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D Holowka

Publications and source records attributed to D Holowka.

At least 73 records · Page 4Linked to original sources
PubMed1988

Molecular crowding on the cell surface.

Strong steric interactions among proteins on crowded living cell surfaces were revealed by measurements of the equilibrium spatial distributions of proteins in applied potential gradients. The fraction of accessible surface occupied by mobile surface proteins can be accurately represented by including steric exclusion in the statistical thermodynamic analysis of the data. The analyses revealed enhanced, concentration-dependent activity coefficients, implying unanticipated thermodynamic activity even at typical cell surface receptor concentrations.

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PubMed1988

Cross-linking of IgE-receptor complexes by rigid bivalent antigens greater than 200 A in length triggers cellular degranulation.

We have examined the effect of cross-linking IgE-receptor complexes with variable receptor-receptor distances on the transmembrane signaling that leads to degranulation of rat basophilic leukemia cells. Linear polymers of the biotin-binding protein avidin were generated with bis biotin-1,12-diamidododecane, and a dinitrophenyl-biotin conjugate was bound at each end of the polymers to form a series of rigid bivalent haptens of well-defined length. The polymers were fractionated by size with nondenaturing PAGE, electro-eluted, and tested for their ability to stimulate degranulation of rat basophilic leukemia cells sensitized with anti-DNP IgE. We found that hexamers of avidin (of length greater than or equal to 240 A) were as effective in triggering degranulation as dimers (of length approximately 80 A), while the monomeric avidin antigen (of length approximately 40 A) elicited a poorer degranulation response from the cells. The mechanism by which aggregation of cell surface receptors can initiate signal transduction is discussed in light of these results.

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PubMed1987

The effect of receptor density on the forward rate constant for binding of ligands to cell surface receptors.

For monovalent ligands interacting with cell surface receptors we have directly observed the functional dependence of the forward rate constant on the number of receptors per cell (N). The experimental system we studied consisted of monovalent ligand, 2,4-dinitrophenyl (DNP)-aminocaproyl-L-tyrosine (DCT), binding to bivalent, monoclonal anti-DNP immunoglobulin E (IgE) anchored to its high affinity receptor on rat basophilic leukemia (RBL) cells. To measure the fractional occupation of antibody combining sites by DNP we employed a recently developed fluorescence technique (Erickson, J., Kane, B. Goldstein, D. Holowka, and B. Baird, 1986, Mol. Immunol., 72:769-781). Our results are well fitted by the equation (Berg and Purcell, 1977, Biophys. J., 20:193-219) konc = 4 pi DaN kappa on/[4 pi Da + N kappa on] where konc is the forward rate constant for binding to the cell, D is the diffusion constant of the ligand, a is the radius of the cell, and kappa on is the intrinsic forward rate constant describing a single IgE combining site-DNP interaction. If D is fixed at 10(-5) cm2/s, the best fit of accumulated data predicts an average cell radius of approximately 4 microns and kappa on of approximately 1.8 x 10(-13) cm3/s [1.1 x 10(8)(M . s)-1]; both in excellent agreement with RBL cell size and the single-site forward rate constant for the binding of DCT to IgE in solution, respectively. We believe this is the first report of experimental evidence that directly illustrates the effect of surface density in determining the rates of binding for small molecules to membrane receptors.

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PubMed1987

Membrane-bound IgE receptor complexes fused with rat basophilic leukemia cells mediate degranulation.

The high affinity receptor for IgE on rat basophilic leukemia (RBL) cells mediates antigen-triggered cellular degranulation. Polyethylene glycol-induced membrane fusion methods were used to introduce exogenous IgE receptors into living RBL cells, and these were tested for normal activities. In cell-cell fusion experiments, RBL cells with fluorescein-labeled rat IgE bound to receptors and containing [5-1,2-3H(N)]hydroxytryptamine binoxalate ([3H]5HT) in their secretory granules were fused to cells with receptors occupied by rhodamine-labeled anti-dinitrophenyl mouse IgE. The fused cells showed a uniform surface distribution of both types of IgE, which could be patched independently by anti-IgE or dinitrophenylated bovine gamma globulin (DNP16BGG). [3H]5HT release could be triggered specifically by DNP16BGG. In vesicle-cell fusion experiments, plasma membrane vesicles, with receptors occupied by fluorescein- and 125I-labeled anti-DNP mouse IgE, were fused to RBL cells containing [3H]5HT. The cells showed substantial associated fluorescein fluorescence and 125I counts, and [3H]5HT release could be triggered specifically by DNP16BGG. These experiments indicate that IgE receptors can be dissociated from their natural cellular interactions and retain the ability to reassociate with another cell's components to deliver the transmembrane signal for degranulation.

