A thrombin receptor in resident rat peritoneal macrophages.

Resident rat peritoneal macrophages possess 6 x 10(2) high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 x 10(4) low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labeled bovine thrombin is achieved after 1 min at 37 degrees C, and after 12 min at 4 degrees C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min-1 at 4 degrees C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radio-activity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. 125I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor size of Mr 120,000.     

Characterization and affinity crosslinking of receptors for tumor necrosis factor on human cells

Receptors for tumor necrosis factor (TNF) were characterized in the U-937 human histiocytic lymphoma cell line with the aid of highly purified recombinant human TNF, radiolabeled with 125I. Saturation binding to specific cell surface receptors occurred with less than 15% nonspecific binding. Analysis of the equilibrium binding data obtained at 4 degrees C revealed a single class of noninteracting binding sites. The mean number of binding sites per cell was calculated to be 12,000, and the apparent dissociation constant (Kd) was 2 X 10(-10) M. Crosslinking of 125I-TNF to the cell surface receptor with disuccinimidyl suberate, followed by NaDodSO4-polyacrylamide gel electrophoresis of the cell lysate, revealed a TNF-receptor complex with a molecular weight of approximately 100,000. Binding to concanavalin A-Sepharose suggested that the TNF receptor is a glycoprotein.     

Platelet-derived growth factor receptors form a high affinity state in membrane preparations. Kinetics and affinity cross-linking studies

The specific binding of 125I-PDGF (platelet-derived growth factor) to intact fibroblasts becomes relatively nondissociable during incubation at 37 degrees C. To characterize the interaction of PDGF with its receptors under conditions in which there is no receptor internalization, we have studied the binding of 125I-PDGF to membrane preparations derived from mouse 3T3 cells and rat liver. The binding sites had the affinity and specificity characteristics expected of PDGF receptors. At 37 degrees C (but not at 4 degrees C) the specific binding of 125I-PDGF to membranes gradually became nondissociable as assessed by either dilution or by addition of excess unlabeled PDGF. This tight binding was not due to a covalent interaction since the polyanionic compound suramin readily dissociated specifically bound 125I-PDGF. This property of suramin was used to expose rat liver PDGF receptors which were occupied by endogenous PDGF. Affinity cross-linking studies demonstrated that the formation of the nondissociable state of 125I-PDGF binding was associated with the binding of 125I-PDGF to a 160,000-dalton protein and to a 110,000-dalton species. The cross-linked binding sites could be adsorbed to wheat germ agglutinin and to anion exchange resins. The isoelectric point of both cross-linked species determined by two-dimensional gel electrophoresis was approximately 4.7. These data demonstrate that in membrane preparations, PDGF binds to an anionic 160,000-dalton glycoprotein which is likely to be the receptor. A high affinity state of PDGF binding, which is formed rapidly at 37 degrees C, can be dissociated by suramin.     

The identification and partial characterization of the fibroblast growth factor receptor of baby hamster kidney cells

The binding of biologically active, 125I-labeled basic fibroblast growth factor (FGF) to baby hamster kidney-derived cell line cells (BHK-21) was studied at 4 degrees C. Unlabeled FGF displaced cell surface bound 125I-FGF, but platelet-derived growth factor, epidermal growth factor, insulin, or transferrin did not. Binding was saturable both as a function of time and as a function of increasing 125I-FGF concentrations. Scatchard analysis of the binding data revealed the presence of about 1.2 X 10(5) binding sites/cell with an apparent KD of 270 pM. The number of the binding sites was down-regulated following preincubation of the cells with FGF. The density of binding sites/cell also decreased as an inverse function of cell density. When 125I-FGF binding was studied in a BHK-21 cell membrane preparation, it was found that the membranal binding site displayed a lower KD of 21 pM. 125I-FGF was covalently cross-linked to its cell surface receptor on intact BHK-21 cells using the homobifunctional agent disuccinimidyl suberate. Two macromolecular species with an apparent molecular weight of 145,000 and 125,000, respectively, were labeled under both reducing and nonreducing conditions. Unlabeled FGF competed with 125I-FGF for binding to both macromolecular species. The labeling of the macromolecules was also inhibited by heparin. No labeling was observed in the absence of the cross-linkers or when heat-inactivated 125I-FGF was used instead of radiolabeled, biologically active FGF.     

