the diffusion limit. In the presence of guanine nucleotide (LR), the kinetic data were adequately fit by a single-step reversible-binding model. In the absence of guanine nucleotides, not all receptors have rapid access to G to form the LRG ternary complex. Mathematically, those R that have rapid access to G are either precoupled to R or the association of G with R is fast compared to the association of L with R. The physiological consequences of coupling heterogeneity are discussed.

Kinetics of N-formyl peptide receptor up-regulation during stimulation in human neutrophils.

The kinetics of receptor up-regulation was examined in isolated neutrophils and in whole blood by flow cytometry during cell activation. Stimulation of neutrophils prepared without exposure to LPS with chemoattractants induced fast up-regulation of N-formyl peptide receptors and C receptor type 3 (CR3). Biphasic N-formyl peptide binding curves were detected for saturating concentrations of N-formyl peptide at 37 degrees C. The bulk of the rapid binding during the first 30 to 60 s is attributed to already expressed binding sites whereas the slow binding over the next 3 to 4 min represents a time course of receptor up-regulation. Support for this interpretation comes from conditions under which the number of binding sites and the progress of the binding curves were affected. Cells treated with LPS, which caused expression of internal N-formyl peptide receptors, exhibited rapid, monophasic binding curves with increased total binding. In LPS-untreated, calcium-depleted cells, N-formyl peptide receptor up-regulation was inhibited and rapid, monophasic binding to a smaller total number of expressed sites was observed. Cytochalasin B enhanced the total number of available N-formyl peptide receptors in LPS-untreated but not LPS-treated cells. In both cases binding was rapid and monophasic suggesting that receptors were either fully or rapidly up-regulated. Although not studied in real-time, C receptor type 3 up-regulation was similar to N-formyl peptide receptor up-regulation in response to LPS, or stimulation by N-formyl peptide, C product C5a, leukotriene B4, and platelet-activating factor in isolated cells and in whole blood. After stimulation with formyl peptide, LPS, or C product 5a, the release of vitamin B12-binding protein paralleled up-regulation of receptors. These data indicate that untreated cells up-regulate N-formyl peptide receptors during cell response at a rate of approximately 10,000/min in a calcium-dependent manner whereas LPS-treated cells already express the bulk of their receptors. In cytochalasin B-treated, degranulating cells 30,000 to 50,000 receptors were up-regulated within a minute.

Conformational dynamics of the formyl peptide receptor: a prototype for studies of receptor dynamics and binding pocket structure.

We have used spectrofluorometric and flow cytometric techniques to examine the interactions of formyl peptide ligands (L) with their cell surface receptors (R). Kinetic studies suggest that L binds to R at a diffusion limited rate and that R undergoes rapid transitions involving three states (LR, LRG, the ternary complex of L and R with the G protein, and a desensitized receptor "LRX" which forms within seconds) prior to internalization. A spectroscopic analysis of the interaction between L and R show that the binding pocket of R is large enough to contain no more than 6 amino acids and that a fluorescein-labelled pentapeptide is quenched upon binding to R. We hypothesize that histidine 90 (putatively located in the extracellular loop connecting the second and third transmembrane domains) protonates L and quenches the probe. New technology will extend the analysis of structure and dynamics to low affinity peptide receptors of living biological systems. Such technology will have implications in the design of peptidomimetic ligand and drug molecules.

Fluorescence analysis of the size of a binding pocket of a peptide receptor at natural abundance.

We have studied the topography of interaction of a family of fluorescent formyl peptides containing four (CHO-Met-Leu-Phe-Lys-fluorescein), five (CHO-Met-Leu-Phe-Phe-Lys- fluorescein), and six (CHO-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and CHO-Met-Leu-Phe-Phe-Phe-Lys- fluorescein) amino acids with their receptor using spectroscopic methods adapted to small sample volumes. Only the fluorescent peptides containing four and five amino acids were quenched upon binding to the receptor, indicating physical contact of the chromophore with the receptor. In contrast, only the hexapeptides were accessible to antibodies to fluorescein. Taken together, these results suggest that the carboxy terminus of the tetrapeptide or the pentapeptide is protected in the receptor binding pocket while the fluorescein on the carboxy terminus of either hexapeptide is exposed and recognized by the antibody to fluorescein. These results indicate that the binding pocket accommodates at least five but no more than six amino acids.

Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases.

Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites.

Eubacteria, halobacteria, and the origin of photosynthesis: the photocytes.

The halobacteria and the photosynthetic members of the eubacteria have previously been classified in two separate urkingdoms--the archaebacteria and the eubacteria, respectively. They were thought to be no more closely related to each other than they each were to the eukaryotes. In accord with this earlier classification, photosynthesis was thought to have originated twice by independent events--once within the eubacteria and once within the archaebacteria. In this paper, however, using three-dimensional ribosome structure as a probe of evolutionary divergences, we show that the eubacteria and the halobacteria are more closely related to each other than they are to any other known organisms. The simplest interpretation of our data is that all extant photosynthetic cells are descended from a single common ancestor that possessed a primeval photosynthetic mechanism. Numerous data on the occurrence of related biochemical processes in halobacteria and eubacteria support this theory. Essential components of the photosynthetic apparatus, such as carotenoids, are present in both halobacteria and in eubacteria, including the nonphotosynthetic eubacteria, suggesting that photosynthesis could be a primitive property of both groups. Our data indicate that together the eubacteria and the halobacteria form a monophyletic group for which we propose the name "photocytes." If other techniques of phylogenetic analysis confirm this evolutionary tree, we propose that the photocytes be given urkingdom status.