Preferential binding of the epidermal growth factor receptor to ganglioside GM3 coated plates

Yednak, Mark A.; Bremer, E.

doi:10.1007/bf02815362

Cited by 13 publications

(9 citation statements)

References 25 publications

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“…For example, GD3 and GD2 coimmunoprecipitate with ␣ v ␤ 3 (40); given the structural similarities between the ␣ 5 ␤ 1 and ␣ v ␤ 3 integrins, it is likely that the ganglioside-␣ v ␤ 3 interaction similarly involves carbohydratecarbohydrate recognition of the extracellular N-terminal region of ␣ v . Studies in 1988 first demonstrated that ganglioside GM3 co-immunoprecipitates with the epidermal growth factor receptor (EGFR) (41), and this ability to complex was further suggested by ELISAs (42). In fact, we have recently provided evidence that the relationship between GM3 and the EGFR that is required for inhibition of EGFR activation involves carbohydrate-carbohydrate interaction.…”

Section: Discussionmentioning

confidence: 98%

Carbohydrate-Carbohydrate Binding of Ganglioside to Integrin α5 Modulates α5β1Function

Wang

Sun

Al-Qamari

et al. 2001

Journal of Biological Chemistry

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Gangliosides GT1b and GD3, components of keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin. Although ganglioside sialylation is known to be important, the mechanism of inhibition is unknown. Using purified insect recombinant ␣ 5 and ␤ 1 proteins and ␣ 5 ␤ 1 integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of ␣ 5 ␤ 1 to fibronectin. Co-immunoprecipitation of GT1b and ␣ 5 ␤ 1 from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified ␣ 5 ␤ 1 from SCC12 cells and insect recombinant ␣ 5 ␤ 1 , particularly the ␣ 5 subunit, further suggest interaction between ganglioside and ␣ 5 ␤ 1 . The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of ␣ 5 ␤ 1 from SCC12 and insect cells or poorly glycosylated recombinant ␣ 5 ␤ 1 from Escherichia coli cells. The GT1b-␣ 5 ␤ 1 interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in ␣ 5 ␤ 1 . The preferential binding of GT1b to high mannose rather than reduced mannose ovalbumin further implicates the binding of GT1b to mannose structures. These data provide evidence that highly sialylated gangliosides regulate ␣ 5 ␤ 1 -mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the ␣ 5 subunit of ␣ 5 ␤ 1 integrin. Human keratinocyte motility on a fibronectin (FN)1 matrix is critical for the re-epithelialization of healing wounds, for the spread of cutaneous malignancy, and in cutaneous embryogenesis. Although the molecular events that influence this migration are poorly understood, the interaction between keratinocyte ␣ 5 ␤ 1 integrin and the arginine-glycine-aspartic acid (RGD) site of FN is known to be key (1). Increased expression of ␣ 5 ␤ 1 has been shown in keratinocytes at the migrating edge of wounds in vivo, in lesional skin of patients with psoriasis, and in cultured keratinocytes (2-4). Both receptor clustering on keratinocytes and ligand occupancy of ␣ 5 ␤ 1 are required to activate intracellular signal transduction components (5), including focal adhesion kinase, phosphatidylinositol 3-kinase, protein kinase C, and integrin-linked kinase, leading to cell adhesion to .Gangliosides are glycosphingolipids characterized by the presence of one or more sialic acid moieties in the oligosaccharide chain (9). The role of gangliosides, which are localized to the outer leaflet of the plasma membrane of eukaryotic cells, is largely unknown, but studies with cultured keratinocytes and keratinocyte-derived cells suggest that gangliosides are involved in regulating cellular proliferation, differentiation, and adhesion (10 -13). The discovery that gangliosides inhibit cell attachment and spreading on a FN matrix (14) led investigators to consider gangliosides to be the cell receptors for FN before integrin ␣ 5 ␤ 1 was identified (15,16). Although these early studies showed that the terminal sialic acid residues of gangliosides were critica...

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Section: Discussionmentioning

confidence: 98%

Carbohydrate-Carbohydrate Binding of Ganglioside to Integrin α5 Modulates α5β1Function

Wang

Sun

Al-Qamari

et al. 2001

Journal of Biological Chemistry

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“…To confirm that gangliosides remained coated on the plates during the assay, anti-ganglioside antibodies directed against GM1 and GM3 were used in place of the LA22 antibody. Equivalent binding of both anti-GM3 and anti-GM1 antibodies was observed, indicating that there was no preferential loss of gangliosides (18).…”

Section: Expression and Purification Of The Recombinant Ecd-inmentioning

confidence: 94%

“…The ganglioside interaction assay was carried out using an assay similar to that described for the full-length EGFR (18). The amounts of gangliosides used for coating the plates varied from 10 to 5000 pmol (Fig.…”