Solubilization and assay of a colony-stimulating factor receptor from murine macrophages

The colony-stimulating factor, CSF-1, selectively stimulates the survival, proliferation, and differentiation of mononuclear phagocytes. The solubilization, assay, and characteristics of the CSF-1 receptor from the J774.2 murine macrophage cell line are described. The recovery of cell-surface receptor in the postnuclear supernatant membrane fraction of hypotonically disrupted cells was 76%. Recovery of the ligand binding activity of the receptor after solubilization of this fraction with 1% Triton X-100 was approximately 150%. The binding of 125I-CSF-1 to intact cells and membrane preparations was consistent with the existence of a single class of high-affinity receptor sites. In contrast, the equilibrium binding of 125I-CSF-1 to the solubilized postnuclear fraction indicated the existence of two distinct classes of binding site (apparent Kds 0.15 nM and 10 nM). A rapid assay was developed for the high-affinity sites, which were shown to be associated with the CSF-1 receptor. The function of the low-affinity sites, which have not been demonstrated on intact cells or cell membranes and which are 13 times more abundant than the high-affinity sites, is unknown. The solubilized high-affinity receptor-CSF-1 complex was stable on storage at 0 degrees C and -70 degrees C but dissociated at 37 degrees C. Dissociation also occurred at 0 degrees C in buffers of low pH (4.0) or high ionic strength (0.7 M NaCl).     

Bivalent binding of an anti-CD3 antibody to Jurkat cells induces association of the T cell receptor complex with the cytoskeleton

Ligand binding and cross-linking of TCR/CD3 complex leads to T cell stimulation in immunologic responses. As a prelude to investigating the dynamic interactions of these receptors, we have characterized binding of the mAb OKT3 specific for CD3 on Jurkat cells. The association of both OKT3 and its Fab' fragment is rapid at 4 degrees C, and dissociation of Fab' is also rapid, but dissociation of OKT3 is slow, indicating bivalent binding in this case. Dissociation of OKT3 is substantially accelerated at 37 degrees C if internalization is prevented. From the concentration dependence, the binding of OKT3 at 4 degrees C appears to be very tight whereas binding of the Fab' fragment is weaker and biphasic. Scatchard analysis of the Fab' equilibrium binding data indicates two binding sites with KD values of 5.1 x 10(-9) M and 2.7 x 10(-8) M. The very tight binding of the bivalent antibody may be caused by inter- or intramolecular cross-linking between these sites. If Jurkat cells are warmed to 37 degrees C, there is an energy-dependent increase by about one-third of sites bound by OKT3 or its Fab' fragment over that seen at 4 degrees C. This increase may be related to a receptor recycling process because internalization of a similar number of the bound ligands occurs at similar rates. Other experiments have revealed that OKT3, but not its Fab' fragment, causes the receptor complex to become associated with the detergent-insoluble cytoskeleton, and there are also insoluble intracellular OKT3-binding sites. These cross-linking-induced receptor-cytoskeletal interactions are sensitive to moderate changes in salt concentration that should allow their molecular basis to be investigated.     

Characterization of the asialoglycoprotein receptor in a continuous hepatoma line

The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells.     

