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Vol. 262, No. 1, Issue of January 5, pp. 135-139,1987 Printed in U.S.A. THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc. Regulation of Ligand-Receptor Dynamics by Guanine Nucleotides REAL-TIME ANALYSIS OF INTERCONVERTINGSTATES PEPTIDE RECEPTOR* FOR THE NEUTROPHIL FORMYL (Received for publication, July 7, 1986) Larry A. SklarS, GaryM. Bokoch, Donald Button, and James E. Smolensll From the Department of Zmmumlogy, Research Znstitute of Scripps Clinic, La Jollu, California 92037 and the $Matt Hospital, Department of Pediatric Hematology, University of Michigan, Ann Arbor, Michigan 41809 tides (1-3), that ligand binding induces GTP hydrolysis as well as guanine nucleotide exchange (2-4), and thatpertussis toxin is a potent inhibitor of cell activation via these receptors (3, 5-10). The ability of neutrophils to release arachidonic acid following stimulation parallels the extent of ADP-ribosylation by pertussis toxin of a 41-kD protein, possibly Gi (10). Phosphoinositide metabolism in membranes isolated from neutrophils is stimulated by guanine nucleotide or by guanine nucleotide in the presence of formyl peptide (11, 12) and is blocked by pertussis toxin. Equilibrium binding studies at 4 “C in neutrophil membranes showed a fraction of high affinity formyl peptide binding sites (95% purity and >95% viability) were digitonin a t 37 “C for 25 permeabilized by incubation in 15 rg/ml . -. min (21). Buffers-Buffers are derived from cell studies of Sklar et al. (22) and permeabilized cell studies of Smolen et al. (21). Sklar’s “standard binding buffer” uses 137 mM NaC1, 5 mM KCI, 1.9 mM KH2P04,1.1 mM Na2HP04,5.5 mM glucose, 1.5 mM CaC12,0.3 mM MgSO,, 1 mM MgC12, 8 pg/ml superoxide dismutase, 8 pg/ml bovine catalase, pH 7.4, and 10 mM NH4Cl to prevent acidification of the lysosomal compartment into which fluorescent formyl peptide is internalized (16). Smolen’s “K buffer” contained 100 mM KCl, 20 mM NaCl, 1 mM EGTA, and 30 mM HEPES, pH 7.0, titrated with KOH. Ionic substitution in the K buffer, including omission of Na+ or K+, is described in the Figure legends. Real-time Analyses of Formyl Peptide Receptor Binding and Dissociation-Binding and dissociation measurements used the fluorimetric methods described by Sklar et al. (22). These measurements were performed on an SLM 8000 photon countingspectrofluorometer (SLM Instruments, Urbana-Champaign, IL). They made use of a fluoresceinated formyl hexapeptide, FLPEP (22), and a high affinity antibody to fluorescein to discriminate between free and receptor bound hexapeptide. The spectrofluorometric binding data are expressed as the specific ligand bound versus time. The spectrofluorometric measurements were verified by analyses which did not use antibody to fluorescein: ligand dissociation in permeabilized cells examined both by flow cytometry (22) and by fluorescence polarization (23) increased following the addition of guanine nucleotide. Similar numbers of receptors for formyl peptides are detected in intact andpermeabilized cells. RESULTS Ligand Dissociation in Intact Cells Is Heterogeneous and Appears to Reflect Interconverting Active and Inactive StatesIn Fig. 1, the specific binding and dissociation of FLPEP on intact neutrophils is displayed on a semilog plot. The experiment examines ligand dissociability as a function of time under conditions where ligand binding was allowedto proceed for 15, 30, 60, or 120 s. Ligand dissociability after this time was assayed. Ligand dissociability following 2 min of binding is roughly linear on a log scale and is characterized by a dissociation half-time of approximately 2-3 min. At shorter periods of binding the dissociability is distinctly heterogeneous with a fast component (tth 10 s) followed by a slow component similar to that observed at longer times. The magnitude of the fast component diminishes with the length of the binding period and is largely absent within 1 min of binding. These observations, combined with parallel measurements of cell function, have been interpreted in termsof transiently active interconverting receptor states (16, 18). Because the slowly dissociating state is found on cells at a time when cell responses have ceased we suggested (16) that this state was - 0.075 36 108 72 144 Time lsscl FIG. 1. The binding and dissociationof FLPEP at 37 OC to receptor on intact neutrophils. The data are plotted as thespecific binding of FLPEP on a log plot versus time. Experimental details (see Refs. 16 and 21): lo’ cells/ml were exposed a t time 0 to 1 nM FLPEP. At 15, 30, 60,or 120 s, antibody to fluorescein is added to each sample. Fluorescence is monitored continuously during the additions. The data are derived from a point by point comparison of the fluorescence measured under conditions of receptor binding and receptor blockade. Data are representative of observations in more than 10 separate experiments. In Fbf. 16, the raw data from a typical experiment (non-log plot treatment of data) are provided. ,075 40 80 120 160 Time lsecsl FIG. 2. The binding and dissociationof FLPEP to receptor on permeabilized neutrophils at37 “C.The data are plotted as the specific binding on a log plot versus time. lo7permeabilized cells/ ml (K buffer) were exposed to 1 nM FLPEP at time 0. After 15 or 120 s, antibody to fluorescein was added to each sample. 