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PubMed1986

Cross-linking of immunoglobulin E-receptor complexes induces their interaction with the cytoskeleton of rat basophilic leukemia cells.

Bridging of immunoglobulin E (IgE)-receptor complexes on rat basophilic leukemia cells by polyclonal anti-IgE antibodies induces a detergent-resistant association of these complexes with the cellular cytoskeleton. In dose-response curves the extent of the cytoskeletal association appears to follow the extent of bridging, continuing to increase beyond where stimulated degranulation is maximal. This stable association is maintained after the aggregated IgE-receptor complexes have been internalized by the cell. Multivalent antigen and trimeric IgE cause less extensive receptor cross-linking than anti-IgE and stimulate degranulation; they also induce receptor association with the cytoskeleton that is revealed only after stabilization by addition of a chemical cross-linking reagent. The ability of a membrane impermeant chemical cross-linker to stabilize this association suggests that the receptor-cytoskeletal interaction may be mediated by a transmembrane protein that is exposed at the cell surface. Monomeric and dimeric IgE bound to receptors fail to induce a stable interaction with the cytoskeleton even in the presence of chemical cross-linkers and are poor (dimers) or insignificant (monomers) stimulators of cellular degranulation. These findings are consistent with a possible relationship between receptor attachment to the cytoskeleton, receptor immobilization as measured by fluorescence photobleaching recovery, and the triggering of cellular degranulation.

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PubMed1986

Cross-linking of IgE-receptor complexes at the cell surface: a fluorescence method for studying the binding of monovalent and bivalent haptens to IgE.

We have developed a method for use in investigating factors controlling the binding and cross-linking by bivalent haptens of immunoglobulin E (IgE) bound to receptors on rat basophilic leukemia (RBL) cells. This method employs monoclonal anti-2,4-dinitrophenyl (DNP) IgE that is labeled with fluorescein-5-isothiocyanate (FITC), and it measures FITC quenching that accompanies DNP occupation of the antibody combining sites in a titration experiment. The validity of this approach is demonstrated using the monovalent hapten DNP-L-lysine. The affinity constant for this ligand obtained by the FITC quenching method is compared with those obtained with previously established methods: equilibrium dialysis and quenching of endogenous tryptophan for IgE in solution and [3H]-DNP-L-lysine binding to IgE on cells. The FITC quenching method has been used to carry out a detailed study of the binding of monovalent DNP-aminocapryol-L-tyrosine (DCT) and bivalent (DCT)2-cystine to FITC-IgE and its Fab fragments in solution. Intrinsic (K) and cross-linking (Kx) affinity constants are obtained by analyzing the binding curves in terms of simple equilibrium equations. With these DCT haptens the ability of this method to assess hapten binding and cross-linking of IgE bound to receptors on RBL cells is shown.

2,4-Dinitrophenol↗
PubMed1986

Cross-linking of IgE-receptor complexes at the cell surface: synthesis and characterization of a long bivalent hapten that is capable of triggering mast cells and rat basophilic leukemia cells.

The ability of a series of bivalent haptens to bind and cross-link immunoglobulin E (IgE) in solution and on the surface of cells was examined. Several short (less than 30 A) dinitrophenyl (DNP) haptens were found to bind tightly to and cross-link a monoclonal anti-DNP IgE in solution, but these failed to trigger substantial release of 3H-serotonin from sensitized rat basophilic leukemia (RBL) cells or rat peritoneal mast cells. A longer bivalent hapten, approximately 50 A in length, consisting of two DNP-aminocaproyl-L-tyrosine (DCT) groups coupled to the alpha-amino groups of L-cystine was synthesized and characterized. This bivalent hapten [(DCT)2-cystine], binds very tightly to the same monoclonal anti-DNP IgE in solution and cross-links these antibodies to form higher mol. wt aggregates as judged by gel filtration and binding studies. It also stimulates degranulation of both RBL and mast cells sensitized with two different monoclonal anti-DNP IgE antibodies, with the mast cells exhibiting generally greater responsiveness to this ligand. The (DCT)2-cystine bivalent hapten appears to have the structural features necessary for carrying out detailed binding studies with receptor-bound IgE on the cell surface.

