The Amino-terminal Immunoglobulin-like Domain of Activated Leukocyte Cell Adhesion Molecule Binds Specifically to the Membrane-proximal Scavenger Receptor Cysteine-rich Domain of CD6 with a 1:1 Stoichiometry

Abstract

Activated leukocyte cell adhesion molecule (ALCAM)was recently identified as a ligand for CD6, a signaling receptor expressed on T cells, a subset of B cells, and some cells in the brain. Receptor-ligand binding assays, antibody blocking experiments, and examination of the tissue distribution of these two cell surface proteins suggest that CD6-ALCAM interactions play an important role in mediating the binding of thymocytes to thymic epithelial cells and of T cells to activated leukocytes. Presently, the details of CD6-ALCAM interactions and of signaling through CD6 are unknown. A series of truncated human ALCAM and CD6 immunoglobulin fusion proteins were produced and tested in different binding assays to analyze ALCAM-CD6 interactions in more detail. In this study, we report that the amino-terminal Ig-like domain of human ALCAM specifically binds to the third membrane-proximal scavenger receptor cysteine-rich (SRCR) domain of human CD6. Using thrombin-cleaved Ig fusion proteins containing single or multiple ALCAM or CD6 domains, we were able to determine that the stoichiometry of the interaction between the amino-terminal ALCAM domains and the membrane-proximal CD6 SRCR domain is 1:1. These results provide the first example of an Ig-like domain mediating an interaction with an SRCR domain. Ig supergene family (IgSF)1 members have been shown to interact with a wide variety of other molecules, including integrins, cytokines, and other IgSF members. Many of these interactions are mediated through protein-protein contacts, although a subset of these proteins, known as sialoadhesins, recognize sialic acid (1). Recently, we have reported on a novel interaction between an IgSF member, activated leukocyte cell adhesion molecule (ALCAM), and a member of the scavenger receptor cysteine-rich (SRCR) family of proteins, CD6 (2). Soluble recombinant proteins consisting of the extracellular domains of either ALCAM or CD6 fused to human IgG1 constant domains were shown to specifically bind to COS cell transfectants expressing CD6 or ALCAM, respectively.ALCAM is a type I membrane protein whose extracellular domain is composed of five Ig-like domains: two amino-terminal V set Ig domains followed by three domains of the C2 set, a hydrophobic transmembrane domain, and a short cytoplasmic anchor sequence (2). ALCAM is also known as SC-1/DM-GRASP/ BEN in the chicken (3-5) and as KG-CAM in the rat (6). The chicken counterpart of ALCAM is a neural adhesion molecule capable of supporting neurite outgrowth (4, 5). Data from the chicken indicate that ALCAM is capable of homophilic interactions (4, 5), and the possibility of such interactions has also been suggested on the basis of molecular modeling (7). We have previously reported that COS cells that expressed CD6 were able to bind to ALCAM positive thymic epithelial cells, which suggested that CD6 and ALCAM binding can mediate adhesive interactions between thymocytes and thymic epithelial cells (2).CD6, also a type I membrane protein (8), is expressed by thymocytes, T cells, a subset of...

CD6 binds to pathogen-associated molecular patterns and protects from LPS-induced septic shock

“…Interestingly, mCD6, but not rsCD6 or nsCD6, was reactive with a polyclonal antiserum raised against the intracytoplasmic region of human CD6 (12) (data not shown). In cell binding experiments, both biotin-labeled rsCD6 and nsCD6 bound to Raji B cells but not to K562 erythroleukemic cells, in accordance with the differential expression of the CD6 ligand (ALCAM/CD166) (17,19) (Fig. 2D).…”

Expression and Characterization of a Novel CD6 Ligand in Cells Derived from Joint and Epithelial Tissues

“…When Abs directed against either the 3A11 Ag or CD166 were used, there was a trend suggesting that adhesion was inhibited, but these results did not reach statistical significance. In our prior assays, we did not detect CD166-dependent binding (16), and this is explained by data suggesting that none of the anti-CD166 Abs available to us recognized the CD6-binding site contained in what is known as CD166 domain 1 (D1) (25,26). The observation that we detected CD6L dependence in our adhesion assays when we used soluble CD6 fusion protein, but that it was difficult to detect when we used Ab against individual CD6L led to our conclusion that neither the 3A11 Ab nor anti-CD166 mAb bind precisely the same epitope of the respective CD6L, as cellular CD6 does (25,37,38).…”

“…Although there was a trend toward inhibition of adhesion by mAbs directed against either 3A11 Ag or CD166, this did not reach statistical significance. Most anti-CD166 mAbs described, including the one we used, bind to domain 2 of CD166, whereas the CD6-binding site is contained in domain 1 of CD166 (25,26).…”

Macrophage Scavenger Receptor Class A

“…A function has been proposed for only 1 of these genes. CD6, containing 3 scavenger receptor cysteine-rich domains, has been shown to be the ligand for the leukocyte adhesion molecule CD166, 21 indicating the involvement of the scavenger receptor cysteine-rich domain in cell-cell interaction. For SRA types I and II, no differences have been shown in ligand interaction between both isoforms.lacking type I SRA.…”