Receptor For Cholera Toxin Research Articles

Specific binding of cholera toxin to rat erythrocytes revealed by analysis with a fluorescence-activated cell sorter.

The direct binding of cholera toxin to the receptor on the native cell surface was analyzed with a fluorescence-activated cell sorter (FACS) by the direct membrane immunofluorescence technique using FITC-conjugated cholera toxin B subunit as a ligand and erythrocytes, but the binding was significantly affected by a change in pH, showing optimum pH of 7.2. The optimum conditions for analysis of the cholera toxin-binding with a FACS were reaction of the target cells with 0.2 M phosphate-buffer (pH 7.2) containing 0.025% of BSA and 0.175 M of NaCl at 4 degrees C for 40 min. The binding of cholera toxin B subunit to rat erythrocytes was linear in the range of 1.2 ng to 80 ng, which corresponded to 2,469 to 163,500 molecules of toxin per cell, and the latter was almost the saturated level of binding. although erythrocytes from different strains of rats possessed equal binding ability for the cholera toxin, no binding was observed with erythrocytes from mouse, guinea pig, cow, pig, man, or rabbit, indicating that the cholera-toxin binding occurs specifically on rat erythrocytes. This is in accord with our previous analytical deta on the absence of GM1 in erythrocytes of these animals except rat, of which erythrocytes contain GM1. Also, the structural specificity of the receptor for cholera toxin was assessed by a binding inhibition experiment using glycolipid-containing liposomes as inhibitors and GM1 was found to be the most potent inhibitor, showing complete inhibition of toxin (40 ng) binding to 5 x 10(6) erythrocytes at 505.6 pmol of GM1.

Structure of the thyrotropin receptor and thyroid adenylate cyclase system as determined by target analysis.

Bovine thyroid plasma membranes were irradiated with high-energy electrons. Analysis of the target size of the thyrotropin (TSH) receptor revealed a complex pattern composed of a TSH binding component of 71 000 daltons and a large component (several hundred thousand daltons) that masked some of the binding. Both components were also observed when binding was assayed in the presence of 50 mM NaCl. Membranes preincubated with Mg2+ and 10 microM guanosine 5'-(beta,gamma-imidotriphosphate) [Gpp(NH)p], a persistent activator of adenylate cyclase, also showed the presence of these same components. Although the receptor for TSH has been reported to have some similarities to the receptor for cholera toxin, target analysis of [125I]iodocholera toxin binding was consistent with a single small component about the size of a ganglioside. Measurement of the target size of ground-state, i.e., not preactivated, adenylate cyclase was also carried out. The basal (Mn2+) adenylate cyclase yielded a Mr of 85 000, the smallest unit capable of producing cAMP. The Gpp(NH)p-responsive adenylate cyclase has a Mr of 150 000, which may reflect the contribution of the guanine nucleotide regulatory component to the mass of the active enzyme. A similar size was previously measured for the Gpp(NH)p-preactivated, detergent-solubilized thyroid enzyme [Asbury, R.F., Cook, G.H., & Wolff, J. (1978) J. Biol. Chem. 253, 5286-5292]. Radiation inactivation of the NaF-responsive enzyme indicated two or more components to this activity, the smaller of which (140 000 daltons) was similar in size to the ground-state Gpp(NH)p-responsive enzyme and the larger of which was greater than 10(6) daltons.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes.

Fluorescent derivatives of gangliosides were prepared by oxidizing the sialyl residues to aldehydes and reacting them with fluorescent hydrazides. When rhodaminyl gangliosides were incubated with lymphocytes, the cells incorporated them in a time- and temperature-dependent manner. Initially, the gangliosides were evenly distributed on the cell surface but were redistributed into patches and caps by antirhodamine antibodies. When the cells were then stained with a second antibody or protein A labeled with fluorescein, the fluorescein stain revealed the coincident movement of both the gangliosides and the antirhodamine antibodies. When the cells were treated with both rhodamine and Lucifer yellow CH-labeled gangliosides, the antirhodamine antibodies induced patching and capping of both fluorescent gangliosides but had no effect on cells incubated only with Lucifer yellow CH-labeled gangliosides. In addition, capping was observed on cells exposed to cholera toxin, antitoxin antibodies, and rhodamine-labeled protein A, indirectly showing the redistribution of endogenous ganglioside GM1, the cholera toxin receptor. By incorporating Lucifer yellow CH-labeled GM1 into the cells and inducing capping as above, we were able to demonstrate directly the coordinate redistribution of the fluorescent GM1 and the toxin. When the lymphocytes were stained first with Lucifer yellow CH-labeled exogenous ganglioside GM3, which is not a toxin receptor, there was co-capping of endogenous GM1 (rhodamine) and exogenous GM3 (Lucifer yellow CH). These results suggest that gangliosides may self-associate in the plasma membrane which may explain the basis for ganglioside redistribution and capping.

