Characterization of CD33 as a new member of the sialoadhesin family of cellular interaction molecules.

CD33 is a member of the Ig superfamily that is restricted to cells of the myelomonocytic lineage but whose functions and binding properties are unknown. It shares sequence similarity with sialoadhesin, CD22, and the myelin-associated glycoprotein, which constitute the Sialoadhesin family of sialic acid-dependent cell adhesion molecules. In the present study, we show that CD33 is a fourth member of this family. As a model for sialic acid-dependent binding, human erythrocytes were derivatized with N-acetylneuraminic acid (NeuAc) in different linkages. A recombinant soluble form of CD33, Fc-CD33, bound red blood cells with a specificity similar to that of sialoadhesin, preferring NeuAc alpha 2,3Gal in N- and O-glycans over NeuAc alpha 2,6Gal in N-glycans. Fc-CD33 also bound selectively to the myeloid cell lines HL-60 and U937. However, CD33 was unable to mediate cell binding after transient expression in COS cells, despite high levels of surface expression. Pretreatment of the CD33-transfected cells with sialidase rendered them capable of mediating sialic acid-dependent binding. These results show that CD33 can function as a sialic acid-dependent cell adhesion molecule and that binding can be modulated by endogenous sialoglycoconjugates when CD33 is expressed in a plasma membrane.

Molecular analysis of the interaction of p56lck with the CD4 and CD8 antigens.

The CD4 and CD8 antigens on the surface of T cells appear to bind to major histocompatibility complex (MHC) class II and I antigens, respectively. These antigens also synergize with the Ti(TcR)/CD3 complex in the potentiation of T-cell proliferation. Our earlier work demonstrated that the CD4 and CD8 receptors are coupled to a protein-tyrosine kinase termed p56lck from normal and transformed T lymphocytes. The p56lck protein is a member of the src family and its homology with receptor-kinases such as the epidermal growth factor receptor (EGFR) make it an important candidate in signal transduction. In this paper, we show in transfectants that p56lck interacts with the cytoplasmic tail of the CD4 antigen. Murine p56lck can interact across species with the human CD4 receptor. Furthermore, peptide competition studies showed that a specific sequence within the cytoplasmic tail of CD4 interacts with the kinase. Cysteine residues also appear to play key roles in this interaction. Lastly, we show biochemically that the CD4:p56lck complex can physically associate with the epsilon chain of the CD3 complex on HPB-ALL transformed T cells. This interaction may provide a bridge by which events related to ligand binding to Ti(TcR)/CD3 may trigger T cells via the CD4/CD8:p56lck complex.

The CD4/CD8:p56lck complex in T lymphocytes: a potential mechanism to regulate T-cell growth.

The CD4 and CD8 antigens on the surface of T cells appear to bind to major histocompatibility complex (MHC) class II and I antigens, respectively. These receptors have also been found to regulate T cell growth in a manner independent of MHC recognition. In this report, we describe recent work showing that the CD4 and CD8 receptors are coupled to a protein-tyrosine kinase, p56lck, from T lymphocytes. The p56lck protein is a member of the src family, which plays a crucial role in the activation and transformation of various mammalian cells. The CD4/CD8:p56lck complex is catalytically active as shown by its ability to phosphorylate at 55-60 kDa. Two-dimensional, nonequilibrium gel electrophoresis demonstrated the similarity of p56lck associated with the CD4 and CD8 antigens. Detergents were found to vary in their ability to solubilize the CD4:p56lck complex in a catalytically active form. We further demonstrated by in vitro phosphorylation that members of the CD3 complex including the gamma, delta, and epsilon chains, as well as a putative zeta subunit can be phosphorylated at tyrosyl residues by the CD4/CD8:p56lck complex. Thus, this interaction may play an important role in the activation of T cells, and may mediate the cooperative interaction between the CD4/CD8 antigens and the Ti(TcR)/CD3 complex. This interaction also represents a possible precedent by which other members of the src family (c-src, c-yes, c-fgr, etc.) may be found to interact with mammalian growth receptors.

The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex.

Many mammalian receptors have been found to regulate cell growth by virtue of a protein-tyrosine kinase domain in their cytoplasmic tail. We recently described an association of the CD4 antigen with a T-cell-specific protein-tyrosine kinase (p56lck; formerly termed pp58lck; EC 2.7.1.112). This interaction represents a potential mechanism by which T-cell growth may be regulated and offers a model by which other members of the src family (products of c-src, c-yes, c-fgr, etc.) may interact with mammalian growth factor receptors. As in the case of the CD4 antigen, the CD8 antigen appears to serve as a receptor for nonpolymorphic regions of products of the major histocompatibility complex and has been implicated in the regulation of T-cell growth. In this study, we reveal that the human CD8 antigen is also associated with the T-cell-specific protein-tyrosine kinase (p56lck). The associated p56lck kinase was detected by use of both in vitro and in vivo labeling regimes using an antiserum to the C terminus of p56lck. Two-dimensional nonequilibrium pH-gradient gel electrophoresis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated the similarity of p56lck to the protein-tyrosine kinase associated with the CD4 antigen. The catalytic activity of p56lck was revealed by the autophosphorylation of the 55- to 60-kDa kinase and the occasional labeling of a 35-kDa protein. Last, we demonstrate directly that members of the CD3 complex, including the gamma, delta, and epsilon chains, as well as a putative zeta subunit, can be phosphorylated at tyrosine residues by the CD4/CD8.p56lck complex.