Cross-linking Of Cell Surface Receptors Research Articles

Monitoring the Dynamics of Ligand−Receptor Complexes on Model Membranes

Ligand-induced cross-linking of cell surface receptors is a basic paradigm of signal activation by many transmembrane receptors. After ligand binding, the receptor complexes formed on the membrane are dynamically maintained by two-dimensional protein-protein interactions on the membrane. The biophysical principles governing the dynamics of such interactions have not been understood, mainly because the measurement of lateral interactions on membranes so far has not been experimentally addressed. Here, we describe a generic approach for measuring two-dimensional dissociation rate constants in vitro using a novel high-affinity chelator lipid for reconstituting a ternary cytokine-receptor complex on solid-supported membranes. While monitoring the interaction between the ligand and one of the receptor subunits on the membrane by fluorescence resonance energy transfer, the equilibrium on the surface was perturbed by rapidly tethering a large excess of the unlabeled receptor subunit. Displacement of labeled by unlabeled protein in the ternary complex was detected as a recovery of the donor quenching. Since the dissociation of the ligand-receptor complex in plane of the membrane was the rate-limiting step under these conditions, the two-dimensional rate constant of this process was determined. Strikingly, the two-dimensional dissociation was much slower than ligand dissociation into solution, suggesting that membrane tethering significantly affects the dissociation process. This result highlights the importance of studying ligand-receptor complexes tethered to membranes for understanding the principles governing signal activation by ligand-induced receptor assembling.

The role of cholesterol and sphigomyelin in tyrosine phosphorylation of proteins and capping of Fcgamma receptor II.

Cross-linking of cell surface receptors by multivalent ligands, e.g. by antibodies, evokes their clustering -- patching. Subsequently, these clusters can be translocated by the acto-myosin machinery toward one pole of the cell and assembly cap. Patching of FcgammaRII in U937 cells correlates with tyrosine phosphorylation of several proteins while cap assembly correlates with their dephosphorylation. To study the mechanism of activation of tyrosine kinases during FcgammaRII activation we disturbed the organization of the putative plasma membrane microdomains by depletion of membrane cholesterol and sphingomyelin. Cholesterol was removed with the use of beta-cyclodextrin while sphingomyelin was decomposed by exogenous sphingomyelinase. Cyclodextrin at 5-10 mM removed about 70% of cholesterol from the cells and abolished the assembly of FcgammaRII caps thereby arresting the receptors at the patching stage. Similarly, 70 mU/ml sphingomyelinase inhibited cap formation by 60%. Cholesterol and sphingomyelin depletion also suppressed the tyrosine phosphorylation of proteins which accompanied cross-linking of FcgammaRII. The observations indicate that cholesterol and sphingomyelin can control the interactions of tyrosine kinases with clustered FcgammaRII.

Localization of lipid peroxidation-derived protein epitopes in the porcine corpus luteum.

Reactive oxygen species (ROS) generated within the corpus luteum (CL) are believed to play an integral role in luteolysis. Unsaturated lipids are susceptible to peroxidation by ROS, leading to the formation of toxic aldehydes such as malondialdehyde (MDA) and 4-hydroxy-nonenal (4-HNE) that can attack cellular and extracellular proteins. Aldehyde conjugation of proteins can have diverse effects on cell function, including cross-linking of cell surface receptors, alteration of enzyme activities, and modification of lipoproteins. The objectives of the present study were to 1) determine by immunological techniques whether lipid peroxidation-derived protein epitopes (i.e., MDA- and 4-HNE-lysine) could be detected in situ during the natural life span of the CL, and 2) determine the temporal and spatial localization of these epitopes. Fresh luteal tissue was collected from young gilts at distinct phases of the estrous cycle: early (Days 4-7), mid (Days 8-12), and late (Days 14-18). Immunocytochemistry was performed on tissue sections using monoclonal antibodies against MDA-lysine and 4-HNE-lysine. Positive immunostaining was evident in porcine CL at all stages of the estrous cycle. Immunoactivity was associated primarily with the steroidogenic cells, most consistently with the large luteal cells, and was most intense in regressing tissue (i.e., late). No staining was observed in sections incubated with nonimmune mouse serum or when anti-4-HNE-lysine was preadsorbed with 4-HNE-protein preparations. Immunoblotting of protein extracts from luteal tissue revealed three major reactive bands. At least two of these bands were closely associated with extracted oxidized human low-density lipoprotein (LDL) and with 4-HNE-conjugated human apoprotein B. These results demonstrate the presence of lipid peroxidation-derived protein epitopes within the porcine CL and show that one or more of these may be an oxidized-LDL moiety. This represents the first direct in situ evidence that protein modification by lipid peroxidation products occurs during the natural luteal life span. The physiological consequences remain to be determined.