The role of gangliosides in the interaction of human chorionic gonadotropin and cholera toxin with murine Leydig tumor cells.

A clonal line of murine Leydig tumor cells (MLTC-1) bound both human chorionic gonadotropin (hCG) and cholera toxin (CT) with high affinity and accumulated cyclic AMP in response to either effector. The major cellular ganglioside was GM3 with small amounts of GM2, GM1, and GD1a. The gangliosides became labeled when the cells were grown in medium containing [3H] galactose or were exposed to galactose oxidase or NaIO4 followed by NaB3H4. CT specifically protected GM1 from surface labeling whereas hCG did not protect any gangliosides from being labeled. When the cells were exposed to sialidase, surface GD1a was eliminated, and GM1 increased with a corresponding increase in CT binding. When sialidase-treated cells were first incubated with the B component of CT, binding and action of CT was blocked. The cells, however, retained their ability to bind and respond to hCG. Addition of purified gangliosides to the medium effectively inhibited the binding and action of CT but not hCG. The cells incorporated the exogenous gangliosides and exhibited increased binding of and responsiveness to CT but not hCG. Both hCG- and CT-receptor complexes were extracted from the cells with nonionic detergent and analyzed by sucrose gradient centrifugation. The hCG-receptor complex had an apparent molecular weight of 190,000 whereas the CT-receptor complex sedimented only slightly faster than CT itself. MLTC-1 gangliosides were separated on thin layer chromatograms which were overlayed with either iodinated CT or hCG. The toxin bound to a ganglioside corresponding to GM1 whereas the hormone did not bind to any of the gangliosides. When the cells were incubated overnight with hCG, they lost their hCG receptors but exhibited an increase in CT binding and gangliosides. Our results indicate that GM1 is the specific receptor for CT whereas gangliosides are not involved in the binding and action of hCG.

Gangliosides, the thyrotropin receptor, and autoimmune thyroid disease.

The thyrotropin (TSH) receptor has been proposed to be composed of a membrane glycoprotein and a membrane ganglioside, the former important in high affinity recognition, the latter vital for message coupling to the adenylate cyclase system. The present study used two approaches, formation of antireceptor monoclonal antibodies and reconstitution, to validate the model and further examine the role of the ganglioside. Three kinds of monoclonal antireceptor antibodies are defined. One group which inhibits TSH binding and TSH functions, i.e., TSH-stimulated adenylate cyclase activity, iodide uptake, and thyroid hormone release, is shown to be directed against the glycoprotein component of the receptor. The second group includes antibodies which mimic TSH in all stimulatory actions, are competitive agonists of TSH, are equivalent to thyroid stimulating antibodies in the sera of patients with Graves' disease, and are directed against the ganglioside component of the receptor. These stimulating monoclonal antibodies are directed against a minor ganglioside membrane component which fractionates as a disialoganglioside. When this ganglioside is incorporated into 1-8 thyroid cells which have a correlated ganglioside deficiency and TSH receptor defect, reconstitution of TSH stimulated adenylate cyclase activity occurs. Whereas the first group of antibodies inhibits TSH-stimulated function, they do not inhibit the stimulatory antibodies which mimic TSH, an observation consistent with the 2 component hypothesis of the receptor model. The third group of antibodies have a mix of properties from the first two groups and suggests that the TSH receptor in situ is an actual complex of the two components or that there are common carbohydrate determinants in the functional sites of each receptor component. Implications of a TSH receptor structure in which its ganglioside and glycoprotein components are in equilibrium with pools of free components and, in turn, components important for cholera toxin, tetanus toxin and interferon receptors are discussed. In regard to the pathogenesis of Graves' disease, the data indicate that thyroid stimulating autoantibodies are autoimmune equivalents of cholera toxin with respect to the importance of ganglioside function. Since antiidiotype studies of antibodies against TSH confirm a structural relationship between receptors for thyrotropin, cholera toxin, and thyroid stimulating autoantibodies, the data establish an unequivocal role for the ganglioside in TSH receptor structure which facilitates interpretation of in vitro experiments aimed at understanding the mechanism of ganglioside-ligand interactions.