Beta nerve growth factor binding to PC12 cells. Association kinetics and cooperative interactions

The association kinetics of 125I beta nerve growth factor (NGF) binding to the PC12 clonal cell line have been examined in detail at 0.5 and 37 degrees C. These data were examined by utilizing a reversible second-order integrated rate equation, and the results were not consistent with a simple bimolecular process. Two association rates were required to explain the results adequately. At 37 degrees C, the faster component was estimated to have a second-order association rate constant of 1.4 X 10(7) M-1 s-1, while the rate constant for the slower component (3.8 X 10(6) M-1 s-1) was about 4-fold lower. As shown by others, the temperature dependence of the dissociation kinetics indicated that while the rapidly dissociating component was only slightly slowed by lowering the chase temperature to 0.5 degrees C, the second component was slowed by about 270-fold, from 8 X 10(-4) s-1 to 3 X 10(-6) s-1. The binding data that describe the slowly dissociating component were obtained by utilizing this differential temperature dependence and revealed a concave downward Scatchard plot. The binding parameters determined from computer analysis using a nonlinear fitting program (LIGAND) suggest that this component consists of (a) an interacting class of about 4000 sites/cell that have a first stoichiometric steady-state dissociation constant of 65 pM and a second stoichiometric interaction constant of 16 pM, indicative of positively cooperative interactions, and (b) a class of sites consistent with a ratio of sites/Kd of about 11.1 sites/(cell X pM). The steady-state binding results at 37 degrees C indicated only one class of binding sites (155,000 +/- 18,000 sites/cell) that had an apparent Kd of 0.52 +/- 0.03 nM. One class of sites was also observed at 0.5 degrees C, and the receptor concentration was found to be reduced (99,000 +/- 7600 sites/cell) while the Kd was increased (1.7 +/- 0.14 nM). A significant level of positively cooperative interactions was observed frequently at 37 degrees C that was not due to a failure to reach steady-state conditions, internalization, or degradation. Since cooperativity of binding was never observed at 0.5 degrees C, a membrane event may be involved. Determination of the contribution of the different classes of NGF receptors found on PC12 cells to the biological actions of NGF requires a clear understanding of their kinetic properties and their relationship to each other. The studies presented here indicate that their interactions are more complex than previously described.     

Binding and structural properties of oxytocin receptors in isolated rat epididymal adipocytes

Oxytocin initiates its insulin-like action in adipocytes through oxytocin-specific receptors. We have studied binding and structural properties of these receptors with the radioligand [3H]oxytocin. Steady-state binding was reached after 45 min, at 21 degrees C, and 10 min at 37 degrees C. Scatchard analyses of equilibrium binding data indicated a single class of oxytocin binding sites at 21 degrees C (KD = 3.3 nM, RT = 6 X 10(4) sites/cell) and 2 binding sites at 37 degrees C (KD = 1.5 nM, RT = 6 X 10(4) sites/cell; and KD = 20 nM, RT = 30 X 10(4) sites/cell). Insulin, insulin-like growth factor I, and epidermal growth factor increased oxytocin binding (approximately 20-40%), whereas adenosine, a regulator of oxytocin action, did not affect oxytocin binding. Binding activity of oxytocin was impaired by pretreatment of the hormone or adipocytes with dithiothreitol. Dithiothreitol treatment of adipocytes preferentially inactivated high-affinity binding sites. N-ethyl maleimide inhibited oxytocin binding in adipocytes more than dithiothreitol. In contrast to the inhibitory effects of dithiothreitol and N-ethyl maleimide, proteases (trypsin, chymotrypsin and papain) were not able to inhibit fat cell binding activity. These results suggested that in isolated adipocytes: there are high-affinity and low-affinity receptors, but the low-affinity receptors are absent at 21 degrees C; the binding of oxytocin can be regulated by insulin, and growth factors; and the oxytocin receptors contain disulfide bridges and free thiols that are essential for the maintenance of oxytocin binding.     

Differential effects of chelating agents on cytosolic glucocorticoid receptor stability and nuclear binding in vitro

The effect of several metal chelators (EDTA, EGTA, and 1,10 phenanthroline) on rat liver glucocorticoid receptor properties in vitro was investigated. At 4 degrees C 10 mM EDTA (unlike 10 mM EGTA and 10 mM 1,10 phenanthroline) had a significant stabilizing effect on unbound hepatic glucocorticoid receptors. At higher temperature (25 degrees C) 10 mM EGTA appeared to act as a chemical stabilizer of unbound receptors. 1,10 Phenanthroline had no stabilizing effect at either temperature. Scatchard analysis indicated that the alteration in receptor binding after incubation at 4 and 25 degrees C in the presence and absence of chelating agents was due to a change in the number of steroid binding sites rather than perturbation of receptor affinity. Unlike results obtained with unbound receptors, all three chelating agents appeared to enhance prebound glucocorticoid-receptor complex inactivation. Interestingly these chelating reagents also significantly altered glucocorticoid-receptor complex binding to isolated nuclei.     

