Ultrastructural studies of endothelial and platelet receptor binding of thrombin-colloidal gold probes

Endothelial cells and platelets are reported to have receptors for alpha-thrombin. To visualize the binding of alpha-thrombin to these cells, we developed a method to label thrombin with colloidal gold. Formed by electrostatic adsorption of thrombin to the negatively charged gold, the resulting probe is stable for weeks and consists of approximately 30 thrombin molecules adsorbed to each 16.5 nm gold particle. The probe retained about 10% of the enzymatic activity (fibrinogen clotting) of the unlabeled native thrombin and 20% of the ability of the native thrombin to aggregate platelets in platelet-rich plasma (PRP). In PRP, approximately 90% of the observed probes were bound to fibrin strands, with the remaining probes (650 per cell) attached to activated platelets. In contrast, washed, paraformaldehyde-fixed human platelets exhibited a marked increase in probe density (4900 per cell). Time-dependent ultrastructural studies (2-240 min) of binding of the thrombin-gold probe to confluent cultures of porcine aortic endothelial cells revealed that the initial binding (7300 probes per cell) occurred randomly at the cell surface. A limited number (25%) of the probes clustered at coated-pit regions and were internalized (60-240 min). The probe induced a limited amount of cellular retraction similar to that achieved with unlabeled thrombin. These results suggest that the thrombin-gold probe is suitable for investigations of the localization of thrombin receptors on cell surfaces and the interaction of thrombin with these receptors during thrombotic events.     

Single high affinity binding of interferon α2 to receptors on human lymphoblastoid cells: Internalization and inactivation of receptors

The interaction of human recombinant interferon (rIFN) α2 with its receptor on lymphoblastoid cells was studied using competitive displacement binding. The data were analysed with the LIGAND program, which tests their fit to one-site or multiple binding site models. The binding at 4° and 37°C fits a one-site model, with a similar K_D for both IFN-sensitive and resistant cells. Binding at 37°C to Daudi cells at high density fits artifactually a two-site mode only when the receptor concentration is close to that of the K_D. The binding of IFN to its receptor, therefore, follows a simple bimolecular interaction. Furthermore, IFN-sensitive and resistant cells internalize IFN at similar rates. We have examined whether IFN receptors are also internalized and whether they subsequently recycle to the cell surface. By measuring cell surface and total receptors, we have observed that after 2 h treatment with IFN total receptors remain constant whereas cell surface receptors decrease. After prolonged treatment with IFN, however, there is a loss of total receptors. By inactivating cell surface receptors with proteinase K, we have shown that a fraction of cell surface receptors becomes resistant to inactivation and is apparently internalized. Moreover, experiments which measure IFN receptors either during incubation in the presence of IFN or after IFN has been removed from the medium, show that receptors do not recycle to the cell surface after internalization. The addition of monensin, a drug which has been shown to inhibit receptor recycling, has no effect on the loss of IFN receptors.     

Transport of vitamin B12 in Escherichia coli: common receptor system for vitamin B12 and bacteriophage BF23 on the outer membrane of the cell envelope.

We showed previously that the outer membrane of the Escherichia coli cell envelope normally contains about 200 to 250 B12 receptors, and that these receptors function both in B12 transport and as receptors for the E colicins. This paper shows that this receptor system is also shared with bacteriophage BF23. A strong positive correlation was observed between the number of B12 receptors per cell and the rate of adsorption of BF23. Cells from mutant strains that lacked B12 receptors did not adsorb BF23 particles. The rate of adsorption of BF23 to cells of a merodiploid strain (RK4151), with about 550 B12 receptors per cell, was approximately double that to cells of a normal, haploid strain. The adsorption of BF23 to hole cells, cell envelopes, outer membrane particles, and solubilized outer membranes was inhibited by vitamin B12, with 50% inhibition at B12 concentrations in the range of 0.5 to 2.0 nM. These values are close to the observed KS for B12 binding to the B12 receptors. Vitamin B12 concentrations as high as 100 nM did not inhibit adsorption of bacteriophages T5, T6, and lambdacI to cells of sensitive strains of E. coli. Bacteriophage BF23 inhibited B12 transport by whole cells and was shown to be a competitive inhibitor of B12 binding to isolated cell envelope particles. The B12/BF23 receptors from E. coli strains KBT069 (btuB69) and RK4104 (btuB69) were fully active, but the number per cell was reduced to an average value of about 0.5.     

