The adaptor protein Grb2 regulates cell surface Fas ligand in Schwann cells.

Fas Ligand (FasL, CD178) is a cytokine that may be secreted or expressed as a transmembrane ligand at the cell surface, and induces apoptosis by binding to the "death receptor" Fas (CD95). Here, we show that Grb2, an SH3 domain-containing adaptor protein, binds to the proline-rich domain of FasL and regulates its cell surface expression. We found that knocking down Grb2 expression decreased the amount of FasL at the cell surface and increased the abundance of intracellular vesicles containing FasL. Furthermore, we showed that Grb2 acts as an adaptor for FasL to interact with adaptin beta, a molecule known to regulate trafficking. Our data reveal that Grb2 facilitates the association of FasL with adaptin beta, and promotes sorting of FasL to the cell surface. As FasL is a potent regulator of cell death, dynamic regulation of its cell surface localization is critical for controlling local tissue remodeling and inflammation.

Constitutive overexpression of a novel 21 kDa protein by Hodgkin lymphoma and aggressive non-Hodgkin lymphomas.

BACKGROUND: CD30, a 120 kDa surface phosphorylated protein is a member of tumour necrosis/nerve growth factor receptor (TNF/NGFR) family and constitutively expressed by Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) and the neoplastic cells of Anaplastic Large Cell Lymphoma (ALCL). A disease-specific protein marker is yet to be identified in Hodgkin lymphoma cells. In order to define HL-specific biomarkers, novel murine monoclonal antibodies were developed in our laboratory. RESULTS: Murine monoclonal antibodies (mabs) were raised against the B3 sub clone of HL-derived cell line KM-H2. Two of these mabs (clone R23.1 mab and clone R24.1 mab) are IgG1 class antibodies that recognize a 21 kDa protein present at the cell membrane and in the cytoplasm in HL-derived cell lines. Clone R24.1 mab recognizes a formalin-resistant epitope and labels HRS cells in tissue samples from patients with HL of the classical type, ALCL, and subsets of T and B cell aggressive Non-Hodgkin Lymphomas (NHL). The antigen recognized by the clone R23.1 mab and clone R24.1 mab does not share epitopes with CD30 cluster regions A, B, or C, and, unlike CD30, is not expressed by phytohemagglutinin (PHA) activated T cells. CONCLUSION: The 21 kDa protein detected by clone R23.1 and clone R24.1 mabs is a novel membrane-associated protein that may be a potential marker for the diagnosis and targeted therapy of HL and aggressive T and B cell NHL.

A proteomic screen reveals novel Fas ligand interacting proteins within nervous system Schwann cells.

Fas ligand (FasL) binds Fas (CD95) to induce apoptosis or activate other signaling pathways. In addition, FasL transduces bidirectional or 'reverse signals'. The intracellular domain of FasL contains consensus sequences for phosphorylation and an extended proline rich region, which regulate its surface expression through undetermined mechanism(s). Here, we used a proteomics approach to identify novel FasL interacting proteins in Schwann cells to investigate signaling through and trafficking of this protein in the nervous system. We identified two novel FasL interacting proteins, sorting nexin 18 and adaptin beta, as well as two proteins previously identified as FasL interacting proteins in T cells, PACSIN2 and PACSIN3. These proteins are all associated with endocytosis and trafficking, highlighting the tight regulation of cell surface expression of FasL in the nervous system.

Targeted mutations of the juxtamembrane tyrosines in the Kit receptor tyrosine kinase selectively affect multiple cell lineages.

Loss-of-function mutations in the murine dominant white spotting/c-kit locus affect a diverse array of biological processes and cell lineages and cause a range of phenotypes, including severe anemia, defective pigmentation, sterility, mast cell deficits, a lack of interstitial cells of Cajal, spatial learning memory deficits, and defects in peripheral nerve regeneration. Here we show that tyrosine residues 567 and 569 in the juxtamembrane (Jx) domain of the murine Kit receptor tyrosine kinase are crucial for the function of Kit in melanogenesis and mast cell development, but are dispensable for the normal development of erythroid, interstitial cells of Cajal and germ cells. Furthermore, adult mice lacking both tyrosines exhibit splenomegaly, dysregulation of B-cell and megakaryocyte development, and enlarged stomachs. Analysis of signal transduction events induced by the mutant receptors after ligand stimulation indicates that Jx tyrosine mutations diminish receptor autophosphorylation and selectively attenuate activation of extracellular signal-regulated kinase/mitogen-activated protein kinases. Together, these observations demonstrate that the Jx domain of Kit plays a cell-type specific regulatory role in vivo and illustrate how engineered mutations in Kit can be used to understand the complex biological and molecular events that result from activating a receptor tyrosine kinase.