Section: Expression and Purification Of The Recombinant Ecd-inmentioning

confidence: 99%

“…The mechanism for GM3 inhibition of the EGFR is not understood. The ability of A431 membrane preparations rich in EGFR to bind to GM3-coated plates (18) or to GM3-coated beads (19) suggests a direct interaction with GM3. But, because the EGFR was not the only component of the membrane preparations, we cannot rule out the possibility that GM3 also binds to other membrane components, such as a tyrosine-specific phosphatase, as suggested by Suarez Pestana et al (20,21).…”

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confidence: 99%

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Interaction of the Extracellular Domain of the Epidermal Growth Factor Receptor with Gangliosides

Miljan¹,

Meuillet²,

Mania-Farnell³

et al. 2002

Journal of Biological Chemistry

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Ganglioside GM3 inhibits epidermal growth factor (EGF)-dependent cell proliferation in a variety of cell lines. Both in vitro and in vivo, this glycosphingolipid inhibits the kinase activity of the EGF receptor (EGFR). Furthermore, membrane preparations containing EGFR can bind to GM3-coated surfaces. These data suggest that GM3 may interact directly with the EGFR. In this study, the interaction of gangliosides with the extracellular domain (ECD) of the EGFR was investigated. The purified human recombinant ECD from insect cells bound directly to ganglioside GM3. The ganglioside interaction site appears to be distinct from the EGF-binding site. In agreement with previous reports on the effects of specific gangliosides on EGFR kinase activity, the ECD preferentially interacted with GM3. The order of relative binding of other gangliosides investigated was as follows: GM3 > > GM2, GD3, GM4 > GM1, GD1a, GD1b, GT1b, GD2, GQ1b > lactosylceramide. These data suggest that NeuAc-lactose is essential for binding and that any sugar substitution reduces binding. In agreement with the specificity of soluble ECD binding to gangliosides, GM3 specifically inhibited EGFR autophosphorylation. Identification of a ganglioside interaction site on the ECD of the EGFR is consistent with the hypothesis that endogenous GM3 may function as a direct modulator of EGFR activity.

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“…A unique transition state III is supported by ligand-binding experiments. GM3 did not affect ligand binding to the full-length EGFR (16,36) and in binding assays with recombinant EGFR the GM3 binding site was separate from the EGF binding site (30). However, in keratinocyte-derived SCC12 cells, EGF binding to the EGFR is inhibited by GM3 (37).…”

Section: Mechanisms Of Growth Factor Receptor Modulation By Gangliosidesmentioning

confidence: 97%

Regulation of Growth Factor Receptors by Gangliosides

2002

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Since their discovery in the 1940s, gangliosides have been associated with a number of biological processes, such as growth, differentiation, and toxin uptake. Hypotheses about regulation of these processes by gangliosides are based on indirect observations and lack a clear definition of their mechanisms within the cell. The first insights were provided when a reduction in cell proliferation in the presence of gangliosides was attributed to inhibition of the epidermal growth factor receptor (EGFR). Since that initial finding, most, if not all, growth factor receptors have been described as regulated by gangliosides. In this review, we describe the effects of gangliosides on growth factor receptors, beginning with a list of known effects of gangliosides on growth factor receptors; we then present three models based on fibroblast growth factor (FGFR), platelet-derived growth factor receptor (PDGFR), and EGFR. We focus first on ganglioside modulation of ligand binding; second, we discuss ganglioside regulation of receptor dimerization; and third, we describe a model that implicates gangliosides with receptor activation state and subcellular localization. The methodology used to develop the three models may be extended to all growth factor receptors, bearing in mind that the three models may not be mutually exclusive. We believe that gangliosides do not act independently of many well-established mechanisms of receptor regulation, such as clathrin-coated pit internalization and ubiquitination, but that gangliosides contribute to these functions and to signal transduction pathways. We hypothesize a role for the diverse structures of gangliosides in biology through the organization of the plasma membrane into lipid raft microdomains of unique ganglioside composition, which directly affect the signal duration and membrane localization of the growth factor receptor.

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Preferential binding of the epidermal growth factor receptor to ganglioside GM3 coated plates

Cited by 13 publications

References 25 publications

Carbohydrate-Carbohydrate Binding of Ganglioside to Integrin α5 Modulates α5β1Function

Carbohydrate-Carbohydrate Binding of Ganglioside to Integrin α5 Modulates α5β1Function

Interaction of the Extracellular Domain of the Epidermal Growth Factor Receptor with Gangliosides

Regulation of Growth Factor Receptors by Gangliosides

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