Differential expression of gangliosides on the surfaces of myelinated nerve fibers

The binding of cholera and tetanus toxins to receptors on the surfaces of teased nerve fibers was used to localize GM1 and G1b-series gangliosides, respectively, by immunocytochemical methods. Native fibers and fibers treated with various hydrolytic enzymes to degrade specific surface components were studied. With native fibers, both toxins bound abundantly to nodes of Ranvier and poorly to the most external, internodal Schwann cell surfaces. Treatment of the fibers with proteases, hyaluronidase, and chondroitin ABC lyase neither eliminated receptors at the nodes nor unmasked receptors over the internodes. The axolemma underlying the paranodal or internodal myelin, exposed by extensive treatment with protease, bound both toxins in large amounts. Neuraminidase action induced cholera toxin receptors on the Schwann cell surface; these receptors were insensitive to protease. The results indicate that GM1 and G1b-series gangliosides are predominantly localized to axonal and glial structures of the node of Ranvier and to paranodal/internodal Axolemma, and that polysialogangliosides not of the G1b-series are present on the internodal Schwann cell surface.

Ganglioside localization on myelinated nerve fibres by cholera toxin binding

GM1 ganglioside has been localized on the surfaces of myelinated, peripheral nerve fibres by using immunofluorescence to detect cholera toxin receptors. Unfixed, mouse sciatic nerves were teased into individual, intact fibres in order to expose their extracellular surfaces. Cholera toxin binding sites were abundant at all nodes of Ranvier; they were scarce on the internodal fibre surfaces. The nodal receptors were resistant to various degradative enzymes, including trypsin and proteinase K. Proteases did not unmask receptors on the internodal surfaces. Exogenous GM1 successfully competed for the toxin binding sites on the fibres. From this evidence and the specificity of cholera toxin binding, we conclude that GM1 ganglioside is abundantly present on the membrane surfaces of peripheral nodes of Ranvier and is not present on the internodal Schwann cell surfaces in an appreciable amount. The patterns of fluorescence within the node suggest that the axon and Schwann cell structures are sites where GM1 is localized. Treatment of the teased fibres with Vibrio cholerae neuraminidase, which is known to reduce polysialogangliosides to the monosialoganglioside GM1, induced cholera toxin binding on the internodal Schwann cell surfaces. The induced receptors, as well as their precursors, were resistant to trypsin and proteinase K. We conclude that the internodal Schwann cell surface is rich in an unidentified polysialoganglioside(s) that can be converted to GM1 by neuraminidase.

Human platelets are defective in processing of cholera toxin.

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.

Ganglioside dependent return of TSH receptor function in a rat thyroid tumor with a TSH receptor defect

The 1–8 rat thyroid tumor line with a thyrotropin and cholera toxin receptor defect and a deficiency in higher order membrane gangliosides is shown to regain both receptor functions with the in vivo resynthesis or the in vitro reconstitution of higher order gangliosides. Reconstitution was achieved by exposing primary cell cultures of the tumor to preparations of gangliosides from thyroid cells with functional thyrotropin receptor activity.

Rabbit intestinal glycoprotein receptor for Escherichia coli heat-labile enterotoxin lacking affinity for cholera toxin.

