Detergent solubilization of the formyl peptide chemotactic receptor. Strategy based on covalent affinity labeling

The formyl peptide chemotactic receptor has been solubilized by digitonin treatment of purified human neutrophil membranes. Of several potential assay methods tested for their ability to separate receptor-bound from free ligand, only gel filtration through an acrylamide cross-linked agarose matrix yielded satisfactory results. Approximately 70% of the receptor initially present in the membrane was recovered in the digitonin extract. Binding of 125I-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys to the soluble receptor was rapid (t 1/2 at 22 degrees C less than 5 min), of high affinity (Kd = 2.2 nM) and saturable. The relative potencies of a small series of peptides as inhibitors of binding to the soluble receptor paralleled their potencies as inhibitors of the membrane-bound receptor. N-Formylation of the peptides was required for high affinity binding. Binding was maximal at pH 6.5 and was sulfhydryl-dependent; 20 microM p-chloromercuriphenylsulfonic acid decreased binding by 50%. 125I-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys was specifically cross-linked to the soluble receptor with ethylene glycol bis(succinimidyl succinate) and an apparent molecular weight of 55,000 to 70,000 and determined for the soluble receptor by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A strategy for obtaining an active, detergent-soluble receptor preparation based on covalent affinity labeling is presented.     

The anti-infective peptide, innate defense-regulator peptide, stimulates neutrophil chemotaxis via a formyl peptide receptor

The anti-infective peptide, innate defense-regulator peptide (IDR-1), has been selectively reported to modulate the innate immune response. We found that IDR-1 stimulates the chemotactic migration in human neutrophils. Moreover, IDR-1-induced neutrophil chemotaxis was completely blocked by pertussis toxin, suggesting the importance of the G_i protein in this process. The mechanism governing the IDR-1-induced neutrophil chemotaxis was found to be completely inhibited by the formyl peptide receptor (FPR) antagonist; cyclosporin H. IDR-1 was also found to induce chemotactic migration in FPR but not in vector-expressing HCT116 cells. Meanwhile, IDR-1 failed to stimulate superoxide anion generation and intracellular calcium increase in human neutrophils. Furthermore, IDR-1 was found to inhibit fMLF (an FPR agonist)-induced superoxide generation and calcium signaling in human neutrophils and FPR-expressing HCT116 cells. Taken together, the results demonstrate that IDR-1 is a partial agonist for FPR and further, stimulates neutrophil chemotaxis without inducing calcium signaling and superoxide generation.     

Shear-induced resistance to neutrophil activation via the formyl peptide receptor

The application of fluid shear stress on leukocytes is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. The formyl peptide receptor (FPR) on neutrophils, which binds to formyl-methionyl-leucyl-phenylalanine (fMLP) and plays a role in neutrophil chemotaxis, has been implicated as a fluid shear stress sensor that controls pseudopod formation. The role of shear forces on earlier indicators of neutrophil activation, such as L-selectin shedding and α(M)β(2) integrin activation, remains unclear. Here, human neutrophils exposed to uniform shear stress (0.1-4.0 dyn/cm(2)) in a cone-and-plate viscometer for 1-120 min showed a significant reduction in both α(M)β(2) integrin activation and L-selectin shedding after stimulation with 0.5 nM of fMLP. Neutrophil resistance to activation was directly linked to fluid shear stress, as the response increased in a shear stress force- and time-dependent manner. Significant shear-induced loss of FPR surface expression on neutrophils was observed, and high-resolution confocal microscopy revealed FPR internalized within neutrophils. These results suggest that physiological shear forces alter neutrophil activation via FPR by reducing L-selectin shedding and α(M)β(2) integrin activation in the presence of soluble ligand.     

Detergent solubilisation of the rabbit neutrophil receptor for chemotactic formyl peptides

Digitonin was found to be the only detergent (out of 24 tested) capable of solubilising the chemotactic formyl peptide receptor from rabbit neutrophil membranes in a form which retained its [3H]fMet-Leu-Phe binding activity. The solubilised material retained many of the characteristics of the membrane-bound receptor. [3H]fMet-Leu-Phe binding to the digitonin extract was measured at 4 degrees C using an equilibrium dialysis assay. Binding was saturable and of high affinity (Kd = 3.5 +/- 0.7 nM). The potencies of a series of synthetic peptides as inhibitors of [3H]fMet-Leu-Phe binding to the soluble receptor showed the same rank order as for inhibition of the membrane-bound receptor. In addition, binding to both preparations was sulphydryl dependent showing a parallel inhibition by p-chloromercuribenzene sulphonate which could be partially reversed by subsequent incubation with dithiothreitol.     