Domain deletion in the extracellular portion of the EGF-receptor reduces ligand binding and impairs cell surface expression.

Cultured NIH-3T3 cells were transfected with cDNA constructs encoding human epidermal growth factor-receptor (EGF-R)* and two deletion mutants in the extracellular portion of the receptor molecule. One mutant is devoid of 124 amino-terminal amino acids, and the other lacks 76 residues. Mutant receptors were not delivered to the cell surface unless the transfected cells contained also endogenous EGF-Rs, suggesting that receptor interaction complements the mutation and allows surface display of mutant receptors. Immunoprecipitation experiments revealed an association between mutant and endogenous EGF-Rs when both proteins were expressed in the same cell. Hence, receptor-oligomers may exist in the plane of the membrane even in the absence of ligand binding, and oligomerization may play a role in normal trafficking of EGF-Rs to the cell surface. Mutant receptors retained partial ligand binding activity as 125I-labeled EGF was covalently cross-linked to both mutant receptors, and EGF stimulated, albeit weakly, their protein tyrosine kinase activity. Both mutant EGF-Rs bind EGF with a 10-fold lower affinity than that of the solubilized wild type EGF-R. These results provide further evidence that the region flanked by the two cysteine-rich domains plays a crucial role in defining ligand-binding specificity of EGF-R.     

Relationships between glycosaminoglycan and receptor binding sites in chemokines-the CXCL12 example

Chemokines are small proteins, promoting directional migration and activation of different cells through binding to specific receptors. Most chemokines also bind to heparan sulfate (HS), a family of complex and highly sulfated glycosaminoglycan (GAG) found at the cell surface and in the extracellular matrix. This class of molecules has recently emerged as critical regulators of many events involving cell response to the external environment. Binding to HS is thought to be functionally important. Current models suggested that HS ensures the correct positioning of chemokines within tissues and maintains haptotactic gradients of the proteins along cell surfaces, thus providing directional cues for migrating cells. On the chemokine surface, the GAG binding epitopes can be displayed on different areas, some of which overlap the receptor binding domain, while others are clearly separated. We review here some structural aspects of the interaction between GAGs or receptors and chemokines. In particular, we will address the case of CXCL12, a chemokine whose receptor binding site is distinct from the GAG binding site and whose different isoforms display different GAG binding abilities. This chemokine system thus offers an unprecedented opportunity to ascertain the importance of chemokine/GAG interaction in the regulation of cell migration.     

The binding of vascular endothelial growth factor to its receptors is dependent on cell surface-associated heparin-like molecules.

Vascular endothelial growth factor (VEGF) induces the proliferation of endothelial cells and is a potent angiogenic factor that binds to heparin. We have therefore studied the effect of heparin upon the interaction of VEGF with its receptors. Heparin, at concentrations ranging from 0.1 to 10 micrograms/ml, strongly potentiated the binding of 125I-VEGF to its receptors on endothelial cells. Scatchard analysis of 125I-VEGF binding indicates that 1 microgram/ml heparin induces an 8-fold increase in the apparent density of high affinity binding sites for VEGF, but does not significantly affect the dissociation constant of VEGF. Cross-linking experiments showed that heparin strongly potentiates the formation of the 170-, 195- and 225-kDa 125I-VEGF-receptor complexes on endothelial cells. At high 125I-VEGF concentrations (4 ng/ml), heparin preferentially enhanced the formation of the 170- and 195-kDa complexes. Preincubation of the cells with heparin, followed by extensive washes, produced a similar enhancement of subsequent 125I-VEGF binding. The binding of 125I-VEGF was completely inhibited following digestion of endothelial cells with heparinase and could be restored by the addition of exogenous heparin to the digested cells. The enhancing effect of heparin facilitated the detection of VEGF receptors on cell types that were not known previously to express such receptors. Our results suggest that cell surface-associated heparin-like molecules are required for the interaction of VEGF with its cell surface receptors.     