60 seconds M G T P r S was added. The data are representative of later, experiments performed on a t least three occasions. inactive; the rapidly dissociating state appearsto be associated with cell activation. Ligand Dissocatwn in Permeabilized Cells Is Homogeneous but Is Regulated by Guanine Nucleotides between States Comparable to the Active (+Guanine Nucleotide) and Inactive (-Guanine Nucleotide) Forms-A binding experiment analogous to thatperformed on intactcells, but using permeabilized cells is illustrated in Fig. 2. After 15 s, 2 min, or 5 min (not shown) of ligand binding, dissociation in the absence of guanine nucleotide is essentially homogeneous (linear on a semilog plot) and characterized by a half-time of -2 min. When guanine nucleotide (loM5M GTPyS) is added, the rate of 10 s ) . Virtually all of dissociation increases markedly ( 4 the receptors (consistently 290%) are sensitive to guanine nucleotide. Homogeneous dissociation was also detected if permeabilized cells were placed into the intact cell buffer (free of Ca2+;see below). The concentration dependence of the guanine nucleotide effect is shown in the upper panel of Fig. 3. As the GTPyS increases, the rateof FLPEP dissociation increases. However, - Receptor Regulation by Guanine Nucleotides 1.o GTPyS 1 .o GTPyS 137 OI 0.5 0 = t z 0 Y 2 0.25 -.- 1.0 > ._ U 0.5 0.25 24 40 12 Timu (sed 96 FIG. 3. The dependence of F L P E P dissociation on the concentration of GTPrS and M 8 + . The data are plotted as the specific binding on a log scale uersus time. lo7permeabilized cells/ml (K buffer +. 10 mM MgClz) were exposed to 1 nM FLPEP for 60 s prior to the addition of antibody to fluorescein (time 0 in the Figure). Dissociation was examined thereafter. GTP+ (10-8-10" M) was added to individual samples at time = 30 s. The data arerepresentative of experiments performed in duplicate on two occasions. Similar results were obtained if 1mM EDTA and 1 mM EGTA were present. the maximal rate of dissociation appears to be established rapidly. The specificity of guanine nucleotide based on the relative rate of induced ligand dissociation was determined (data not shown). The order of potency, GTPyS > GTP > GDP > Gpp(NH)p >> GMP, is similar to that observed in nucleotide binding to Gi (24). Adenine nucleotides were inactive. Guanine Nucleotide-dependentStates Are Regulated by Monovalent and Divalent Cations--In the lower panel of Fig. 3, we compare the effectiveness of GTPyS to induce ligand dissociation in thepresence of 10 mM MgC12. MgCl, increases the sensitivity of FLPEP dissociation to GTPySby approximately 1% orders of magnitude. A complex of aluminum and fluoride has been shown to cause activation and dissociation of purified G proteins and is an effective G protein activator in cells or membranes (25). We observed, however, that AIR had no effect on FLPEPdissociation, however, neither in the presence or absence of Mg", nor in Na+ or K+ containing buffers (not shown). Ligand dissociation is sensitive to thepresence of Na+, K', and Mg2+ (Fig. 4). We find that in the absence of guanine nucleotide, the formyl peptide dissociation rate issensitive to M$+ and decreases by approximately half when 10 mM M e is present. In the presence of saturating nucleotide, the dissociability of the receptor is sensitive to Na+ and K+. The half-time is approximately 4 s in the presence of Na+ and 10 s in the presence of K+, but not affected by 10 mMM$'. Thus in theabsence of guanine nucleotide, the slowly dissociating state is sensitive to Mg2'. In thepresence of nucleotide, the rapidly dissociating state is sensitive to Na+ or K+. The addition of Caz+a t levels above 10 NM suppresses the impact of guanine nucleotide (Fig. 5). Caz+ must be present for 1 min or more, prior to nucleotide addition in order to suppress the nucleotide effect (time course not shown). Pertussis ToxinProduces a RapidlyDissociating State, Comparable to the Active Form, in the Absence of Guanine Nucleo- Time Isec) FIG. 4. The ionic dependence of receptorstates for FLPEP. The data are plotted as thespecific binding on a log scale uersus time. lo7permeabilized cells were equilibrated for 1 