Molecular Cloning and Characterization of DdCAD-1, a Ca2+-dependent Cell-Cell Adhesion Molecule, in Dictyostelium discoideum

doi:10.1074/jbc.271.27.16399

Molecular Cloning and Characterization of DdCAD-1, a Ca²⁺-dependent Cell-Cell Adhesion Molecule, in *Dictyostelium discoideum**

From the Banting and Best Department of Medical Research and Department of Biochemistry University of Toronto, Toronto, Ontario M5G 1L6, Canada

^¶ To whom correspondence should be addressed:
Charles H. Best Institute, University of Toronto, 112 College St., Toronto, Ontario M5G 1L6, Canada
. Tel.: 416-978-8766; Fax: 416-978-8528; E-mail: chi.hung.siu{at}utoronto.ca

↵^§ Present address: Department of Medicine, University of Toronto, Toronto, Ontario, M56 1L6 Canada.

Abstract

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 (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.

Footnotes

↵^‡ Recipient of a studentship award from the Medical Research Council of Canada.
↵* This work was supported in part by Operating Grant MT-6140 from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) BankIt35881 U49650[GenBank].
↵¹ The abbreviations used are:

gp

glycoprotein

GST

glutathione S-transferase

PBS

phosphate-buffered saline

kb

kilobase pair(s).
↵² C. Yang and C.-H. Siu, manuscript in preparation.
↵³ H. Sesaki and C.-H. Siu, manuscript in preparation.
- Received October 25, 1995.
- Revision received March 11, 1996.