Identification and characterization of the endothelial cell surface lipoprotein lipase receptor.

The hydrolysis of triglycerides in plasma lipoproteins is mediated by lipoprotein lipase (LPL) that is bound to vascular endothelial cells. The specific endothelial cell surface protein(s) with which LPL associates has not been characterized. To identify this LPL binding protein(s), radioiodinated cell surface proteins from cultured bovine aortic endothelial cells were chromatographed using bovine LPL-Sepharose. A single radioiodinated protein of apparent molecular mass 220 kDa was specifically retained by the gel and eluted with 0.4 M NaCl. A LPL-binding protein of similar size was obtained after metabolic labeling of the cellular proteoglycans with 35SO4, indicating that the 220-kDa protein is a proteoglycan. After heparitinase or nitrous acid treatments the molecular mass of the LPL-binding protein decreased to approximately 50 kDa, suggesting that it contains heparin sulfate chains. A 220-kDa protein from the basal cell surface was also identified using LPL-Sepharose chromatography. 125I-LPL was cross-linked to the endothelial cell surface using ethylene glycobis (succinimidylsuccinate). A single ligand-receptor complex, approximately 350 kDa, was obtained. Heparin and unlabeled LPL decreased the cross-linking of radioiodinated LPL to the cell surface receptor. To examine whether the receptor mediates the internalization of cross-linked 125I-LPL, cells containing 125I-LPL complexed to the surface were incubated at either 37 or at 4 degrees C. The amount of 125I-LPL internalized by the cells was 74% greater at 37 degrees C than at 4 degrees C. This suggested that LPL cross-linked to the receptor was internalized in a temperature-dependent manner. Thus, a 220-kDa heparan sulfate proteoglycan functions as an endothelial cell surface receptor for LPL.     

Identification of functional platelet-activating factor receptors in Raji lymphoblasts

The binding and metabolism of platelet-activating factor (PAF) were characterized in Raji, a human Burkitt's lymphoma-derived cell line. Raji lymphoblasts readily metabolized PAF by deacetylation-reacylation at 37 degrees C, but not at 4 degrees C. Binding studies conducted at 4 degrees C demonstrated specific binding that reached saturation within 80 min. This binding was only partially reversible. Scatchard analysis of PAF binding data revealed a single class of PAF binding sites (17,800 +/- 3,600/cell) with a K of 2.3 +/- 0.3 nM. These high-affinity PAF binding sites were shown to be functional receptors, as 100 pM to 1 microM PAF increased free intracellular calcium in a dose-dependent manner. The dose of PAF necessary to achieve half maximal calcium mobilization response was 6.3 nM, which was in the range of the K for the receptor calculated from the binding studies. The structurally dissimilar PAF receptor antagonists CV-3988 and BN52021 inhibited the PAF-induced calcium changes at doses that competed with PAF binding. These studies provide the first evidence for a functional PAF receptor expressed on a lymphocyte cell line.     

