Characterization of the cell adhesion molecule gp24 in Dictyostelium discoideum. Mediation of cell-cell adhesion via a Ca(2+)-dependent mechanism.
Dictyostelium discoideum cells express EDTA-sensitive cell-cell adhesion sites soon after the initiation of development and EDTA-resistant adhesion sites later at the aggregation stage. A glycoprotein of M(r) 24,000 (gp24) has been implicated in the mediation of the EDTA-sensitive type of intercellular cohesiveness (Knecht, D. A., Fuller, D. L., and Loomis, W. F. (1987) Dev. Biol. 121, 277-283). In this study, a relatively simple procedure was developed to purify gp24 to homogeneity. A highly specific rabbit antiserum was raised against gp24, and the localization of gp24 at the cell surface was shown by quantitative binding of the anti-gp24 antibodies to intact cells. To demonstrate the cell binding activity of gp24, the binding of solubilized gp24 to intact cells was examined. 125I-Labeled gp24 bound to cells in a dose-dependent and saturable manner, and the binding was displaced specifically by unlabeled gp24. Purified gp24 was capable of inhibiting the reassociation of dispersed cells previously undergoing EDTA-sensitive aggregation. Moreover, precoating cells with anti-gp24 IgG and Fab fragments blocked the binding of 125I-labeled gp24 to cells. Collectively, these in vitro assays provide direct evidence that gp24 is a cell adhesion molecule that most likely functions through a homophilic mode of interaction. The binding of gp24 to cells was sensitive to EGTA, suggesting that the activity of gp24 may involve calcium ions. Binding studies showed that 45Ca2+ could bind to gp24 blotted onto nitrocellulose membrane. In addition, preincubation of the native protein with calcium ions resulted in a shift in its gel mobility. It is therefore likely that gp24 mediates cell-cell interactions via a Ca(2+)-dependent mechanism, rendering gp24 the first cell adhesion molecule in D. discoideum to utilize a Ca(2+)-based adhesion system.
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Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004023:Dictyostelium / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data
D000596:Amino Acids; D015815:Cell Adhesion Molecules; D005656:Fungal Proteins; D006023:Glycoproteins; D004533:Egtazic Acid; D002118:Calcium