Characterization of the calcium -dependent cell adhesion molecule DdCAD -1 in Dictyostelium discoideum

DdCAD-1 is a Ca2+-dependent cell adhesion molecule in Dictyostelium discoideum. DdCAD-1 mediates EDTA-sensitive homophilic cell-cell adhesion during early stages of development. The cDNA sequence encoding DdCAD-1 has been cloned and the predicted primary sequence shows sequence similarity to the cadherin family of Ca2+-dependent cell adhesion molecules. Furthermore, the carboxy-terminal portion of DdCAD-1 is similar to the 12-residue Ca2+-binding loop of the EF hand motif. Structural/functional studies were performed using GST-fusion proteins of full length as well as deletions of DdCAD-1. Equilibrium binding studies suggest that DdCAD-1 binds four Ca2+ ions with an average Kd of 2.4 x 10-5 M. Ca2+-binding activity is demonstrated in the carboxy-terminus (exon 3) and two or more Ca 2+-binding sites are found to be associated with the rest of the molecule (encoded by exon 1 and 2) by Ca2+ overlay assays. Inhibition of cell reassociation by the various DdCAD-1 GST-fusion proteins shows that sequences encoded by exon 1 and 2 are involved in mediating cell-cell adhesion. The DdCAD-1 knockout mutant was generated by disruption of the cadA gene. cadA- cells show a 50% reduction in the level of cell reassociation. These cells complete the developmental cycle and form fruiting bodies with viable spores. However, the percentage of prestalk cells in cadA- slugs is >60% as compared to 25% in wild-type slugs. In addition, prestalk cadA- cells do not occupy the anterior one-quarter of the slug as in the case of wild-type cells. Only 25% of cadA - slugs display the normal sorting pattern versus ∼80% in wild-type slugs. These results suggest that in addition to cell-cell adhesion, DdCAD-1 may be involved in cell type differentiation as well as cell sorting during morphogenesis. The full length and the carboxy-terminal deleted version of DdCAD-1 were expressed in cadA- cells for in vivo studies. Analysis of the cell-cell adhesiveness of these transformants reveals that the DdCAD-1 deletion mutant can only partially restore the cell-binding activity of cadA- cells. However, the defects in cell type proportioning and cell sorting are rescued in both types of transformants, indicating that the carboxy-terminal Ca2+-binding site is dispensable in cell type differentiation.