Calcium signaling through the beta 2-cytoplasmic domain of LFA-1 requires intracellular elements of the T cell receptor complex.

The beta(2) integrin LFA-1 is an important cell-cell adhesion receptor of the immune system. Evidence suggests that the molecule also participates in signaling and co-stimulatory function. We show here that clustering of the intracellular domain of the beta(2) chain but not of the alpha(L)- or beta(1)-cytoplasmic domains, respectively, triggers intracellular Ca(2+) mobilization in Jurkat cells. A beta(2)-specific NPXF motif, located in the C-terminal portion of the beta(2) tail, is required for Ca(2+) signaling, and we show that this motif is important for the induction of allo-specific target cell lysis by cytotoxic T cells in vitro. Significantly, the Ca(2+)-signaling capacity of the beta(2) integrin is abrogated in T cells that do not express the T cell receptor but may be reconstituted by co-expression of the T cell receptor-zeta chain. Our data suggest a specific function of the cytoplasmic domain of the beta(2) integrin chain in T cell signaling.

The contactin-related protein FAR-2 defines purkinje cell clusters and labels subpopulations of climbing fibers in the developing cerebellum.

FAR-2 is a novel neural member of the Ig superfamily, which is related to F11/F3/contactin and axonin-1/TAG-1. This protein is expressed by subpopulations of Purkinje cells in the chicken cerebellum and FAR-2-positive clusters of these neurons alternate with FAR-2-negative clusters in both tangential dimensions of the cerebellar cortex. Furthermore, FAR-2 is also expressed by one type of Purkinje cell afferents, namely, the climbing fibers, and different subpopulations of these axons show distinct levels of FAR-2 expression. Homology modeling using axonin-1 as a template reveals that the four aminoterminal Ig domains of FAR-2 form a compact U-shaped structure, which is likely to contain functionally important ligand-binding sites. FAR-2 is binding to the Ig superfamily protein NgCAM/L1, but not to the related receptor NrCAM, and it is also interacting with the modular ECM protein tenascin-R. These results suggest that FAR-2 may contribute to the formation of somatotopic maps of cerebellar afferents during the development of the nervous system.

Cloning of ACP33 as a novel intracellular ligand of CD4.

CD4 recruitment to T cell receptor (TCR)-peptide-major histocompatibility class II complexes is required for stabilization of low affinity antigen recognition by T lymphocytes. The cytoplasmic portion of CD4 is thought to amplify TCR-initiated signal transduction via its association with the protein tyrosine kinase p56(lck). Here we describe a novel functional determinant in the cytosolic tail of CD4 that inhibits TCR-induced T cell activation. Deletion of two conserved hydrophobic amino acids from the CD4 carboxyl terminus resulted in a pronounced enhancement of CD4-mediated T cell costimulation. This effect was observed in the presence or absence of p56(lck), implying involvement of alternative cytosolic ligands of CD4. A two-hybrid screen with the intracellular portion of CD4 identified a previously unknown 33-kDa protein, ACP33 (acidic cluster protein 33), as a novel intracellular binding partner of CD4. Since interaction with ACP33 is abolished by deletion of the hydrophobic CD4 C-terminal amino acids mediating repression of T cell activation, we propose that ACP33 modulates the stimulatory activity of CD4. Furthermore, we demonstrate that interaction with CD4 is mediated by the noncatalytic alpha/beta hydrolase fold domain of ACP33. This suggests a previously unrecognized function for alpha/beta hydrolase fold domains as a peptide binding module mediating protein-protein interactions.

Neurotractin, a novel neurite outgrowth-promoting Ig-like protein that interacts with CEPU-1 and LAMP.

The formation of axon tracts in nervous system histogenesis is the result of selective axon fasciculation and specific growth cone guidance in embryonic development. One group of proteins implicated in neurite outgrowth, fasciculation, and guidance is the neural members of the Ig superfamily (IgSF). In an attempt to identify and characterize new proteins of this superfamily in the developing nervous system, we used a PCR-based strategy with degenerated primers that represent conserved sequences around the characteristic cysteine residues of Ig-like domains. Using this approach, we identified a novel neural IgSF member, termed neurotractin. This GPI-linked cell surface glycoprotein is composed of three Ig-like domains and belongs to the IgLON subgroup of neural IgSF members. It is expressed in two isoforms with apparent molecular masses of 50 and 37 kD, termed L-form and S-form, respectively. Monoclonal antibodies were used to analyze its biochemical features and histological distribution. Neurotractin is restricted to subsets of developing commissural and longitudinal axon tracts in the chick central nervous system. Recombinant neurotractin promotes neurite outgrowth of telencephalic neurons and interacts with the IgSF members CEPU-1 (KD = 3 x 10(-8) M) and LAMP. Our data suggest that neurotractin participates in the regulation of neurite outgrowth in the developing brain.

T cell activation induced by novel gain-of-function mutants of Syk and ZAP-70.

The Syk family tyrosine kinases play a crucial role in antigen receptor-mediated signal transduction, but their regulation and cellular targets remain incompletely defined. Following receptor engagement, phosphorylation of tyrosine residues within ZAP-70 and Syk is thought to control both kinase activity and recruitment of modulatory factors. We report here the characterization of novel mutants of ZAP-70 and Syk, in which conserved C-terminal tyrosine residues have been replaced by phenylalanines (ZAP YF-C, Syk YF-C). Both mutant kinases display a prominent gain-of-function phenotype in Jurkat T cells, as demonstrated by lymphokine promoter activation, tyrosine phosphorylation of potential targets in vivo, and elevated intracellular calcium mobilization. While the presence of p56-Lck was required for ZAP YF-C-induced signaling, Syk YF-C showed enhanced functional activity in Lck-deficient JCaM1 Jurkat cells. Our results implicate the C terminus of Syk family kinases as an important regulatory region modulating T cell activation.