Characterization of a receptor for C5a anaphylatoxin on human eosinophils

The complement anaphylatoxin peptide C5a is well known to activate human polymorphonuclear leukocytes through receptor-mediated processes. C5a has also been reported to activate eosinophils for both chemotaxis and hexose uptake. We characterized the receptor molecule for human C5a on human eosinophils and compared it with the receptor on human neutrophils. At 4 degrees C, uptake of 1 nM 125I-C5a reaches equilibrium within 10 min on both cell types. Binding of 125I-C5a occurs over a concentration range comparable to that which stimulates lysosomal enzyme release and hexose uptake in both cell types. Scatchard analyses of the data indicate the presence of two receptor populations on eosinophils; a high affinity receptor with 15,000-20,000 sites/cell and a Kd of 3.1 +/- 0.6 x 10(-11) M, and a low affinity receptor with approximately 375,000 sites/cell and a Kd of 1 x 10(-7) M. Parallel experiments with neutrophils indicate the presence of a single receptor population with approximately 90,000 sites/cell and a Kd of 4.8 +/- 0.1 x 10(-10)M. The eosinophil receptor molecule was further characterized by covalently cross-linking 125I-C5a to cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized material. Autoradiography indicates the presence of a dominant C5a-eosinophil receptor complex with an apparent mass of 60-65 kDa. The corresponding neutrophil-C5a receptor complex has an apparent mass of 50-52 kDa as observed by others. When the cross-linked 125I-C5a-receptor complex was treated with cyanogen bromide, different patterns were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis for neutrophils and eosinophils. Thus, human eosinophils have a receptor for C5a anaphylatoxin which appears to be distinct from the C5a receptor present on human neutrophils.     

Phaseolus vulgaris isolectin binding to human erythrocytes.

The Phaseolus vulgaris isolectins L4,L3E1, L2E2, L1E3, and E4 were isolated by affinity and ion exchange chromatography. Pure isolectins were radiolabeled by the chloramine-T method with Na125IO4 and their binding to human erythrocytes was studied. A normal erythrocyte has approximately 8 times 10(5) receptor sites for each isolectin; however, the association constants (Ka) of binding increased from 1.1 times 10(7) M-1 to 3.8 times 10(8) M-1, with increasing number of E subunits per tetrameric isolectin molecule. Isolectin to erythrocyte binding reached equilibrium rapidly and was reversed by fetuin. All isolectins competed with 125I-E4 for erythrocyte binding sites, with a constant (KI) similar to the Ka calculated for each respective radiolabeled isolectin. When isolectin binding at 0 degrees C, 4 degrees C, or 8 degrees C was compared to that at 25 degrees C, there was no reduction in the number of binding sites per cell, but the Ka of E4 was reduced to 3 times 10(7) M-1. Fixed erythrocytes displayed similar isolectin binding characteristics.     

Characterization of the receptors for vascular endothelial growth factor

Vascular endothelial growth factor (vEGF) is a recently discovered mitogen for endothelial cells. It is also a potent angiogenic factor. We have characterized the vEGF receptors of endothelial cells using both binding and cross-linking techniques. Scatchard analysis of equilibrium binding experiments revealed two types of high-affinity binding sites on the cell surfaces of bovine endothelial cells. One of the sites has a dissociation constant of 10(-12) M and is present at a density of 3 x 10(3) receptors/cell. The other has a dissociation constant of 10(-11) M, with 4 x 10(4) receptors/cell. A high molecular weight complex containing 125I-vEGF is formed when 125I-vEGF is cross-linked to bovine endothelial cells. This complex has an apparent molecular mass of 225 kDa. Two other faintly labeled complexes with apparent molecular masses of 170 and 195 kDa also are detected. Reduction in the presence of dithiothreitol causes a substantial increase in the labeling intensity of the 170- and 195-kDa complexes, suggesting that these complexes are derived from the 225-kDa complex by reduction of disulfide bonds. The labeling of the vEGF receptors was inhibited by an excess of unlabeled vEGF but not by high concentrations of several other growth factors. Suramin and protamine, as well as several species of lectins, inhibited the binding. The expression of functional vEGF receptors was inhibited when the cells were preincubated with tunicamycin, indicating that glycosylation of the receptor is important for the expression of functional vEGF receptors. Pretreatment with swainsonine on the other hand, did not prevent formation of functional receptors. However, the mass of the 225-kDa complex is decreased by 20 kDa when 125I-vEGF is cross-linked to swainsonine-treated endothelial cells.     