Cell-to-cell binding induced by different lectins

The cell-to-cell binding induced by concanavalin A (Con A) and the lectins from wheatgerm, soybean, and waxbean has been analyzed by measuring the ability of single cells to bind to lectin-coated cells immobilized on nylon fibers. The cells used were lymphoma, myeloid leukemia, and normal fibroblast cells. With all lectins, cell-to-cell binding was inhibited if both cells were prefixed with glutaraldehyde. However, in most cases cell-to-cell binding was enhanced when only the lectin-coated cell was prefixed. With normal fibroblasts, treatment of either one or both cells with trypsin enhanced the cell-to-cell binding induced by Con A and the wheatgerm lectin. Neuraminidase, which increases the number of receptors for soybean agglutinin, increased cell-to-cell binding only if both cells were treated. Although cell-to- cell binding induced by the lectins from soybean and wheatgerm could be partially reversed by the appropriate competitive saccharide inhibitor, binding induced by Con A could not be reversed. The experiments indicate that cell-to-cell binding induced by a lectin can be prevented by an insufficient density of receptors for the lectin, insufficient receptor mobility, or induced clustering of receptors. These effects can explain the differences in cell-to-cell binding and agglutination observed with different cell types and lectins. They also suggest that cell-to-cell binding induced by different lectins with a variety of cell types is initiated by a mechanism involving the alignment of complementary receptors on the colliding cells for the formation of multiple cell-to-lectin-to-cell bridges.     

Demonstration of binding of dengue virus envelope protein to target cells.

The nature of the initial interaction of dengue virus with target cells and the extent to which this interaction defines tropism are unknown. Infection of some cells may involve antidengue antibody-mediated immune adherence to cells bearing immunoglobulin Fc receptors; however, this mechanism does not explain primary infection or the infection of cells without Fc receptors. We hypothesized that dengue virus envelope protein mediates initial binding to target cells. To test this hypothesis, a recombinant chimeric form of dengue type 2 virus envelope protein was used as a probe to investigate binding to the surfaces of potential target cells. Envelope protein was expressed amino terminal to the heavy-chain constant region of human immunoglobulin G containing the Fc receptor binding motif; the binding mediated by envelope determinants was distinguishable from the binding mediated by immunoglobulin Fc determinants. We found that the recombinant chimera bound to Vero, CHO, endothelial, and glial cells through envelope protein determinants and to monocytes and U937 cells by Fc-Fc receptor interactions. The highest level of binding was to Vero cells; binding was dose and time dependent and saturable. Examination of partial-length recombinant envelope proteins indicated that the binding motif was expressed between amino acids 281 and 423. Recombinant envelope protein inhibited infection of Vero cells by dengue virus, indicating the functional significance of the interaction of envelope protein and target cells in infectivity. These results suggest that envelope protein binding to a non-Fc receptor could explain the cell and tissue tropism of primary dengue virus infection.     

Initial interaction of herpes simplex virus with cells is binding to heparan sulfate.

We have shown that cell surface heparan sulfate serves as the initial receptor for both serotypes of herpes simplex virus (HSV). We found that virions could bind to heparin, a related glycosaminoglycan, and that heparin blocked virus adsorption. Agents known to bind to cell surface heparan sulfate blocked viral adsorption and infection. Enzymatic digestion of cell surface heparan sulfate but not of dermatan sulfate or chondroitin sulfate concomitantly reduced the binding of virus to the cells and rendered the cells resistant to infection. Although cell surface heparan sulfate was required for infection by HSV types 1 and 2, the two serotypes may bind to heparan sulfate with different affinities or may recognize different structural features of heparan sulfate. Consistent with their broad host ranges, the two HSV serotypes use as primary receptors ubiquitous cell surface components known to participate in interactions with the extracellular matrix and with other cell surfaces.     

Antigen binding to receptors on immunocompetent cells. I. Simple models and interpretation of experiments

We have developed simple mathematical models for treating the kinetics of binding of multivalent antigen to immune cell receptors when the binding may be in competition with nonspecific binding of antigen to cell surfaces and with the binding of hapten to the receptors. All three kinds of binding are treated as reversible bimolecular reactions. In general, the resulting equations must be solved numerically. When, however, the antigen and hapten concentrations are large compared to receptor concentrations, approximate algebraic solutions are found. It is shown that the most important effect of the hapten-receptor binding and of the nonspecific antigen binding is to slow down the antigen-receptor association; this may be viewed as a decrease in the antigen-receptor association rate constant.We have applied these models to analyze experiments of Davie and Paul on the binding of antigen to receptors on immunocompetent cells. Many difficulties have been found to arise from nonspecific binding. In particular, the association rate constant and equilibrium constant will appear reduced by nonspecific binding and the association rate constant will appear anomalously temperature dependent. We interpret hapten inhibition of antigen binding as a nonequilibrium effect in which hapten reduces the rate of antigen-cell association. In this way the concentration of hapten required to give 50% inhibition of antigen binding is found to decrease, as observed, with time after immunization. If equilibrium were to be achieved we predict that the required concentrations of hapten would be found to increase with time.     