Human neutrophil Fc gamma RIIIB and formyl peptide receptors are functionally linked during formyl-methionyl-leucyl-phenylalanine-induced chemotaxis.

The formyl peptide receptor (FPR) and the glycosyl-phosphatidylinositol-linked type III receptor for the Fc portion of IgG (Fc gamma RIIIB; CD16) play important roles in various inflammatory responses in human neutrophils. The mechanisms of signaling by the glycosyl phosphatidylinositol-anchored Fc gamma RIIIB are not known. Therefore, we investigated the possibility that Fc gamma RIIIB and FPR may act in concert to mediate neutrophil functions. We observed that pretreatment of normal human neutrophils with Fab fragments of a mAb to the Fc gamma RIII (3G8) specifically inhibited their chemotaxis into micropore filters in response to the formylated peptides FMLP or formyl-norleucyl-leucyl-phenylalanine. Pretreatment of neutrophils with a saturating concentration of 3G8 Fab (100 nM or 5 micrograms/ml) followed by exposure to FMLP (0.5 to 500 nM) indicated that significant inhibition of chemotaxis was observed at peptide concentrations greater than 5 nM. However, 3G8 Fab had no effect on the neutrophil response to a wide range (0.05 to 500 nM) of other chemotactic factors, including C5a, leukotriene B4, IL-8 (neutrophil-activating peptide-1), and platelet-activating factor. Moreover, pretreatment of neutrophils with mAb to other cell surface molecules (decay-accelerating factor, Fc gamma RII, and HLA class I) did not affect chemotaxis to FMLP. Inhibition of movement was not due to degradation of FMLP by the cell surface endopeptidase 24.11 (CD10), because neutrophils pretreated with the CD10 inhibitor phosphoramidone and 3G8 Fab displayed the same altered response to FMLP as cells pretreated with 3G8 Fab alone. Ligation of the Fc binding site of Fc gamma RIIIB appears to be essential for altering the FMLP-induced response, since soluble aggregated IgG and other anti-Fc gamma RIII antibodies, all of which recognize the ligand binding site, mimic the inhibitory effect of the 3G8 Fab on FMLP-induced chemotaxis. In contrast, a mAb (214.1) that does not recognize the Fc binding site of Fc gamma RIIIB had no effect on FMLP-induced chemotaxis. Not only did anti-Fc gamma RIII inhibit neutrophil chemotaxis to FMLP in a filter-based migration assay, but 3G8 Fab also inhibited FMLP-induced neutrophil transendothelial migration. Scatchard plot analysis of radioligand binding experiments indicated that 3G8 Fab did not significantly alter the number of FMLP binding sites on neutrophils but significantly increased the affinity of the FPR for [3H]FMLP. Removal of greater than 80% of cell surface Fc gamma RIIIB by phospholipase C abolished the neutrophil chemotactic response to FMLP but did not affect movement toward C5a, IL-8, or leukotriene B4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular Leydig cells

The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antimicrobial peptide scolopendrasin VII,derived from the centipede Scolopendra subspinipes mutilans,stimulates macrophage chemotaxis via formyl peptide receptor 1

In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. [BMB Reports 2015; 48(8): 479-484]     

Substance P binds to the formylpeptide chemotaxis receptor on the rabbit neutrophil

Substance P, a potent vasodilatory and smooth muscle contracting agent, binds specificially to the formyl peptide receptor on the rabbit neutrophil. Substance P stimulates chemotaxis and induces lysosomal enzyme secretion in concentrations which similarily inhibit f Met-Leu-(³H)Phe receptor binding. Competitive antagonists of the formyl peptide receptor also inhibit the activity of Substance P. The finding of a naturally occurring eukaryotic peptide interacting with the neutrophil formyl peptide receptor is of importance.     

