Molecular cloning and characterization of DdCAD-1, a Ca²⁺-dependent cell-cell adhesion molecule, in Dictyostelium discoideum

Dictyostelium discoideum expresses EDTA-sensitive cell-cell adhesion sites soon after the initiation of development, and a Ca²⁺-binding protein of M(r) 24,000 (designated DdCAD-1) has been implicated in this type of adhesiveness. We have previously purified DdCAD-1 to homogeneity and characterized its cell binding activity (Brat, S. K., and Siu, C.-H. (1992) J. Biol. Chem. 268, 24902-24909). In this report, we describe the cloning of DdCAD-1 cDNAs. DNA sequencing revealed a single open reading frame coding for a polypeptide containing 213 amine acids. The identity of the cDNA was confirmed by amine acid sequences of two cyanogen bromide peptides. The deduced amine acid sequence of DdCAD-1 exhibits a relatively high degree of sequence similarity with members of the cadherin family and protein S of Myxococcus xanthus. Unlike the other cadherins, the carboxyl-terminal region of DdCAD-1 contains a Ca²⁺-binding motif. Although analyses of the sequence suggest that the polypeptide lacks a signal peptide sequence and a transmembrane domain, immunofluorescence microscopy demonstrates the association of DdCAD-1 with the ecto-surface of the plasma membrane. To investigate the structure/function relationships of DdCAD-1, glutathione S- transferase fusion proteins containing different DdCAD-1 fragments were expressed and assayed for their ⁴⁵Ca²⁺ and cell binding activities. These studies revealed that the cell binding activity is dependent on the amino- terminal segment and not the carboxyl-terminal Ca²⁺-binding domain and showed additional Ca²⁺-binding site(s) within the amino-terminal segment.