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PubMed1986

Clustering, mobility, and triggering activity of small oligomers of immunoglobulin E on rat basophilic leukemia cells.

We have recently shown that small oligomers of IgE bound to univalent receptors for IgE on the surface of rat basophilic leukemia cells induce extensive aggregation of the receptors at 4 degrees C into patches resolvable by fluorescence microscopy and that this does not occur with monomeric IgE (Menon, A. K., D. Holowka, and B. Baird, 1984, J. Cell Biol. 98:577-583). Here we use fluorescence photobleaching recovery measurements to show that receptor oligomerization by this means is accompanied by a dramatic reduction of receptor lateral mobility, and that this immobilization occurs even when the clustering is not microscopically detectable. Furthermore, the degree of immobility induced by a particular oligomer fraction from a gel filtration column correlates positively with its ability to trigger cellular degranulation, whereas receptors labeled with monomeric IgE have no triggering activity and exhibit typical membrane protein mobility. The slow, large-scale oligomer-induced clustering appears to be a long term consequence of earlier selective interactions that result in receptor immobilization, and this highly clustered state provides a competent, noninhibitory triggering signal resulting in cellular degranulation upon warming to 37 degrees C. We conclude that even limited clustering of IgE receptors on rat basophilic leukemia cells induces interactions with other cellular components that constrain receptor mobility and eventually cause massive coalescence of the clusters. These primary selective interactions occurring at the level of receptor oligomers or small clusters of oligomers that result in immobilization may play a role in triggering cellular degranulation.

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PubMed1986

Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization.

Controlled cross-linking of IgE-receptor complexes on the surface of rat basophilic leukemia cells and mast cells has allowed a comparison of the lateral mobility and cell triggering activity of monomers, dimers, and higher oligomers of receptors. Addition of a monoclonal anti-IgE(Fc) antibody to IgE-sensitized cells in stoichiometric amounts relative to IgE produces IgE-receptor dimers with high efficiency. These dimers are nearly as mobile as IgE-receptor monomers and trigger cellular degranulation poorly, but in the presence of 30% D2O, substantial immobilization of the dimers is seen and degranulation activity doubles. Addition of this monoclonal antibody in larger amounts results in the formation of larger oligomeric receptor clusters which are immobile and effectively trigger the cells. Thus, small receptor clusters that are active in stimulating degranulation are immobilized in a process that is not anticipated by simple hydrodynamic theories. Further experiments involving cross-linking of receptor-bound IgE by multivalent antigen demonstrate that immobilization of receptors occurs rapidly (less than 2 min) upon cross-linking and is fully and rapidly reversible by the addition of excess monovalent hapten. The rapidity and reversibility of the immobilization process are entirely consistent with the possibility that immobilization represents a recognition event between clustered receptors and cytoskeleton-associated components that plays an important role early in the cell triggering mechanism.

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PubMed1985

Segmental flexibility of receptor-bound immunoglobulin E.

The segmental flexibility of mouse immunoglobulin E (IgE) bound to its high-affinity receptor on membrane vesicles from rat basophilic leukemia cells was compared to that of IgE in solution by measuring the steady-state anisotropy as a function of temperature and viscosity. A monoclonal IgE was used to bind the fluorescent probe N-[5-(dimethylamino)naphthalene-1-sulfonyl]-L-lysine (DNS-Lys) rigidly and specifically in the antigen combining site at the tip of the Fab region. The average rotational correlation time, phi, of 74-89 ns for the receptor-bound IgE is only slightly longer than that for IgE in solution where phi = 54 ns. Another mouse monoclonal IgE was covalently labeled in the Fab region with N-(1-pyrenyl)maleimide. Anisotropy measurements with this derivative yielded results that are very similar to those found with anti-DNS IgE and DNS-Lys. These findings are strikingly different from that expected for a rigid IgE bound to its receptor since in this case phi is likely to be very much larger. Evidently, the segmental flexibility of IgE is not greatly altered upon binding to its receptor.