Receptor-mediated binding of C1q on pulmonary endothelial cells

Normal undamaged pulmonary endothelial cells appear to be immunologically privileged in that they do not express receptors for the Fc portion of IgG nor for C3b. However, these receptors become unmasked on endothelial cells injured by viral infection or exposure to white cell lysates. We now present evidence to indicate that C1q binds to specific receptors on the surface of normal healthy endothelial cells. The binding is dose-dependent, reversible and saturable. Furthermore our data show that binding of C1q to endothelial cells is via the collagenous portion of the molecule not via the globular head regions. Thus binding of C1q to endothelium would have the effect of exposing Fc receptors that could then bind to IgG of circulating immune complexes. That Fc receptors are in fact exposed is shown by rosette formation with antibody sensitized erythrocytes. With 2C1r-2C1s-associated C1q, no binding occurred using C1 fixation and transfer assays. Our results indicate that C1q binding to endothelium provides a means for localizing immune complexes on pulmonary vessels and may be important in the initiation and progression of the inflammatory response.     

The interaction of nominal antigen with T cell antigen receptors. I. Specific binding of multivalent nominal antigen to cytolytic T cell clones

In this report, we describe an experimental strategy for analyzing the interaction of nominal antigen with antigen-specific T cell clones. Our approach was based on the notion that low affinity interactions between nominal antigen and T cell antigen receptors might be detected by using a highly multivalent form of the antigen in which a large number of identical, appropriately spaced epitopes are attached to a polymer backbone. Antigens of this kind should be capable of multivalent binding to receptors on the T cell, resulting in a marked enhancement of the overall avidity of the interaction. To examine this possibility, we established a series of murine cytolytic T cell (Tc) clones specific for the readily detectable hapten fluorescein isothiocyanate (FL). These clones lysed FL-conjugated target cells in an antigen-specific fashion and also showed specificity for target cell MHC gene products. The interaction of these clones with the nominal antigen FL was assessed by flow cytometry, using a series of water-soluble FL-conjugated polymers varying in polymer backbone and FL isomer. High m.w. (600 to 2000 Kd) polymers of acrylamide, dextran, or Ficoll conjugated with 300 to 800 FL groups/molecule bound specifically to anti-FL Tc clones. There was little binding to syngeneic spleen cells, thymocytes, noncytolytic T cell clones, or T cell clones specific for other haptens such as NIP. Polymer concentrations in the 1 to 10 micrograms/ml range produced readily detectable binding within minutes at 20 degrees C, and the binding approached plateau levels at polymer concentrations of between 100 and 300 micrograms/ml. Studies with closely related FL isomers showed that the same antigen fine specificity was operative in both lysis of FL-conjugated target cells and in binding of FL-conjugated polymers. The functional significance of the observed binding was assessed by measuring the effect of FL-conjugated polymers on lymphokine secretion by the clones. High m.w. FL-conjugated polymers caused a dose-dependent increase in the production of macrophage activation factor (MAF) by anti-FL Tc clones, but did not increase MAF production by an NIP-specific clone. In contrast, concanavalin A induced MAF production by both FL-specific and NIP-specific clones. Thus, the observed binding is both specific and functionally significant. These results suggest that soluble nominal antigen, in an appropriately multivalent form, can bind specifically to antigen receptors on Tc clones.     