The interaction of 125I-colony-stimulating factor-1 with bone marrow-derived macrophages

The colony-stimulating factor, CSF-1, stimulates cultured quiescent murine bone marrow-derived macrophages (BMM) to enter DNA synthesis with a lag phase of 10-12 h. The binding, dissociation, internalization, and degradation of 125I-CSF-1 by BMM during the lag phase were investigated. Quiescent BMM express approximately 5 X 10(4) cell surface receptor sites/cell but contain additional cryptic sites (approximately 10(5)/cell) that can appear at the cell surface within 10 min at 37 degrees C. Studies of the binding reaction at both 2 degrees C (Kd less than or equal to 2 X 10(-13) M) and 37 degrees C (Kd approximately 4 X 10(-10) M) are consistent with the existence of a single class of cell surface sites. The disappearance of cell surface 125I-CSF-1 following a 2-37 degrees C temperature shift results from two, competitive, first order processes, internalization and dissociation. Internalization (t1/2 = 1.6 min) is 6 times more frequent than dissociation (t1/2 = 9.6 min). Following internalization, 10-15% of the intracellular CSF-1 is rapidly degraded whereas the remaining 85-90% is slowly degraded by a chloroquin-sensitive first order process (t1/2 greater than 3.5 h). These findings were confirmed and extended by studies of the uptake of 125I-CSF-1 at 37 degrees C. Following addition of 125I-CSF-1, cell surface receptors are rapidly down-regulated (t1/2 approximately 7 min) and their replacement does not commence until 20-60% of pre-existing surface receptor sites have disappeared. Despite receptor replacement, initially from the cryptic pool and later by de novo synthesis and/or receptor recycling (4 molecules/cell/s at steady state), the number of receptors at the cell surface remains low. The process results in the intracellular accumulation of large amounts of 125I-CSF-1 (greater than 10(5) molecules/cell) by BMM. Thus, whereas the kinetics of association, dissociation, and internalization of CSF-1 with BMM and peritoneal exudate macrophages are similar, BMM, which exhibit a higher proliferative response, degrade growth factor 12 times more slowly.     

Evidence for the rapid internalization and recycling of lutropin receptors in rat testis Leydig cells.

A study into the binding of 125I-human chorionic gonadotropin (hCG) to the lutropin (LH) receptor in rat testis Leydig cells, and subsequent internalization of the hormone-receptor complex, has been carried out. The results show that there is rapid internalization of the hormone-receptor complex; 240 receptors/cell (from a total of approx. 4000 receptors/cell) were internalized each minute in the first hour after exposure to hCG. Radioactivity was released from the cell 1 h after internalization and was found to be associated with highly degraded hCG. The endocytic process was found to have two temperature-sensitive steps. At 4 degrees C, movement of the hormone-receptor complex inside the cell did not occur, and at 21 degrees C hormone accumulated within the cytoplasm but was not degraded or released from the cell. At 34 degrees C, internalization, degradation and loss of the degraded hormone from the cell occurred. These processes appeared to reach a steady state after 2 h. Even though there is rapid internalization of the hormone-receptor complex following exposure to hCG, the binding sites on the cell surface were maintained for at least 4 h. The number of binding sites on the cell surface was not decreased by a protein synthesis inhibitor but was reduced to undetectable levels by monensin. This compound inhibits acidification of endocytic vesicles, which is known to be an important prerequisite to receptor cycling. It is concluded that, in the rat testis Leydig cells, following binding of hCG to the LH receptor there is rapid internalization of the complex and that recycling of the receptor occurs to the cell surface. This process may be essential in maintaining the capacity of the Leydig cell to bind fresh hormone.     