The receptors for cholera toxin and Escherichia coli heat-labile toxin (LT) in rabbit small intestinal epithelium were characterized and compared. (i) In vivo studies in ligated intestinal loops showed that whereas LT B subunits could block the fluid secretogenic action of purified LT as well as cholera toxin, cholera toxin B subunits did not inhibit the LT response even when tested in a concentration 100-fold higher than one which gave complete blocking of cholera toxin action. (ii) In vitro studies indicated that isolated intestinal epithelial cells or brush-border membranes could bind about 10-fold more of E. coli LT than of cholera toxin. (iii) All binding sites for cholera toxin in duodenal, jejunal, or ileal mucosal cells or brush-border membranes were extracted by chloroform-methanol-water (4:8:3), which removed lipids quantitatively but did not extract glycoproteins. The extracted cholera toxin binding sites were to greater than 95% recovered in a monosialoganglioside fraction; quantitatively these sites closely corresponded to the concentration of chromatographically identified mucosal GM1 ganglioside (1 nmol of cholera toxin was bound per 1 to 2 nmol of GM1). In contrast, a substantial fraction of mucosal binding sites for E. coli LT remained in the delipidized tissue residue, and these sites had properties consistent with a glycoprotein nature. Thus, whereas cholera toxin appeared to bind highly selectively to GM1 ganglioside receptor sites of rabbit small intestine, E. coli LT bound both to GM1 ganglioside and to a main glycoprotein receptor for which cholera toxin lacks affinity.

Immunocytochemical localization of cell type-specific markers in reaggregating cell cultures of mouse cerebellum.

Reaggregate cultures were obtained from single-cell suspensions of fetal and early postnatal cerebellum, and fetal telencephalon and mesencephalon from C57BL/6J and NMRI mice and maintained in suspension under constant rotation as described previously (Seeds 1971). The percentage of dead cells in the aggregates as measured by the uptake of the fluorescent dye propidium iodide was always less than 5% of all cells. During the initial phase of reaggregation up to 20 h in vitro (hiv) several immunocytochemically defined cell types had a random distribution within the aggregate. Astrocytes were identified by indirect immunofluorescence by the use of the markers glial fibrillary acidic protein (GFAP), C1 and M1 antigens; neurons by NS-4 antigen and tetanus-toxin receptors; fibroblasts or fibroblast-like cells by fibronectin and laminin; and oligodendrocytes by myelin basic protein (MBP). Choleratoxin receptors and M 2 antigen served to distinguish the more mature from the less mature neurons. In reaggregates of early postnatal cerebellar cells neurons had started to redistribute after 40 hiv, forming an outer region containing more immature neurons and a core with more mature neurons. After 5 days in vitro (div) immature neurons were no longer detectable. From 3–8 div M1-and GFAP-positive astrocytic processes in the outer region showed a tendency for radial orientation. At later stages the processes appeared more randomly distributed and formed a dense glial network. Few oligodendrocytes and fibronectin-positive cells were present in the reaggregates. When reaggregates were prepared from 15 day-old embryonic cerebella, formation of radially oriented astrocytic processes and redistribution of neurons proceeded more slowly, but in a similar pattern as described for early postnatal cerebellum. GFAP was detectable at earlier ages than in situ. In reaggregates of 15 to 17 day old embryonic telencephalic anlage or midbrain, radially oriented astrocytic processes were not detectable. Similar to cerebellar reaggregates, accumulation of neurons in the inner region was observed.

Role of membrane gangliosides in the binding and action of bacterial toxins.

Gangliosides are complex glycosphingolipids that contain from one to several residues of sialic acid. They are present in the plasma membrane of vertebrate cells with their oligosaccharide chains exposed to the external environment. They have been implicated as cell surface receptors and several bacterial toxins have been shown to interact with them. Cholera toxin, which mediates its effects on cells by activating adenylate cyclase, bind with high affinity and specificity to ganglioside GM1. Toxin-resistant cells which lack GM1 can be sensitized to cholera toxin by treating them with GM1. Cholera toxin specifically protects GM1 from cell surface labeling procedures and only GM1 is recovered when toxin-receptor complexes are isolated by immunoadsorption. These results clearly demonstrate that GM1 is the specific and only receptor for cholera toxin. Although cholera toxin binds to GM1 on the external side of the plasma membrane, it activates adenylate cyclase on the cytoplasmic side of the membrane by ADP-ribosylation of the regulatory component of the cyclase. GM1 in addition to functioning as a binding site for the toxin appears to facilitate its transmembrane movement. The heat-labile enterotoxin of E. coli is very similar to cholera toxin in both form and function and can also use GM1 as a cell surface receptor. The potent neurotoxin, tetanus toxin, has a high affinity for gangliosides GD1b and GT1b and binds to neurons which contain these gangliosides. It is not yet clear whether these gangliosides are the physiological receptors for tetanus toxin. By applying the techniques that established GM1 as the receptor for cholera toxin, the role of gangliosides as receptors for tetanus toxin as well as physiological effectors may be elucidated.

Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.

Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].

Ultracytochemistry of cholera-toxin binding sites in ciliary processes.

Cholera toxin reduces the rate of aqueous humor in concentrations (10-11M) that do not disturb the morphology of the aqueous-humor forming epithelial cells of the ciliary processes of the rabbit eye. The search for an endogenous mediator of aqueous-humor formation comparable to cholera toxin in its mode of operation prompted us to map the distribution of cell surface receptors for cholera toxin in the ciliary processes of the eyes of rabbits. Cytochemical studies were carried out with the use of conjugates of cholera toxin to fluorescein isothiocyanate (CT-FITC) and to horseradish peroxidase (CT-HRP), and of the B subunit of cholera toxin to horseradish peroxidase (B-HRP). Multiple fluorescent CT-FITC binding sites were observed on the outer nonpigmented epithelial layer near the crests of the processes. Processes incubated with CT-HRP in vitro showed surface staining of 30-40% of the nonpigmented epithelial cells. A prominent reaction product was observed along the basal and lateral plasma membranes of these cells. In vivo studies carried out after arterial infusion of B-HRP showed a reproducible dense reaction product between the apical surfaces of the pigmented epithelium (PE) and of the nonpigmented epithelium (NPE) facing each other. Aggregations of reaction product were observed with the electron microscope in the extracellular space between the apices of PE and NPE. The apical plasma membrane of the endothelium of the blood vessels near the crests of the ciliary processes was stained after either in vivo or in vitro exposure to peroxidase conjugates. These findings indicate that the cell-surface receptors which mediate the action of cholera toxin on aqueous humor formation are very likely localized in the apical plasma membranes of the epithelium of the ciliary processes.

Cyclic AMP as a mitotic signal for epidermal keratinocytes, but not for dermal fibroblasts.

The function of cyclic AMP in the growth of epidermal and dermal cells was investigated. Cholera toxin was used to increase the amount of intracellular cyclic AMP. This toxin had a stimulatory effect on human epidermal cells only when growth was limited; i.e., when a small number of cells was plated for colony formation, when cells from frozen stock were cultured, and when cells were in the initial phase of primary culture. Cholera toxin, however, had no significant effect on the growth rate during exponential growth or on the saturation density during the stationary phase.The stimulatory effect of the toxin was specific to epidermal keratinocytes. In other types of cells, the effect varied : with human dermal fibroblasts there was no ffeect, or some inhibition.A membrane receptor for cholera toxin, GM 1 ganglioside, was isolated from human epidermal cells. Cyclic AMP was induced markedly by cholera toxin in human epidermal keratinocytes, which suggests that an increase in the amount of cyclic AMP may act as a mitotic signal in these cells. Cholera toxin also induced cyclic AMP in human dermal fibroblasts irrespective of the growth response, an indication that in dermal fibroblasts the content of cyclic AMP is not necessarily related to the proliferation of the cells.

14] Detection of glycolipid ligands by direct binding of carbohydrate-binding proteins to thin-layer chromatograms

Publisher Summary Glycolipid ligands of carbohydrate-binding proteins can be detected by binding of the I-labeled protein to thin-layer chromatograms of total lipid extracts of cells followed by autoradiography. This method, which has been used to detect glycolipid receptors for cholera toxin and monoclonal antibodies, may also be useful in defining the receptors of other proteins, such as some lectins or hormones that bind to glycolipids. Glycolipid antigens of monoclonal antibodies can be detected as follows: A total lipid extract from approximately 1 mg wet weight of tissue is chromatographed, and the chromatogram is dried and soaked in buffer A for 10 min at 4 °. The wet chromatogram was laid horizontally as before and overlayed with hybridoma culture medium containing about 10 μg of antibody per milliliter diluted 1:4 with buffer. After incubation in a humid atmosphere for 6 hr at 4 °, the chromatogram is washed by dipping in six successive changes of buffer, overlayed with 125 I-labeled F(ab') of rabbit IgG antibodies to mouse immunoglobulins in buffer A (1×10 6 cpm/ml), and incubated for an additional 12 hr in a humid atmosphere at 4 °. The chromatogram is then washed six times in buffer B, air-dried, and exposed to X-ray film.