Dictyostelium discoideum: Cell-type proportioning, cell-differentiation preference, cell fate, and the behavior of anterior-like cells in Hs1/Hs2 and G+/G− mixtures

Abstract. Conjugation in Tetrahymena is a cell interaction that involves the formation of pairs of cells of complementary mating types that are joined with opposite polarity at their anterior ends. Characteristically, it takes 1 h from the time cells are mixed until they begin to pair. We have previously shown that, during this time, the anterior tips of both mating types undergo morphogenetic transformation. The tips, which are normally pointed and ciliated, become truncated and cilia-free, and the cortical ridges disappear, leaving a smooth surface. We have also shown that the conjugation junction is formed during pairing by the apposition and alignment of two transformed surfaces. In the present study, we examined the binding of fluorescein-conjugated concanavalin A (F-Con A) in paraformalde-hyde-fixed cells at a stage when most cells have transformed tips but few are paired. We observed binding of Con A at anterior tips in a manner that was correlated with the extent of tip transformation. We further mapped the distribution of Con A receptors in conjugant pairs by orienting pairs with the plane of the junction perpendicular to the axis of illumination. It was observed that the distribution of Con A receptors formed a heart-shaped ring around the conjugation junction, in perfect accordance with the boundary line between the normal cortex and the transformed cortex. Additional experiments indicated that this binding pattern reflects the true distribution of receptors, suggesting that the receptors migrate from transformed tips to the junction ring in association with cell pairing. There is evidence that Con A receptors may have a function in cell adhesion in this system. Therefore, during this highly programmed cell interaction, the spatial redistribution of surface receptors and the morphological differentiation of the cell surface — both of wich may have a function in cell-cell attachment — are closely coordinated processes.     

Plasmin binding to the plasminogen receptor enhances catalytic efficiency and activates the receptor for subsequent ligand binding.

Specific cell surface receptors for plasminogen (Pg) are expressed by a wide variety of cell types. The colocalization of receptors for Pg and its activators restricts plasmin (Pm) activity to specific sites and serves to promote fibrinolysis and local Pg activation. These studies show that both Pg and Pm bind to cellular receptors on monocytoid U937 cells. Limited Pm pretreatment of the cells enhances total Pg binding and alters the kinetics of Pm binding. Furthermore, surface-bound Pg is converted to Pm in the absence of exogenous activators. Cell-bound Pm exhibits a 12-fold increase in catalytic efficiency (kcat/Km) relative to Pm free in solution. These studies demonstrate that Pg/Pm receptor occupancy can be regulated by Pm in the microenvironment and may play a significant regulatory role in fibrinolysis and extravascular proteolysis.     

Factor-assisted DNA binding as a possible general mechanism for steroid receptors. Functional heterogeneity among activated receptor-steroid complexes

We previously reported that activated glucocorticoid receptor-steroid complexes from rat HTC cell cytosol exist as at least two sub-populations, one of which requires a low molecular weight (700–3000 Da) factor(s) for binding to DNA. This factor is removed by Sephadex G-50 chromatography and is found predominantly in extracts of crude HTC cell nuclei. We have now determined that factor is not limited to HTC cells since an apparently identical factor(s) was found in nuclear extracts of rat kidney and liver as well as human HeLa and MCF-7 cells. Furthermore, the DNA binding of a sub-population of human glucocorticoid receptors depends on factor. While these results were obtained with agonist (dexamethasone) bound receptors, a sub-population of HTC cell receptors covalently labeled by the antiglucocorticoid dexamethasone 21-mesylate also displayed factor-dependent DNA binding. This receptor heterogeneity was not an artifact of cell-free activation since the cell-free nuclear binding of dexamethasone mesylate labeled complexes was, as in intact cells, less than that for dexamethasone bound complexes. Earlier results suggested that the increased DNA binding with factor involved a direct interaction of receptor with factor(s). We now find that the factor-induced DNA binding is retained by amino terminal truncated (42 kDa) glucocorticoid receptors from HTC cells. Thus the ability of receptor to interact with factor(s) is encoded by the DNA and/or steroid binding domains. Two dimensional gel electrophoresis analysis of dexamethasone-mesylate labeled 98 kDa receptors revealed multiple charged isoforms for both sub-populations but no differences in the amount of the various isoforms in each sub-population. Finally, activated progesterone and estrogen receptor complexes were also found to be heterogeneous, with a similar, if not identical, small molecular weight factor(s) being required for the DNA binding of one sub-population. The observations that functional heterogeneity of receptors is not unique to glucocorticoid receptors, whether bound by an agonist or antagonist, and that the factor(s) is neither species nor tissue specific suggests that factor-assisted DNA binding may be a general mechanism for all steroid receptors.     