Appearance of specific leukotriene B4 binding sites in myeloid differentiated HL-60 cells

Exposure of HL-60 cells for 6 days to a combination of 1.25% (v/v) dimethyl sulfoxide and 10 microM dexamethasone induces myeloid differentiation which results in a cell with many of the characteristics of a mature granulocyte. At 4 degrees C myeloid differentiated, but not undifferentiated, monocytic differentiated or eosinophilic differentiated HL-60 cells display marked specific leukotriene B4 binding. Leukotriene B4 binding at 4 degrees C reaches a maximum within 10 min, is readily reversed by unlabeled leukotriene B4, and is stereospecific. Only molecules with structural and biological similarity to leukotriene B4 can competitively inhibit leukotriene B4 binding. Scatchard analysis at 4 degrees C in differentiated cells shows two classes of binding sites. The high affinity sites have a Kd of 0.27 nM and a Bmax of 14.8 fmol/10(7) cells; the low affinity sites have a Kd of 0.58 microM and a Bmax of 2453 fmol/10(7) cells. The appearance of specific leukotriene B4 binding sites in the myeloid differentiated cells correlates with their ability to chemotax in response to leukotriene B4. Undifferentiated cells do not chemotax to leukotriene B4. At 37 degrees C leukotriene B4 is incorporated into phospholipid and triglyceride species in both undifferentiated and myeloid differentiated HL-60 cells making binding studies at 37 degrees C in intact cells impossible. No evidence of omega-hydroxylase activity was found in HL-60 cells. These data suggest that the HL-60 cell may be an excellent model system for the study of leukotriene B4 receptor binding, processing, and gene expression.     

The characterization of the receptor for endothelial cell growth factor by covalent ligand attachment

Cellular receptors for endothelial cell growth factor (ECGF) have been demonstrated on several cell types by binding of 125I-ECGF in a specific and saturable manner (Schreiber, A. B., Kennedy, J., Kowalski, J., Friesel, R., Mehlman, T., and Maciag, T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6138-6142). Here we report the covalent cross-linking of 125I-ECGF to a polypeptide present on the surface of the plasma membrane of murine lung capillary endothelial cells by the homobifunctional reagent, disuccinimidyl suberate. Cross-linking of cell surface associated 125I-ECGF yields a major polypeptide with an apparent molecular weight of 150,000. Experiments demonstrated that the cross-linked polypeptide complex represents 125I-ECGF covalently bound specifically to a cell surface receptor because: covalent modification of the polypeptide was inhibited by excess, unlabeled ECGF; preincubation of cells with unlabeled ECGF at 37 degrees C significantly reduced cross-linking while incubation at 4 degrees C did not; other polypeptide growth factors do not compete with 125I-ECGF for cross-linking to the ECGF receptor; labeling of the polypeptide did not take place in the absence of DSS; and cells previously shown to have a paucity of ECGF receptors did not yield a cross-linked labeled receptor. These data suggest that the mitogenic events mediated by ECGF occur after occupancy of the specific cell surface polypeptide and suggest that these events are relevant to ECGF-induced signal transduction across the endothelial cell plasma membrane.     

Cell surface receptor for hemopexin in human leukemia HL60 cells. Specific binding, affinity labeling, and fate of heme

The binding of 125I-labeled human hemopexin to human leukemia HL60 cell at 4 degrees C was saturable with time and with increasing concentrations of 125I-hemopexin. Scatchard analysis of the binding data revealed the presence of approximately 42,000 binding sites/cell with an apparent dissociation constant (Kd) of 1.0 X 10(-9) M. When cells were incubated with radioactive hemopexin at 37 degrees C, 125I-hemopexin was rapidly bound and then was dissociated after the release of heme. Treatment of surface-bound 125I-hemopexin with divalent lysine-directed cross-linking disuccinimidyl suberate revealed a membrane polypeptide of about 80,000 Da, to which hemopexin is cross-linked. To examine the fate of the internalized heme, lysates from the cells previously incubated with [59Fe]heme-hemopexin complex were analyzed by CM-cellulose and Sephacryl S-200 column chromatography. A considerable amount of the radioactivity was present in the fraction which co-eluted with the myeloperoxidase activity. When myeloperoxidase was isolated from the cells incubated with [59Fe]heme-hemopexin complex by immunoprecipitation with anti-myeloperoxidase antibody, radiolabeled iron associated with myeloperoxidase increased with time, and more than 30% of the radioactivity in the cells was present in the myeloperoxidase. These results indicate that the binding of hemopexin to the surface receptors triggers a release of heme and that this heme is incorporated into the intracellular myeloperoxidase.