Cross-linking reconsidered: binding and cross-linking fields and the cellular response

We analyze a model for the reversible cross-linking of cell surface receptors by a collection of bivalent ligands with different affinities for the receptor as would be found in a polyclonal anti-receptor serum. We assume that the amount of cross-linking determines, via a monotonic function, the rate at which cells become activated and divide. In addition to the density of receptors on the cell surface, two quantities, the binding field and the cross-linking field, are needed to characterize the cross-linking curve, i.e., the equilibrium concentration of cross-linked receptors plotted as a function of the total ligand site concentration. The binding field is the sum of all ligand site concentrations weighted by their respective binding affinities, and the cross-linking field is the sum of all ligand site concentrations weighted by the product of their respective binding and cross-linking affinity and the total receptor density. Assuming that the cross-linking affinity decreases if the binding affinity decreases, we find that the height of the cross-linking curve decreases, its width narrows, and its center shifts to higher ligand site concentrations as the affinities decrease. Moreover, when we consider cross-linking-induced proliferation, we find that there is a minimum cross-linking affinity that must be surpassed before a clone can expand. We also show that under many circumstances a polyclonal antiserum would be more likely than a monoclonal antibody to lead to cross-linking-induced proliferation.

Inhibition of CD44, CD45 and LFA-3 mediated cytokine release from human monocytes by SK&F 86002 and pentoxifylline

Compounds from two distinct pharmacological classes namely, SK&F 86002 and pentoxifylline, were examined for their effects on TNFα and IL-1β release by human monocytes stimulated with LPS or monoclonal antibodies to three cell surface glycoproteins, CD44, CD45 and LFA-3 (LFA-3 is also known as CD58). SK&F 86002, an inhibitor of 5-LO and CO in arachidonic acid metabolism, inhibited LPS-induced release of TNFα and IL-1β with an IC 50 of 1 μM. At this dose, it also inhibited by >50%, release of both cytokines induced by the three monoclonal antibodies. Pentoxifylline, a methylxanthine derivative with phosphodiesterase inhibitory activity, selectivity inhibited LPS-induced TNFα release with an IC 50 of 100 μM. TNFα and IL-1β release mediated by the monoclonal antibodies were inhibited by less than 30% in the presence of 100 μM pentoxifylline. These results suggest that (a) LPS induced cytokine release shares a common step with the physiologically relevant stimuli (involving cross-linking of cell surface receptors), and that this pathway is sensitive to inhibition by SK&F 86002 and, (b) SK&F 86002 is more potent than pentoxifylline in inhibiting TNFα and IL-1β release induced by both stimuli.

Differential binding of transforming growth factor-beta 1, -beta 2, and -beta 3 by fibroblasts and epithelial cells measured by affinity cross-linking of cell surface receptors.

A murine fibroblast cell line (AKR-2B clone 84A) and an epithelial cell line (BALB/MK) were compared for their ability to bind different transforming growth factor-beta (TGF beta) species. The results of competitive binding assays indicated that the epithelial cells had a higher affinity for TGF beta than the fibroblasts. This difference may be the basis for the sensitivity of epithelial cells to much lower concentrations of TGF beta than fibroblasts. Affinity cross-linking studies showed that both cell types express the three cell surface TGF beta-binding molecules that have been previously described for a variety of cell types. The complexity of these cell surface binding proteins was further evaluated using all possible combinations of radiolabeled ligands in competition with each of the three unlabeled TGF beta species. Differences in the ability of specific TGF beta types to compete with radiolabeled TGF beta 2 for binding to the type I and II receptors were observed, with TGF beta 1 being more potent for epithelial cells, and TGF beta 2 being more potent for fibroblasts. In addition, a difference in the ability of different TGF beta species to compete the [125I]TGF beta 3 from epithelial cell surface receptors was apparent. TGF beta 2 was not able to compete with [125I]TGF beta 3 for binding to the type II receptor at any concentration tested, while TGF beta 1 and TGF beta 3 were about equally potent in competition for this receptor type. These differences in cell surface receptor binding of structurally and biologically similar molecules may reflect different functions for these molecules.

