CHARACTERIZATION OF MOUSE ALCAM (CD166) - THE CD6-BINDING DOMAIN IS CONSERVED IN DIFFERENT HOMOLOGS AND MEDIATES CROSS-SPECIES BINDING

Activated leukocyte cell adhesion molecule (ALCAM; CD166) is a member of the immunoglobulin gene superfamily (IgSF) which is expressed by ac tivated leukocytes and thymic epithelial cells and is a ligand for the lymphocyte antigen CD6. Herein, we report on the isolation and charac terization of cDNA clones encoding mouse ALCAM (mALCAM). Comparison of the predicted amino acid sequence of mALCAM and human ALCAM (hALCAM) showed an overall identity of 93 %. Binding studies with truncated for ms of the extracellular region of mALCAM showed that the CD6 binding s ite is located in the N-terminal Ig-like domain and that mALCAM is cap able of binding both human and mouse CD6. Mutagenesis studies on hALCA M suggested that residues critical for CD6 binding map to the predicte d A'GFCC'C'' beta-sheet of ALCAM's N-terminal binding domain. Residue differences in the N-terminal domains of mALCAM and hALCAM were analyz ed with the aid of a molecular model of ALCAM. All residues critical f or CD6 binding are conserved in both mALCAM and hALCAM, whereas residu e differences map to the predicted BED face which is opposite the CD6 binding site on hALCAM. These findings provide a molecular rationale f or the observed cross-species CD6/ALCAM interaction and the apparent i nability to generate monoclonal antibodies (mAb) against the CD6 bindi ng site. RNA blot analysis showed that mRNA transcripts encoding mALCA M are expressed in the brain, lung, liver, and the kidney, as well as by activated leukocytes and a number of cell lines. A rat mAb specific for mALCAM was produced and by two-color immunofluorescence studies w as shown to bind to both activated CD4(+) and CD8(+) T cells.