Receptor binding oscillations]

A kinetic analysis of the delta-opiate receptor agonist, [3H] DADLE, binding to cell (NG108-15) suspensions has led to the discovery of a new periodic biological phenomenon, namely, receptor activity oscillations. The absence of oscillations for the antagonist binding to the receptors suggests that oscillations are generated only as a result of receptor signal transformation. The correlation between the quantitative characteristics of the kinetic curves and the experimental conditions points to the fact that receptor binding oscillations may be due either to the receptor binding to the G-protein or the interaction of the receptor (or G-protein) with microtubules.     

Cholesterol modulates cellular TGF-beta responsiveness by altering TGF-beta binding to TGF-beta receptors

Transforming growth factor-beta (TGF-beta) responsiveness in cultured cells can be modulated by TGF-beta partitioning between lipid raft/caveolae- and clathrin-mediated endocytosis pathways. The TbetaR-II/TbetaR-I binding ratio of TGF-beta on the cell surface has recently been found to be a signal that controls TGF-beta partitioning between these pathways. Since cholesterol is a structural component in lipid rafts/caveolae, we have studied the effects of cholesterol on TGF-beta binding to TGF-beta receptors and TGF-beta responsiveness in cultured cells and in animals. Here we demonstrate that treatment with cholesterol, alone or complexed in lipoproteins, decreases the TbetaR-II/TbetaR-I binding ratio of TGF-beta while treatment with cholesterol-lowering or cholesterol-depleting agents increases the TbetaR-II/TbetaR-I binding ratio of TGF-beta in all cell types studied. Among cholesterol derivatives and analogs examined, cholesterol is the most potent agent for decreasing the TbetaR-II/TbetaR-I binding ratio of TGF-beta. Cholesterol treatment increases accumulation of the TGF-beta receptors in lipid rafts/caveolae as determined by sucrose density gradient ultracentrifugation analysis of cell lysates. Cholesterol/LDL suppresses TGF-beta responsiveness and statins/beta-CD enhances it, as measured by the levels of P-Smad2 and PAI-1 expression in cells stimulated with TGF-beta. Furthermore, the cholesterol effects observed in cultured cells are also found in the aortic endothelium of atherosclerotic ApoE-null mice fed a high cholesterol diet. These results indicate that high plasma cholesterol levels may contribute to the pathogenesis of certain diseases (e.g., atherosclerosis) by suppressing TGF-beta responsiveness.     

33-kDa C-terminal heparin binding fragment of fibronectin promotes attachment and spreading of hepatocytes

The possibility of interaction of hepatocytes with the heparin binding domain of Fibronectin was examined. Rat hepatocytes adhered to coverslips coated with the 33-kDa heparin binding fragment of the C-terminal region of plasma fibronectin. When different concentrations of the heparin binding fragment were used to coat coverslips and used as substratum, cell attachment showed saturation kinetics. Half the maximum attachment was observed at 30–40 min after seeding of cells. The cells became flat after 2–3 h indicating that they spread on the heparin binding domain as they do on intact fibronectin. Among the different glycosaminoglycans tested, maximum inhibition of attachment was observed for heparin. However it was not possible to completely inhibit attachment even at high concentrations. These results indicate that hepatocytes interact with fibronectin not only through the Arg-Gly-Asp-containing cell binding fragment, but also through the heparin binding domain of fibronectin and, further, that there exist heparin-dependent and heparin-independent mechanisms of interaction of cells with the 33-kDa heparin binding fragment of fibronectin     

Receptors for IgG and complement in human spleen lymphoid cells. Preferential binding of particulate immune complexes through complement receptors.

Human lymphoid spleen cells attached to Petri dishes by poly-L-lysine bind 51Cr-labeled erythrocytes coated with IgG antibodies or complement but not uncoated erythrocytes or those coated with IgM antibodies. The number of erythrocytes bound through complement receptors is several times larger than that bound through IgG receptors. Increasing up to five times the number of IgG molecules on the red blood cells only leads to a slight increase of binding. However, the addition of complement to the IgG-coated erthrocytes increases 10 times the binding to spleen cells, even in the presence of an excess of normal IgG. These results can be explained by postulating that there is a larger number (or greater affinity) of spleen cell receptors for complement than that of spleen cell receptors for IgG.