Interferon (IFN)-like antiviral effect is induced by unspecific cross-linking of cell surface receptors

Treatment of human amniotic cells (UAC) with Cytodex 1 (DEAE-dextran) results in the development of an antiviral state of the cells, as proven by studying (i) the cytopathic effect and (ii) [3H]uridine incorporation into the RNA of vesicular stomatitis virus (VSV) after VSV infection. The same treatment transiently triggers the breakdown of inositol phospholipids and activates the translocation of protein kinase C (PKC). On the basis of these data it can be suggested that cross-linking of cell surface receptors by a solid carrier bearing covalently bound positive charges may result in IFN-like effects.

Inhibition of cell growth by monoclonal anti-transferrin receptor antibodies.

Five anti-murine transferrin receptor monoclonal antibodies have been characterized with respect to immunoglobulin class, effects on binding of transferrin, and effects on AKR1 lymphoma cell growth in vitro. The immunoglobulin M (IgM) antibodies, but not the IgG antibodies, prevent cell growth. We suggest that the profound effects of the IgM antibodies on cell growth are probably due to extensive cross-linking of cell surface receptors. In support of this, we are able to mimic the growth-inhibiting effects of the IgM antibodies by adding antiimmunoglobulin to an IgG antibody. By flow microfluorimetry, we show that an IgG antibody by itself induces up to a 10-fold downward regulation in the cell surface transferrin receptor, which is accompanied by accelerated receptor degradation. A similar downward regulation is seen in mutant cells resistant to growth inhibition by an IgM antibody, when grown in the selecting antibody. Wild-type cells grown in the presence of IgM antibody do not show receptor downward regulation. Inhibitory effects of antibody plus antiimmuoglobulin on mutant cells are also consistent with extensive cross-linking causing inhibition of growth.

Inhibition of cell growth by monoclonal anti-transferrin receptor antibodies.

Five anti-murine transferrin receptor monoclonal antibodies have been characterized with respect to immunoglobulin class, effects on binding of transferrin, and effects on AKR1 lymphoma cell growth in vitro. The immunoglobulin M (IgM) antibodies, but not the IgG antibodies, prevent cell growth. We suggest that the profound effects of the IgM antibodies on cell growth are probably due to extensive cross-linking of cell surface receptors. In support of this, we are able to mimic the growth-inhibiting effects of the IgM antibodies by adding antiimmunoglobulin to an IgG antibody. By flow microfluorimetry, we show that an IgG antibody by itself induces up to a 10-fold downward regulation in the cell surface transferrin receptor, which is accompanied by accelerated receptor degradation. A similar downward regulation is seen in mutant cells resistant to growth inhibition by an IgM antibody, when grown in the selecting antibody. Wild-type cells grown in the presence of IgM antibody do not show receptor downward regulation. Inhibitory effects of antibody plus antiimmuoglobulin on mutant cells are also consistent with extensive cross-linking causing inhibition of growth.

Order of events leading to surface immunoglobulin capping: analysis of a transmembrane signal.

Capping provides a rapid assay for the transduction of 1 type of membrane signal generated by the cross-linking of cell surface receptors. The order of the steps comprising this signal was determined by employing reversible inhibitors of lymphocyte surface Ig capping in a sequential incubation protocol. The results demonstrated that surface Ig cross-linking leads to capping by a linear series of discrete events. Although the steps inhibited by the calcium ionophore A23187 and the tranquilizer chlorpromazine could not be distinguished, other agents also thought to influence calcium distribution in the cell acted at different steps. The order of inhibited steps was shown to be: 1) hydrocortisone; 2) calcium ionophore or chlorpromazine; 3) cytochalasins; 4) dibucaine; 5) propranolol; 6) fluoride; 7) azide. These results suggest a model wherein cross-linked membrane Ig aggregates engage the preassembled microfilament system by means of a calcium-dependent linkage. Further calcium redistribution within the cell then leads to an energy-consuming contractile event.