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 Mr 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 (Brar, S. K., and Siu, C.-H. (1993) 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 amino acids. The identity of the cDNA was confirmed by amino acid sequences of two cyanogen bromide peptides. The deduced amino 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.
Wong
E F
EF
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Brar
S K
SK
Sesaki
H
H
Yang
C
C
Siu
C H
CH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015820:Cadherins / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000737:chemistry; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018744:DNA, Plant / Q000737:chemistry / Q000378:metabolism; D004023:Dictyostelium / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016941:Myxococcus xanthus; D017293:Protein S / Q000737:chemistry; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid
D015820:Cadherins; D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D018744:DNA, Plant; C100604:DdCAD-1 protein, Dictyostelium discoideum; D017293:Protein S; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D002118:Calcium