Isolation and partial characterization of the formyl peptide receptor components on human neutrophils

The receptor for formylated peptides such as FMLP has been reported to consist of glycoprotein components ranging from 24-95 kDa, and to exhibit both high and low affinity for ligand. Controversy exists on the molecular size and number of these components, and whether the different affinities represent distinct ligand binding sites. In this study, the receptor was found to be comprised of components, of 94, 68, and approximately 40 kDa molecular size. Competitive binding inhibition experiments showed that FMLP bound to the components in the following order from highest to lowest affinity: 68 kDa greater than approximately 40 kDa greater than 94 kDa. Our findings suggest that the FMLP receptor of human neutrophils contains at least three components, and that each component has a different affinity for FMLP.     

Regulation of formyl peptide receptor agonist affinity by reconstitution with arrestins and heterotrimeric G proteins

Although heptahelical chemoattractant and chemokine receptors are known to play a significant role in the host immune response and the pathophysiology of disease, the molecular mechanisms and transient macroassemblies underlying their activation and regulation remain largely uncharacterized. We report herein real time analyses of molecular assemblies involving the formyl peptide receptor (FPR), a well described member of the chemoattractant subfamily of G protein-coupled receptors (GPCRs), with both arrestins and heterotrimeric G proteins. In our system, the ability to define and discriminate distinct, in vitro receptor complexes relies on quantitative differences in the dissociation rate of a fluorescent agonist as well as the guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) sensitivity of the complex, as recently described for FPR-G protein interactions. In the current study, we demonstrate a concentration- and time-dependent reconstitution of liganded, phosphorylated FPR with exogenous arrestin-2 and -3 to form a high agonist affinity, nucleotide-insensitive complex with EC(50) values of 0.5 and 0.9 microm, respectively. In contrast, neither arrestin-2 nor arrestin-3 altered the ligand dissociation kinetics of activated, nonphosphorylated FPR. Moreover, we demonstrated that the addition of G proteins was unable to alter the ligand dissociation kinetics or induce a GTP gamma S-sensitive state of the phosphorylated FPR. The properties of the phosphorylated FPR were entirely reversible upon treatment of the receptor preparation with phosphatase. These results represent to our knowledge the first report of the reconstitution of a detergent-solubilized, phosphorylated GPCR with arrestins and, furthermore, the first demonstration that phosphorylation of a nonvisual GPCR is capable of efficiently blocking G protein binding in the absence of arrestin. The significance of these results with respect to receptor desensitization and internalization are discussed.     

Regulation of formyl peptide receptor binding to rabbit neutrophil plasma membranes. Use of monovalent cations, guanine nucleotides, and bacterial toxins to discriminate among different states of the receptor

The regulation by monovalent cations, guanine nucleotides, and bacterial toxins of [3H]FMLP binding to rabbit neutrophil plasma membranes was studied by using dissociation techniques to identify regulatory effects on separate receptor states. Under conditions of low receptor occupancy (1 nM [3H]FMLP) and in both Na+ and K+ buffers, dissociation is heterogenous, displaying two distinct, statistically significant off rates. [3H]FMLP binding was enhanced by substituting other monovalent cations for Na+. In particular, enhanced binding in the presence of K+ relative to Na+ was caused by additional binding to both rapidly and slowly dissociating receptors. Three receptor dissociation rates, two of which appear to correspond to the two affinity states detected in equilibrium binding studies, were defined by specific GTP and pertussis toxin (PT) treatments. Neither GTP, nor PT or cholera toxins (CT) had an effect on the rate of dissociation of [3H]FMLP from the rapidly dissociating form of the receptor. Both 100 microM GTP and PT treatments increased the percentage of rapidly dissociating receptors, correspondingly decreasing the percentage of slowly dissociating receptors. The observed changes in the rapidly and slowly dissociating receptors after GTP, PT, and CT treatments were caused by an absolute decrease in the amount of binding to the slowly dissociating receptors. However, complete inhibition of slowly dissociating receptor binding by GTP, PT, or both was never observed. Both GTP and PT treatments, but not CT treatment, increased by two-fold the rate of dissociation of 1 nM [3H]FMLP from the slowly dissociating form of the receptor, resulting in a third dissociation rate. Thus, slowly dissociating receptors comprise two different receptor states, a G protein-associated guanine nucleotide and PT-sensitive state and a guanine nucleotide-insensitive state.     