min with 1nM F L P E P antibody to fluorescein was added (time 0 in the Figure) and ligand dissociation was measured thereafter. Saturating GTPrS M) was added as indicated at time = 30 s to the samples indicated by the solid line. Experiments in the upper p a n e l were performed in K+only buffer f 10 m M MgC1,; in the lower p a n e l , experiments were performed in Na+ only buffer f 10 mMMgC1,. The data are respresentative of observations made on three separate donors. GThS 0.25' 20 60 40 80 Tm i (ad Flc. 5. The inhibitionof guanine nucleotideeffects by Cap+. The data are plotted as the specific ligand binding on a log scale versus time. lo' permeabilized cells were exposed to Ca*+ for 1 min andthen equilibrated with 1 nM FLPEP for 30 s. Antibody to fluorescein was added (time 0 in theFigure). Dissociation was monitored thereafter. lo-' M GTP+ was added as indicated. Ca2+concentrations were monitored in parallel suspensions as described in Ref. 21. The data are representative of observations repeated on six individual preparations of permeabilized cells. tide (Fig. 6)"When permeabilized cells are treated with pertussis toxin, there is anincrease in the average rate of ligand dissociation which is a function of the duration of the incubation period with pertussis toxin. The ligand-receptor complex exhibits dissociability similar to theactive form following pertussis toxin treatmentwhile cells which underwent a comparable incubation did not show an altered rate of dissociation. The ADP-ribosylation of the a subunit of the neutrophil G protein mimics the binding of guanine nucleotide to this subunit in the sense that it results in the formation of the rapidly dissociating receptor state. 138 Receptor Regulation by Guanine Nucleotides affinity"; t% -2 min) observed in the absence of guanine nucleotides (kMp"') and two rapidly dissociating states ("low affinity"; t% 4-10 s ) observed in the presence of guanine nucleotides (+ Na+ and K+). A relationship appears to exist between these receptor affinity states observed in permeabilized cells and states of the receptor in intact neutrophils. In the intact cell at 37"C, dissociation of formyl peptide is heterogenous, with half-times of -10 s and 2 min, respectively, and time-dependent with disappearance of the rapidly dissociating state in 1-2 min (Fig. 1).This heterogeneity has been interpreted as a rapid interconversion between the rapidly and slowly dissociating forms (18,23). Interpreted in light of the data from other receptor-(; protein systems, the present experimental data lead to predictions about the regulation of formyl peptide receptor dynamics by G proteins. slowly Dissociating State-Based on the observation that Gi reconstitutes high affinity formyl peptide binding (2) and on analogy with the @-adrenergicreceptor system, the slowly dissociating state of the formyl peptide receptor detected in 20 40 60 ' EO the absence of guanine nucleotide should reflect ligand release Time (sed from an R - G complex. The inability to detect the rapidly FIG. 6. The effect of pertussis toxin on ligand-receptor in- dissociating form of receptor in the permeabilized neutrophil teractions in permeabilized cells. The data are plotted as the specific binding on a log scale versus time. Permeabilized cells (lo7/ even after brief periods of ligand binding (15 s; Fig. 2)implies ml) were exposed to 1nM FLPEP for 30 s and antibody to fluorescein that R. G interaction is rapid (I seconds) following formyl was added (time 0 in the Figure). Dissociation was monitored there- peptide binding or that R and G are preassociated even in the after. lo" M GTPyS was added as indicated. Upper panel, control absence of formyl peptide. cells; lower panel, toxin-treated cells. The permeabiliid cells (lo7/ Rapidly Dissociating State-In the @-adrenergicreceptor ml) were incubated at 37 'C with pertussis toxin (1pglml), ATP (1 system, the so-called high affinity ternary complex of hormM), dithiothreitol (1 mM), and NAD (20 p ~ ) Control . cells were incubated without toxin. Sample aliquots were removed at 30-min mone-receptor-(; protein is dissociated by guanine nucleotide intervals for the binding determinations. Data are representative of and converted to a low affinity state of the receptor. We detect a rapidly dissociating receptor form in permeabilized cells three separate experiments. following the addition of a saturating concentration by guanine nucleotide. A similar or identical rapidly dissociating DISCUSSION form of the receptor can be induced by ADP-ribosylation of the neutrophil G protein by pertussis toxin; both effects G Protein-Receptor Interactions presumably due to uncoupling of the R G complex. The rapid Neutrophil G protein appearsto be a member of the family onset (