The cholera toxin receptor ganglioside GM 1 remains associated with Triton X-100 cytoskeletons of BALB/c-3T3 cells

A substantial amount of the cholera toxin which binds to the surface of mouse fibroblasts resists solubilization by neutral detergents and remains associated with Triton X-100 cytoskeletons prepared by extraction of monolayer cultures. The observation is surprising given that the receptor for cholera toxin is a ganglioside (GM 1), and that membrane lipids are often assumed to be quantitatively extracted from Triton X-100 cytoskeletons. Indeed such preparations from mouse fibroblasts contain GM 1, and approx. 20% of the total cellular phospholipid and ganglioside. The observations are discussed in terms of the current trend to assume that detergent insolubility implies an association with the cytoskeleton.

Cytoskeletal association of the cholera toxin receptor in rat erythrocytes

Summary Cholera toxin receptors are associated with the cytoskeletal residue of Triton X-100 extracts of rat erythrocytes. The solubilized cytoskeletal toxin-receptor complex appears to be a large macromolecular entity whose formation is facilitated by increased receptor occupancy. These findings suggest a role for GM 1 -gangliosides in transmembrane signalling and in the mechanism of action of cholera toxin.

Reevaluation of the role of gangliosides in the binding and action of thyrotropin.

A cloned line of normal rat thyroid cells, FRTL, contained a small number of high-affinity binding sites for thyrotropin (TSH) when measured under physiological conditions. The cells also bound small amounts of cholera toxin, and both hormone and toxin stimulated cyclic AMP production by the cells. The major ganglioside of FRTL cells was N-acetylneuraminylgalactosylglucosylceramide (GM3), with minor amounts of gangliosides corresponding to galactosyl-N-acetylgalactosaminyl-[N-acetyl-neuraminyl]-galactosylglucosylceramide (GM1) and N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GD1a). Treatment of these cells with neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) converted most of the GD1a to GM1. After neuraminidase treatment, the binding of cholera toxin, which binds to GM1, was increased, but there was no change in the binding of TSH. Preincubation of neuraminidase-treated FRTL cells with the B (binding) component of cholera toxin completely prevented cholera toxin binding but had no effect on the binding of TSH. Neuraminidase treatment also somewhat enhanced, rather than decreased, the cyclic AMP response to TSH. Pretreatment of FRTL cells with mixed brain gangliosides resulted in a 10-fold increase in cholera toxin binding. Again there was no enhancement of TSH binding or adenylate cyclase stimulation. Finally, prolonged exposure of FRTL cells to TSH induced down-regulation of TSH receptors but had no effect on gangliosides or cholera toxin receptors. The results indicate that more complex gangliosides do not serve as a component of the TSH receptor nor are they involved in the transmission of the hormone signal across the cell membrane of these cultured rat thyroid cells.

Identification of cholera toxin binding glycoproteins in rat intestinal microvillus membranes.

Glycoproteins of the microvillus membranes from rat small intestinal epithelial cells were examined for their ability to bind cholera toxin. Membrane glycoproteins prepared by Ricinus communis agglutinin-Sepharose affinity chromatography, which were free of glycolipids, were shown to be able to form complexes with iodinated cholera toxin by gel filtration column chromatography. These glycoproteins were further characterized by subjecting the complex of tritiated cholera toxin receptors, cholera toxin, and anti-cholera toxin antibody to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography and by localizing cholera toxin binding glycoproteins on sodium dodecyl sulfate-gels using iodinated cholera toxin. In addition to glycolipids, at least 5 glycoproteins with molecular weights of 69,000, 90,000, 100,000, 114,000, and 132,000 were identified by both methods. These results suggest that microvillus membranes contain glycoprotein and glycolipid receptors for cholera toxin.