During nervous system development, spinal commissural axons project toward and across the ventral midline. They are guided in part by netrin-1, made by midline cells, which attracts the axons by activating the netrin receptor DCC. However, previous studies suggest that additional receptor components are required. Here, we report that the Down's syndrome Cell Adhesion Molecule (DSCAM), a candidate gene implicated in the mental retardation phenotype of Down's syndrome, is expressed on spinal commissural axons, binds netrin-1, and is necessary for commissural axons to grow toward and across the midline. DSCAM and DCC can each mediate a turning response of these neurons to netrin-1. Similarly, Xenopus spinal neurons exogenously expressing DSCAM can be attracted by netrin-1 independently of DCC. These results show that DSCAM is a receptor that can mediate turning responses to netrin-1 and support a key role for netrin/DSCAM signaling in commissural axon guidance in vertebrates.

Tumor necrosis factor-␣ (TNF␣), a potent proinflammatory cytokine, is released from cells by proteolytic cleavage of a membrane-anchored precursor. The TNF-␣ converting enzyme (TACE; a disintegrin and metalloprotease17; ADAM17) is known to have a key role in the ectodomain shedding of TNF␣ in several cell types. However, because purified ADAMs 9, 10, and 19 can also cleave a peptide corresponding to the TNF␣ cleavage site in vitro, these enzymes are considered to be candidate TNF␣ sheddases as well. In this study we used cells lacking ADAMs 9, 10, 17 (TACE), or 19 to address the relative contribution of these ADAMs to TNF␣ shedding in cell-based assays. Our results corroborate that ADAM17, but not ADAM9, -10, or -19, is critical for phorbol ester-and pervanadate-stimulated release of TNF␣ in mouse embryonic fibroblasts. However, overexpression of ADAM19 increased the constitutive release of TNF␣, whereas overexpression of ADAM9 or ADAM10 did not. This suggests that ADAM19 may contribute to TNF␣ shedding, especially in cells or tissues where it is highly expressed. Furthermore, we used mutagenesis of TNF␣ to explore which domains are important for its stimulated processing by ADAM17. We found that the cleavage site of TNF␣ is necessary and sufficient for cleavage by ADAM17. In addition, the ectodomain of TNF␣ makes an unexpected contribution to the selective cleavage of TNF␣ by ADAM17: it prevents one or more other enzymes from cleaving TNF␣ following PMA stimulation. Thus, selective stimulated processing of TNF␣ by ADAM17 in cells depends on the presence of an appropriate cleavage site as well as the inhibitory role of the TNF ectodomain toward other enzymes that can process this site. TNF␣1 is a pro-inflammatory cytokine that has a critical role in autoimmune disorders such as rheumatoid arthritis and Crohn's disease (1, 2). TNF␣ is synthesized as a trimeric type II membrane-anchored precursor referred to as pro-TNF␣ (3). Upon cleavage in the juxtamembrane domain, the mature form of TNF␣ is released from the cell and can enter the blood stream (4, 5). This proteolytic release of TNF␣ from the membrane is referred to as "protein ectodomain shedding" (6, 7). Protein ectodomain shedding also affects the function of a variety of other structurally and functionally diverse molecules on the cell surface, including cytokines and growth factors, their receptors, adhesion proteins, and other molecules, such as the amyloid precursor protein, Notch and Delta (6 -9).Because of the critical role of TNF␣ in rheumatoid arthritis, considerable efforts have been made to identify the TNF␣ convertase. ADAM17 (a disintegrin and metalloprotease 17, also referred to as TNF␣ converting enzyme or TACE) is considered to be an important, if not the major, sheddase for TNF␣ (10, 11). ADAM17 was initially purified based on its ability to process a peptide, which mimics the physiological cleavage site of TNF␣, in exactly the same position that is used by the TNF␣ converting activity in cells (see Table I and Refs. 10 and 11). A targeted de...

Cellular senescence has been viewed as a tumor suppression mechanism and also as a contributor to individual aging. Widespread shortening of 3 ′ untranslated regions (3 ′ UTRs) in messenger RNAs (mRNAs) by alternative polyadenylation (APA) has recently been discovered in cancer cells. However, the role of APA in the process of cellular senescence remains elusive. Here, we found that hundreds of genes in senescent cells tended to use distal poly(A) (pA) sites, leading to a global lengthening of 3 ′ UTRs and reduced gene expression. Genes that harbor longer 3 ′ UTRs in senescent cells were enriched in senescence-related pathways. Rras2, a member of the Ras superfamily that participates in multiple signal transduction pathways, preferred longer 3 ′ UTR usage and exhibited decreased expression in senescent cells. Depletion of Rras2 promoted senescence, while rescue of Rras2 reversed senescence-associated phenotypes. Mechanistically, splicing factor TRA2B bound to a core "AGAA" motif located in the alternative 3 ′ UTR of Rras2, thereby reducing the RRAS2 protein level and causing senescence. Both proximal and distal poly(A) signals showed strong sequence conservation, highlighting the vital role of APA regulation during evolution. Our results revealed APA as a novel mechanism in regulating cellular senescence.