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PubMed1985

Structural mapping of Fc receptor bound immunoglobulin E: proximity to the membrane surface of the antibody combining site and another site in the Fab segments.

Resonance energy-transfer methods have been used to investigate the structure of immunoglobulin E (IgE) bound to its high-affinity receptor on plasma membrane vesicles derived from rat basophilic leukemia cells. The structural mapping of receptor-bound IgE was initiated in an earlier study [Holowka, D., & Baird, B. (1983) Biochemistry 22, 3475], and it is based on measuring the minimal distance from IgE sites that are selectively labeled with donor probes to a plane of amphipathic acceptors at the membrane surface. This paper describes the use of monoclonal IgE specific for 5-(dimethylamino)naphthalene-1-sulfonyl (DNS) to place a donor probe, DNS-L-Lys, in the antibody combining sites. The distance from these sites to the membrane surface was determined to be greater than 100 A with two different amphipathic acceptor probes. Another site in the Fab segments of monoclonal IgE (anti-dinitrophenyl) could be labeled selectively with N-[4-[7-(diethylamino)-4-methylcoumarin-3-yl]phenyl]maleimide (CPM) in the absence of reducing agents [CPM(-)], and the reaction could not be blocked by prereaction with N-ethylmaleimide. The pattern of CPM(-)-labeled proteolytic fragments and the lack of fluorescence quenching by (trinitrophenyl)lysine in the antibody combining sites suggested the CPM(-)-labeled site to be in the C epsilon 1 domain of IgE. The distance between this site on receptor-bound IgE and the membrane surface was determined to be 75-87 A with two different amphipathic acceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1985

Structural mapping of membrane-bound immunoglobulin E-receptor complexes: use of monoclonal anti-IgE antibodies to probe the conformation of receptor-bound IgE.

Previous resonance energy-transfer measurements have suggested that immunoglobulin E (IgE) may bend near the junction of its Fc and Fab segments in order to bind to its high-affinity receptor on rat basophilic leukemia cells. In order to test this possibility, two monoclonal antibodies were employed that bind specifically to rat IgE (IgER) when IgER is in solution and when it is bound to receptors on the plasma membrane. The F(ab')2 fragment of one monoclonal (B5) that is specific for the Fab region of IgER was labeled with donor probes and bound to IgER, and the quenching of the fluorescence of these donors due to simultaneous binding of the Fab' fragment of an anti-Fc monoclonal (A2) that was labeled with an acceptor probe at its interchain disulfide bond was measured. Significantly less energy transfer between these probes was observed when IgER was bound to its receptor on membrane vesicles than when it was free in solution, and this result is interpreted in light of other energy-transfer measurements using A2 and B5 that were preferentially labeled near their combining sites with donors and acceptors, respectively, as well as measurements of the distance of closest approach between these sites and the membrane surface. These results along with previous energy-transfer measurements and other biochemical information form the basis for a working model of the conformation and orientation of receptor-bound IgE. This study demonstrates the use of fluorescently labeled monoclonal antibodies as highly selective energy-transfer probes in assessing structures of macromolecular complexes on the plasma membrane.

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PubMed1984

Lactoperoxidase-catalyzed iodination of the receptor for immunoglobulin E at the cytoplasmic side of the plasma membrane.

The structural organization of the high affinity receptor for immunoglobulin E has been investigated in plasma membrane vesicles from rat basophilic leukemia cells using lactoperoxidase-catalyzed 125I-iodination to label exposed polypeptide regions. Intact vesicles are predominantly right-side-out in orientation, and lactoperoxidase iodination of these vesicles results in labeling of the alpha subunit of receptor but not the beta and gamma subunits. Lysis of these vesicles to expose the cytoplasmic face of the membrane by two different methods permits labeling of the beta and gamma subunits with no increase in labeling of alpha. The results indicate that both the beta and gamma subunits of the receptor have segments exposed at the cytoplasmic side of the plasma membrane. These studies have also revealed a previously unidentified IgE binding component in the membrane vesicles; its 125I-labeling characteristics and some other properties are described.

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PubMed1984

Small oligomers of immunoglobulin E (IgE) cause large-scale clustering of IgE receptors on the surface of rat basophilic leukemia cells.