Differential activation of polymorphisms of the formyl peptide receptor by formyl peptides

We have investigated the role of two polymorphic sites (R190W and N192K) on the binding and activation of the formyl peptide receptor (FPR) by viral and formyl peptides. WEDWVGWI, a peptide with antiviral activity derived from the membrane proximal region of feline immunodeficiency virus, binds with high affinity to FPR. The three tryptophans in the peptide are all essential for FPR binding, just as they were essential for antiviral activity [S. Giannecchini, A. Di Fenza, A.M. D'Ursi, D. Matteucci, P. Rovero, M. Bendinelli, Antiviral activity and conformational features of an octapeptide derived from the membrane-proximal ectodomain of the feline immunodeficiency virus transmembrane glycoprotein, J. Virol. 77 (2003) 3724]. Formyl-NleWEDWVGWI behaved as a weak partial agonist with FPR W190/N192 but a stronger partial agonist with FPR R190/K192 and FPR R190/N192. Formyl-NleNleWEDWVGWI behaved as a full agonist toward all three FPRs but exhibited a much higher EC(50) with W190/N192 FPR (300+/-45 nM) than for R190/K192 FPR (40+/-3 nM) or R190/N192 (60+/-8 nM). Formyl-MYKWPWYVWL preferentially activated R190/K192 and R190/N192 FPRs by>5 fold compared to W190/N192 FPR. Formyl-MFEDAVAWF, a peptide derived from a protein in Mycobacterium avium subsp. paratuberculosis and formyl-MFTFEPFPTN, a peptide derived from the N-terminus of chemotaxis inhibitory protein of Staphylococcus aureus with an added N-terminal formyl-methionine exhibited the greatest selectivity for R190/K192 and R190/N192 FPRs with approximately 10 fold lower EC(50)s than that observed with FPR W190/N192. Thus, individuals with the W190 polymorphism may display a reduced ability to detect certain formyl peptides.     

Pharmacologic characterization of the rabbit neutrophil receptor for platelet-activating factor

The characteristics of receptors for platelet-activating factor (PAF) on rabbit neutrophils are investigated in this report. The presence of PAF-specific binding to rabbit neutrophils was confirmed using radiolabeled ligand binding assays and a rabbit peritoneal neutrophil membrane preparation. Binding of PAF to the neutrophil membranes was reversible and reached equilibrium within 30 min. Scatchard analysis of PAF-specific binding to the rabbit neutrophil membranes revealed a dissociation constant (Kd) for PAF of 0.41 +/- 0.045 nM and a Bmax of 0.32 +/- 0.11 pmol of PAF receptor/mg of protein. The order of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF to rabbit peritoneal neutrophil membranes was determined. For the competition assays, 100 micrograms of neutrophil or platelet membrane protein, 0.18 nM 3H-PAF, and varying amounts of PAF antagonist were incubated at room temperature for 1 hr. PAF receptor antagonists tested were ONO-6240, brotizolam, kadsurenone, WEB-2086, L-652-731, BN-52021, CV-3988, triazolam, alprazolam, and verapamil. The orders of potencies of these PAF receptor antagonists were similar for inhibition of 3H-PAF binding to rabbit peritoneal neutrophil and platelet membranes (correlation coefficient, r = 0.97). PAF had a significantly higher affinity for rabbit neutrophil membranes (Kd = 0.41 +/- 0.045 nM), as compared with its affinity for rabbit platelet membranes (Kd = 0.87 +/- 0.092 nM). In addition, sodium was found to inhibit 3H-PAF specific binding to rabbit platelet membranes and not to affect 3H-PAF binding to neutrophil membranes. These data indicate that, although PAF receptors on rabbit platelets and neutrophils exhibit similar orders of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF, the disparity in Kd of PAF for the receptors and the effect of NaCl on the binding of 3H-PAF reveal subtle differences between the cell types.     

The uncoupled state of the human formyl peptide receptor

The formyl peptide receptor (FPR) has been widely used to study the kinetics of the interaction between ligand, receptor and G protein with real-time fluorescence methods. Because the wild type receptor rapidly signals, and is then desensitized and internalized once occupied by ligand, it has been difficult to study the uncoupled receptor form. We have examined a mutant form of the FPR expressed in U937 cells that does not bind G protein and is thus ideal to study the uncoupled form of the FPR in the intact cell. Using kinetic flow cytometry, we have measured the dissociation kinetics of a fluorescent ligand from this mutant in intact, permeabilized and fixed cells. We observed a novel uncoupled receptor form in the intact cell with a dramatically reduced off-rate (approximately 0.02 s-1) from LR in a broken cell preparation (approximately 0.2 s-1). Both receptor forms are retained in the presence of formaldehyde. We also observed this novel receptor form coexisting with the LRG complex when the wild type receptor is fixed in neutrophils or transfectants. These results complex when the wild type receptor is fixed in neutrophils o transfectants. These results lead us to suggest that there are distinct receptor structures in cells and membranes and that only a fraction of receptors in intact cells exist in the uncoupled form.     

