Mapping of a cell-binding domain in the cell adhesion molecule gp80 of Dictyostelium discoideum.
At the aggregation stage of Dictyostelium discoideum development, a cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate the EDTA-resistant type of cell-cell adhesion via homophilic interaction (Siu, C.-H., A. Cho, and A. H. C. Choi. 1987. J. Cell Biol. 105:2523-2533). To investigate the structure-function relationships of gp80, we have isolated full length cDNA clones for gp80 and determined the DNA sequence. The deduced structure of gp80 showed three major domains. An amino-terminal globular domain composed of the bulk of the protein is supported by a short stalk region, which is followed by a membrane anchor at the carboxy terminus. Structural analysis suggested that the cell-binding domain of gp80 resides within the globular domain near the amino terminus. To investigate the relationship of the cell-binding activity to this region of the polypeptide, three protein A/gp80 (PA80) gene fusions were constructed using the expression vector pRIT2T. These PA80 fusion proteins were assayed for their ability to bind to aggregation stage cells. Binding of 125I-labeled fusion proteins PA80I (containing the Val123 to Ile514 fragment of gp80) and PA80II (Val123 to Ala258) was dosage dependent and could be inhibited by precoating cells with the cell cohesion-blocking mAb 80L5C4. On the other hand, there was no appreciable binding of PA80III (Ile174 to Ile514) to cells. Reassociation of cells was significantly inhibited in the presence of PA80I or PA80II. In addition, 125I-labeled PA80II exhibited homophilic interaction with immobilized PA80I, PA80II, or gp80. The results of these studies lead to the mapping of a cell-binding domain in the region between Val123 and Leu173 of gp80 and provide direct evidence that the cell-binding activity of gp80 resides in the protein moiety.
Kamboj
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Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, Canada.
Wong
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Lam
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Siu
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D000595:Amino Acid Sequence; D000954:Antigens, Surface / Q000378:metabolism*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D001667:Binding, Competitive; D050260:Carbohydrate Metabolism; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004023:Dictyostelium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013329:Structure-Activity Relationship
D000954:Antigens, Surface; D015815:Cell Adhesion Molecules; D004271:DNA, Fungal; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C052291:cell cohesion molecule, Dictyostelium