In polarized cells, the multidrug resistance protein MRP2 is localized in the apical plasma membrane, whereas MRP1, another multidrug resistance protein (MRP) family member, is localized in the basolateral membrane. MRP1 and MRP2 are thought to contain an N-terminal region of five transmembrane segments (TMD 0 ) coupled to 2 times six transmembrane segments via an intracellular loop (L 0 ). We previously demonstrated for MRP1 that a mutant lacking TMD 0 but still containing L 0 , called L 0 ⌬MRP1, was functional and routed to the lateral plasma membrane. To investigate the role of the TMD 0 L 0 region of MRP2 in routing to the apical membrane, we generated mutants similar to those made for MRP1. In contrast to L 0 ⌬MRP1, L 0 ⌬MRP2 was associated with an intracellular compartment, most likely endosomes. Co-expression with TMD 0 , however, resulted in apical localization of L 0 ⌬MRP2 and transport activity. Uptake experiments with vesicles containing L 0 ⌬MRP2 demonstrated that the molecule is able to transport LTC 4 . An MRP2 mutant without TMD 0 L 0 , ⌬MRP2, was only core-glycosylated and localized intracellularly. Co-expression of ⌬MRP2 with TMD 0 L 0 resulted in an increased protein level of ⌬MRP2, full glycosylation of the protein, routing to the apical membrane, and transport activity. Our results suggest that the TMD 0 region is required for routing to or stable association with the apical membrane.Several members of the ATP-binding cassette superfamily of transporter proteins are able to confer multidrug resistance to tumor cells. Examples are MDR1 P-glycoprotein (1) and members of the multidrug resistance protein (MRP) 1 family, MRP1and MRP2 (2, 3). Whereas MDR1 Pgp preferably transports large amphipathic molecules (4), MRP1 and MRP2 transport in addition acidic compounds with a large hydrophobic moiety such as drugs conjugated with glutathione, glucuronide, or sulfate (5-7). MRP1-and MRP2-mediated transport of cytotoxic drugs not known to be conjugated to negatively charged ligands is most likely due to co-transport with reduced glutathione (8 -12). Besides MRP1 and MRP2, there are seven other MRP homologs expressed in humans, designated MRP3 to -9 (13-15). MRP3 to -5 can confer resistance against some anticancer drugs and are able to transport certain organic anions (16 -21).The predicted membrane topology of MDR1 Pgp and MRPs is different. Whereas MDR1 Pgp and MRPs share a similar core region consisting of two times six transmembrane regions and two intracellular ATP-binding cassettes, MRP1 and MRP2, and several other members of the MRP family contain an extra amino-terminal domain of about 280 amino acids (22-26) (see also Fig. 1). This latter domain is thought to consist of five transmembrane segments and is bound to the core region via an intracellular loop (L 0 ) of ϳ80 amino acids. By producing various mutants of MRP1 in baculovirus-infected insect cells, we and others previously showed that the MRP1 core, called ⌬MRP1, was inactive. Co-expression of the core (⌬MRP1) with TMD 0 L 0 , however, resu...

The tumor necrosis factor family member Fas ligand (FasL) induces apoptosis in Fas receptor-expressing target cells and is an important cytotoxic effector molecule used by CTL-and NK-cells. In these hematopoietic cells, newly synthesized FasL is stored in specialized secretory lysosomes and only delivered to the cell surface upon activation and target cell recognition. FasL contains an 80-amino acid-long cytoplasmic tail, which includes a proline-rich domain as a bona fide Src homology 3 domain-binding site. This proline-rich domain has been implicated in FasL sorting to secretory lysosomes, and it may also be important for reverse signaling via FasL, which has been described to influence T-cell activation. Here we report the identification of the Src homology 3 domaincontaining adaptor protein PSTPIP as a FasL-interacting partner, which binds to the proline-rich domain. PSTPIP co-expression leads to an increased intracellular localization of Fas ligand, thereby regulating extracellular availability and cytotoxic activity of the molecule. In addition, we demonstrate recruitment of the tyrosine phosphatase PTP-PEST by PSTPIP into FasL⅐PSTPIP⅐PTP-PEST complexes which may contribute to FasL reverse signaling.Fas ligand (FasL 3 ; CD95/APO-1 ligand, CD178, TNFSF6) is a 281-amino acid (aa)-long type II transmembrane molecule belonging to the large TNF family of proteins that bind to and activate members of the TNF receptor family (1, 2). FasL and its corresponding receptor Fas (CD95/APO-1) both interact as oligomers (3), and the activated Fas receptor complex initiates a proapoptotic death signal in the receptorbearing cell (4, 5).Although mainly known for its death-promoting activity, FasL has also been studied in the context of further nonapoptotic functions (1, 2, 6). Such experiments are motivated by the special structure of this type II transmembrane protein. In addition to its hydrophobic transmembrane domain, which anchors the molecule within the plasma membrane, and to its extracellular ectodomain, responsible for binding to its receptor, the FasL protein possesses an 80-aa-long N-terminal intracellular part that is responsible for the transduction of (extracellular) signals into FasL-bearing cells and/or that may fulfill regulatory functions. Such a "reverse signaling" has been described in mouse T-cells, which display an altered proliferative behavior upon triggering of their FasL surface molecules (7-9). Results obtained with mouse Sertoli cells, in which FasL engagement leads to mitogen-activated protein kinase activation, as measured by an increase in Erk phosphorylation, also imply stimulatory FasL reverse signaling (10).The importance of the intracellular domain for FasL function and regulation is underlined by its high homology among different species (7,11,12). Several signaling motives within the FasL intracellular domain are highly conserved, including a tandem casein kinase I phosphorylation site (aa 17-21 (11)), a phosphorylatable tyrosine at position 7, a proline-rich region (aa 40 -70) with bona...

The ability to escape apoptosis is a hallmark of cancer-initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous, evolutionarily conserved apoptosome-activating protein and investigate its potential pro-apoptotic tumor suppressor function in gastrointestinal stromal tumors (GISTs). Interaction between FAM96A and apoptotic peptidase activating factor 1 (APAF1) was identified in yeast two-hybrid screen and further studied by deletion mutants, glutathione-S-transferase pull-down, co-immunoprecipitation and immunofluorescence. Effects of FAM96A overexpression and knock-down on apoptosis sensitivity were examined in cancer cells and zebrafish embryos. Expression of FAM96A in GIST and histogenetically related cells including interstitial cells of Cajal (ICCs), ‘fibroblast-like cells’ (FLCs) and ICC stem cells (ICC-SCs) was investigated by Northern blotting, reverse transcription—polymerase chain reaction, immunohistochemistry and Western immunoblotting. Tumorigenicity of GIST cells and transformed murine ICC-SC stably transduced to re-express FAM96A was studied by xeno- and allografting into immunocompromised mice. FAM96A was found to bind APAF1 and to enhance the induction of mitochondrial apoptosis. FAM96A protein or mRNA was dramatically reduced or lost in 106 of 108 GIST samples representing three independent patient cohorts. Whereas ICCs, ICC-SCs and FLCs, the presumed normal counterparts of GIST, were found to robustly express FAM96A protein and mRNA, FAM96A expression was much reduced in tumorigenic ICC-SCs. Re-expression of FAM96A in GIST cells and transformed ICC-SCs increased apoptosis sensitivity and diminished tumorigenicity. Our data suggest FAM96A is a novel pro-apoptotic tumor suppressor that is lost during GIST tumorigenesis.