We examined the distribution of small oligomers of IgE bound to rat basophilic leukemia cells using fluorescence microscopy. The oligomers were seen to cluster into visible patches on the cell surface at 4 degrees C; at higher temperatures internalization also was observed. In contrast, cells labeled with IgE monomers remained predominantly ring-stained. Evidence is provided that the observed clustering of IgE oligomers is a cell-induced phenomenon, and the possible significance of this clustering is discussed in the context of the oligomer-triggered degranulation of rat basophilic leukemia cells.

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PubMed1983

Structural studies on the membrane-bound immunoglobulin E-receptor complex. 1. Characterization of large plasma membrane vesicles from rat basophilic leukemia cells and insertion of amphipathic fluorescent probes.

In order to investigate the properties of the membrane-bound IgE-receptor complex, a simple procedure has been adapted for preparing large plasma membrane vesicles from rat basophilic leukemia cells. These vesicles pinch off from the adherent cells after treatment with 2 mM N-ethylmaleimide or 50 mM formaldehyde and 1 mM dithiothreitol, and they are isolated from the supernatant after two centrifugation steps with yields of 20-25% of the initial cell-bound 125I-IgE. With phase and fluorescence microscopy, micron-size vesicles are seen which are unilamellar and spherically shaped and devoid of intracellular organelles. On dextran gradients at least 70% of the 125I-IgE is bound to membranes which band at low density, indicating large, intact vesicles that are impermeable to macromolecules. Between 60 and 75% of the bound 125I-IgE is accessible to the external medium, showing the vesicles to be predominantly right side out. This preparation was found to be suitable for resonance energy-transfer measurements. We have determined that amphipathic, fluorescent donor and acceptor probes partition into the vesicle bilayer in a randomly distributed, noninteracting manner. The densities of the probes can be ascertained directly from the amount of energy transfer that is observed as a function of acceptor concentration. This experimental system will allow energy-transfer measurements to determine distances between sites on receptor-bound IgE and the membrane surface.

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PubMed1982

Further characterization of the beta-component of the receptor for immunoglobulin E.

A 30,000 mol. wt component (beta) is associated in a 1:1 ratio with the 50,000 mol. wt glycoprotein (alpha) which binds immunoglobulin E (IgE) on mast cells and related tumor cells. We show that alpha and beta are associated in membrane preparations. This is consistent with previous results which showed labeling of beta with the hydrophobic probe 5-iodonaphthyl-1-azide (INA). The beta-polypeptide is susceptible to proteolytic cleavage during preparation of the membranes and when this occurs a 20,000 fragment can be labeled with INA and remains associated with alpha. No incorporation of carbohydrate precursors into beta was observed. Since beta is also not modified when cells are surface-labeled, it may not be exposed on the cell surface. Rigorous washing of IgE-receptor complexes with non-ionic detergent results in dissociation of beta from the alpha-IgE complex. The latter will then not reassociate with beta when exposed to crude detergent extracts of the tumor cells.

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PubMed1982

Structure of the high-affinity mast cell receptor for IgE.

Mast cells and related rat tumor basophils have a surface receptor that binds monomeric IgE with high avidity. The receptor in situ is unclustered, mobile, and univalent, and its aggregation into dimers and higher oligomers triggers degranulation. It has two subunits, alpha and beta. The alpha chain has a molecular weight of about 50,000, of which 30% is carbohydrate. Heterogeneity in the latter accounts at least in part and perhaps fully for the heterogeneity of the alpha chain. Studies with proteases have delineated two domains: alpha 1 appears slightly larger on sizing gels, and incorporation studies suggest it has more carbohydrate than alpha 2. The alpha 2 domain and a 24,000-dalton subfragment of it contain the principal site whose surface labeling is blocked by the presence of IgE. The alpha subunit is firmly but noncovalently bound to beta in a 1:1 complex in situ and in detergent extracts. Nevertheless, during thorough washing of IgE-receptor complexes with detergent, beta dissociates. A portion of beta, beta 1, is integrated in the membrane; it is this domain that is labeled when the receptor is isolated from cells reacted with the intramembranous probe iodonapthylnitrene and that interacts with the alpha chain. These results and others have been used to draw a provisional model of the receptor.

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