Signal transduction and ligand-receptor dynamics in the human neutrophil. Transient responses and occupancy-response relations at the formyl peptide receptor

The responses of neutrophils to formyl peptides are initiated and in many cases achieve a maximal level prior to equilibrium receptor occupancy. In order to begin to understand the linkage between receptor occupancy and cell response we have used a pulsed binding procedure to analyze: 1) the number of receptors contributing to three potential signalling events and six functional responses and 2) the evolution of these responses once ligand binding is interrupted. We find that the half-optimal elevations of the potential signals are produced by less than 1% occupancy (Ca2+) or 1-3% occupancy (cAMP, membrane depolarization). In contrast, actin polymerization and a rapid light scatter response are elicited by less than 0.1% occupancy. Half-optimal elastase release and degranulation require approximately 3% occupancy. While half-optimal O2- production and aggregation require approximately 30% occupancy, the half-optimal rate of O2- production requires less than 10% occupancy. To resolve the apparent lack of correlation between the responses and the signals we examined their time courses following the pulse of stimulation. At least four responses and one signal are transient and decay while occupied receptors remain on the membrane surface. These include the Quin 2-Ca2+ signal, actin polymerization, the light scatter response, O2- generation, and aggregation. Ca2+ elevation is correlated with the responses in that: 1) each of these responses is transient unless new receptors are occupied; 2) occupancy of nearly all of the receptors contributes to the time course of these responses; 3) when binding is interrupted, the responses decay with a half-time of 15 s, following a latency of approximately 10 s or less (except for disaggregation where latency is 30-40 s). We discuss evidence in support of the hypothesis that transient cell responses arise from transient receptor activation.     

A novel ligand of the formyl peptide receptor: annexin I regulates neutrophil extravasation by interacting with the FPR

The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner. Lower peptide concentrations possibly found in inflammatory situations elicit Ca2+ transients without fully activating the MAP kinase pathway. This causes a specific inhibition of the transendothelial migration of neutrophils and a desensitization of neutrophils toward a chemoattractant challenge. These findings identify annexin I peptides as novel, endogenous FPR ligands and establish a mechanistic basis of annexin I-mediated antiinflammatory effects.     

Identification of functional domains in the formyl peptide receptor-like 1 for agonist-induced cell chemotaxis

Formyl peptide receptor-like 1 (FPRL1) is a seven transmembrane domain, G protein-coupled receptor that interacts with a variety of exogenous and host-derived agonists. In order to identify domains crucial for ligand recognition by FPRL1, we used chimeric receptors with segments in FPRL1 replaced by corresponding amino acid sequences derived from the prototype formyl peptide receptor FPR. The chimeric receptors were stably transfected into human embryonic kidney epithelial cells and the capacity of the cells to migrate in response to formyl peptide receptor agonists was evaluated. Our results showed that multiple domains in FPRL1 are involved in the receptor response to chemotactic agonists with the sixth transmembrane domain and the third extracellular loop playing a prominent role. Interestingly, the N-terminus and a segment between the fourth transmembrane domain and the third intracellular loop of FPRL1 are important for receptor interaction with a 42 amino acid amyloid beta peptide (Abeta42), an Alzheimer's disease-associated FPRL1 agonist, but not with MMK-1, a synthetic FPRL1 agonist, suggesting that diverse agonists may use different domains in FPRL1. Considering the potential importance of FPRL1 in inflammation and neurodegenerative diseases, the identification of functional domains in this receptor will provide valuable information for the design of specific receptor antagonists.     