Tumor necrosis factor ␣-convertase (TACE) is a metalloprotease-disintegrin involved in the ectodomain shedding of several proteins and is critical for proper murine development. TACE-mediated ectodomain shedding is regulated, and the cytoplasmic domain of TACE contains several potential signaling motifs, suggesting that this domain may play a role in regulating the metalloprotease activity. Here we report that the proteintyrosine phosphatase PTPH1, which contains both a band 4.1 domain and a single PDZ domain, can interact with the cytoplasmic domain of TACE. The interaction was initially observed in a yeast two-hybrid screen and was confirmed using an in vitro binding assay and coimmunoprecipitations from eukaryotic cell extracts. The interaction is mediated via binding of the PDZ domain of PTPH1 to the COOH terminus of TACE. The latter represents a novel group I PDZ binding sequence characterized by a terminal cysteine residue. In co-expression experiments, significantly lower levels of TACE were observed in the presence of catalytically active forms of PTPH1 compared with catalytically inactive forms of PTPH1. Furthermore, phorbol ester-stimulated shedding of the TACE substrate tumor necrosis factor-␣ was decreased in cells expressing catalytically active PTPH1 compared with inactive PTPH1. Taken together, these results suggest that PTPH1 may be a negative regulator of TACE levels and function, and thus provide the first evidence for the regulation of TACE through a cytoplasmic protein.

ADAMs are membrane-anchored glycoproteins with functions in fertilization, heart development, neurogenesis, and protein ectodomain shedding. Here we report an evaluation of the catalytic activity of recombinantly expressed soluble forms of ADAM19, a protein that is essential for cardiovascular morphogenesis. Proteolytic activity of soluble forms of ADAM19 was first demonstrated by their autocatalytic removal of a purification tag (Myc-His) and their ability to cleave myelin basic protein and the insulin B chain. The metalloprotease activity of ADAM19 is sensitive to the hydroxamic acid-type metalloprotease inhibitor BB94 (batimastat) but not to tissue inhibitors of metalloproteases (TIMPs) 1-3. Moreover, ADAM19 cleaves peptides corresponding to the known cleavage sites of tumor necrosis factor-alpha (TNF-alpha), TNF-related activation-induced cytokine (TRANCE, also referred to as osteoprotegerin ligand), and kit ligand-1 (KL-1) in vitro. Although ADAM19 is not required for shedding of TNFalpha and TRANCE in mouse embryonic fibroblasts, its overexpression in COS-7 cells results in strongly increased TRANCE shedding. This suggests a potential role for ADAM19 in shedding TRANCE in cells where both molecules are highly expressed, such as in osteoblasts. Interestingly, our results also indicate that ADAM19 can function as a negative regulator of KL-1 shedding in both COS-7 cells and mouse embryonic fibroblasts, instead of acting directly on KL-1. The identification of potential in vitro substrates offers the basis for further functional studies of ADAM19 in cells and in mice.

Chemotherapy is an important treatment for ovarian cancer. However, conventional chemotherapy has inevitable drawbacks due to side effects from nonspecific biodistribution of the chemotherapeutic drugs. To solve such problem, targeted delivery approaches were developed. The targeted delivery approaches combine drug carriers with the targeting system and can preferentially bring drugs to the targeted sites. Follicle-stimulating hormone receptor (FSHR) is an ovarian cancer-specific receptor. By using a peptide derived from FSH (amino acids 33-53 of the FSH B chain, named as FSH33), we developed a conjugated nanoparticle, FSH33-NP, to target FSHR in ovarian cancer. FSH33-NP was tested for recognition specificity and uptake efficiency on FSHR-expressing cells. Then, the antitumor efficiency of paclitaxel (PTX)-loaded FSH33-NP (FSH33-NP-PTX) was determined. FSH33-NP-PTX displayed stronger antiproliferation and antitumor effects compared with free PTX or naked PTX-loaded nanoparticles (NP-PTX) both in vitro and in vivo. In summary, this novel FSH33-NP delivery system showed very high selectivity and efficacy for FSHR-expressing tumor tissues. Therefore, it has good potential to become a new therapeutic approach for patients with ovarian cancer.

All ligands of the epidermal growth factor receptor (EGFR) are made as membrane anchored precursors that can be proteolytically processed and released from the plasma membrane. This process, which is referred to as protein ectodomain shedding, is emerging as a key regulator of the function of EGFR ligands. In light of the important roles of EGFR signaling in development and disease, it will be important to understand more about the regulation of proteolytic processing of EGFR ligands. This chapter describes a sensitive and semiquantitative method to measure ectodomain shedding of EGFR ligands that was designed to facilitate studies of this process in cells.

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