Characterization, solubilization, affinity labeling and purification of the cardiac Na+ channel using Tityus toxin gamma

Saturable, high-affinity binding of iodinated toxin gamma from Tityus serrulatus scorpion venom (TiTx gamma) to Na+ channel receptor was identified in sarcolemma membrane of chick heart. A binding capacity of 450-600 fmol/mg of protein was found similar to that of tetrodotoxin-binding component. The enrichment of these membrane-bound toxin binding sites follows that of other sarcolemma markers. Kinetic data and displacement of 125I-TiTx gamma from its binding sites by unlabeled TiTx gamma gave an equilibrium dissociation constant (Kd) of 1-3 pM. The gating component and the selectivity filter of the voltage-sensitive Na+ channel, identified as binding sites of TiTx gamma and of tetrodotoxin respectively, have been efficiently solubilized with Nonidet P-40. Purification was achieved by ion-exchange chromatography on DEAE-Sephadex A-25, affinity chromatography on wheat-germ-agglutinin-Sepharose and sucrose density gradient centrifugation. An enrichment of 1400-fold from the original detergent extract was measured for both toxin binding sites (1120-1230 pmol/mg of protein). Sodium dodecyl sulfate gel electrophoresis reveals a single large polypeptide component of Mr230000-270000. The purified material exhibits an apparent sedimentation coefficient of 8.8S. Covalent cross-linking of 125I-TiTx gamma to its membrane-embedded cardiac receptor shows that the cross-linked material, solubilized and purified by the same procedure comprises a single polypeptide chain of the same Mr of 230000-270000. Furthermore, as seen for Electrophorus electricus electroplax and rat brain, the tetrodotoxin-binding component and the TiTx gamma-binding component are carried by the same polypeptide chain. The functional Na+ channel might be an oligomer of this subunit of Mr23000-270000.     

Partial characterization and detergent solubilization of the putative glutathione chemoreceptor from hydra.

Feeding behavior in hydra is initiated by the association of glutathione (GSH) with a putative external chemoreceptor. In the present study, the binding of [35S]GSH to hydra membranes has been characterized. Nondisplaceable [35S]GSH binding which compromised previous analyses [Grosvenor, W., Bellis, S., Kass-Simon, G., & Rhoads, D. (1992) Biochim. Biophys. Acta (in press)] was eliminated by treating membranes with an inhibitor of GSH metabolism, borate in combination with L-serine. The specific binding which was not inhibited by borate/serine demonstrated many of the characteristics expected of a ligand/receptor interaction. The binding was rapid, reversible, and saturable. A Scatchard analysis of saturation isotherms indicated a dissociation constant (KD) of 3.4 microM, a value which is in good agreement with concentrations of glutathione which are known to induce feeding behavior. Hydra membranes were detergent-solubilized with 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 100 mM KCl, and 10% glycerol. The soluble fraction contained 40% of the original saturable, reversible GSH binding activity. The KD for GSH binding to the solubilized preparation was estimated as 2.7 microM, a valuable which is not appreciably different from the KD for binding to intact membranes. The fidelity of GSH binding in the solubilized preparation suggests that this preparation will be useful in further characterization of the putative glutathione chemoreceptor.     

Regulation of ligand-receptor dynamics by guanine nucleotides. Real-time analysis of interconverting states for the neutrophil formyl peptide receptor

Intact neutrophils exhibit interconverting active and inactive receptor states with half-times for dissociation of 10 s and 2 min, respectively. We examined the effect of guanine nucleotides on ligand-receptor dynamics at 37 degrees C in neutrophils permeabilized with digitonin using continuous fluorometric measurements. The permeabilized cells exhibit a single class of slowly dissociating receptors with a half-time similar to the inactive state. The slowly dissociating state is lengthened in the presence of 10 mM by Mg2+ about two-fold but is relatively insensitive to substitutions of Na+ or K+. When guanine nucleotide is added the receptors dissociate uniformly with a half-time similar to the active state but are sensitive to the substitution of Na+ or K+ (K+ or K+/Mg2+ approximately 10 s; Na+ or Na+/Mg2+ approximately 4 s). When receptors in permeabilized cells are ADP-ribosylated with pertussis toxin the rapidly dissociating state is detected. In the presence of nonsaturating nucleotide or incomplete ribosylation, complex rates of ligand dissociation intermediate between the active and inactive forms are observed. Micromolar concentrations of Ca2+ block the effect of guanine nucleotide on the receptor. The relationships between ligand-receptor dynamics in intact neutrophils and interconverting states regulated by guanine nucleotides and ions in permeabilized cells are discussed.