Hydrogen peroxide and endothelin-1 are novel activators of betacellulin ectodomain shedding

The betacellulin precursor (pro-BTC) is a novel substrate for ADAM10-mediated ectodomain shedding. In this report, we investigated the ability of novel physiologically relevant stimuli, including G-protein coupled receptor (GPCR) agonists and reactive oxygen species (ROS), to stimulate pro-BTC shedding. We found that in breast adenocarcinoma MCF7 cells overexpressing pro-BTC, hydrogen peroxide (H2O2) was a powerful stimulator of ectodomain shedding. The stimulation of pro-BTC shedding by H2O2 was blocked by the broad-spectrum metalloprotease inhibitor TAPI-0 but was still functional in ADAM17 (TACE)-deficient stomach epithelial cells indicating the involvement of a distinct metalloprotease. H2O2-induced pro-BTC shedding was blocked by co-culturing cells in the anti-oxidant N-acetyl-L-cysteine but was unaffected by culture in calcium-deficient media. By contrast, calcium ionophore, which is a previously characterized activator of pro-BTC shedding, was sensitive to calcium depletion but was unaffected by co-culture with the anti-oxidant, identifying a clear distinction between these stimuli. We found that in vascular smooth muscle cells overexpressing pro-BTC, the GPCR agonist endothelin-1 (ET-1) was a strong inducer of ectodomain shedding. This was blocked by a metalloprotease inhibitor and by overexpression of catalytically inactive E385A ADAM10. However, overexpression of wild-type ADAM10 or ADAM17 led to an increase in ET-1-induced pro-BTC shedding providing evidence for an involvement of both enzymes in this process. This study identifies ROS and ET-1 as two novel inducers of pro-BTC shedding and lends support to the notion of activated shedding occurring under the control of physiologically relevant stimuli.     

Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor alpha, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17-/- knockout mice corroborated the essential role of adam17-/- in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.     

Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain-truncated form (HBdeltatm) of the molecule. HB(uc/uc) mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBdeltatm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.     

Dopamine-dependent ectodomain shedding and release of epidermal growth factor in developing striatum: target-derived neurotrophic signaling (Part 2)

Epidermal growth factor (EGF) and structurally related peptides promote neuronal survival and the development of midbrain dopaminergic neurons; however, the regulation of their production has not been fully elucidated. In this study, we found that the treatment of striatal cells with dopamine agonists enhances EGF release both in vivo and in vitro. We prepared neuron-enriched and non-neuronal cell-enriched cultures from the striatum of rat embryos and challenged those with various neurotransmitters or dopamine receptor agonists. Dopamine and a dopamine D(1) -like receptor agonist (SKF38393) triggered EGF release from neuron-enriched cultures in a dose-dependent manner. A D(2) -like agonist (quinpirole) increased EGF release only from non-neuronal cell-enriched cultures. The EGF release from striatal neurons and non-neuronal cells was concomitant with ErbB1 phosphorylation and/or with the activation of a disintegrin and metalloproteinase and matrix metalloproteinase. The EGF release from neurons was attenuated by an a disintegrin and metalloproteinase/matrix metalloproteinase inhibitor, GM6001, and a calcium ion chelator, BAPTA/AM. Transfection of cultured striatal neurons with alkaline phosphatase-tagged EGF precursor cDNA confirmed that dopamine D(1) -like receptor stimulation promoted both ectodomain shedding of the precursor and EGF release. Therefore, the activation of striatal dopamine receptors induces shedding and release of EGF to provide a retrograde neurotrophic signal to midbrain dopaminergic neurons.     

Laminin-1 and epidermal growth factor family members co-stimulate fetal pancreas cell proliferation and colony formation

The epidermal growth factor (EGF) family is implicated in the development and function of multiple cells and organs, including the pancreas. We used a serum-free, low-cell density culture system to investigate the effect of EGFs on fetal pancreas cells. By RT-PCR, the EGF receptors ErbB 1-3 were detected in the developing mouse pancreas between embryonic day (E) 13.5 and E17.5, whereas ErbB4 was not detected until E17.5. The presence but not absence of the basement membrane glycoprotein laminin-1, betacellulin, and to a lesser extent EGF, transforming growth factor alpha, heparin binding EGF, and epiregulin induced E15.5 pancreatic cells to proliferate and form cystoid and solid colonies. These results demonstrate that laminin-1 and EGF signaling pathways interact to promote pancreas development.     

Ectodomain shedding of nectin-1 regulates the maintenance of dendritic spine density

Synaptic remodeling has been postulated as a mechanism underlying synaptic plasticity and cell adhesion molecules are thought to contribute to this process. We examined the role of nectin-1 ectodomain shedding on synaptogenesis in cultured rat hippocampal neurons. Nectins are Ca(2+) -independent immunoglobulin-like adhesion molecules, involved in cell-cell adherens junctions. Herein, we show that the processing of nectin-1 occurs by multiple endoproteolytic steps both in vivo and in vitro. We identified regions containing two distinct cleavage sites within the ectodomain of nectin-1. By alanine scanning mutagenesis, two point mutations that disrupt nectin-1 ectodomain cleavage events were identified. Expression of these mutants significantly alters the density of dendritic spines. These findings suggest that ectodomain shedding of nectin-1 regulates dendritic spine density and related synaptic functions.     

An Intracellular Fragment of Osteoactivin Formed by Ectodomain Shedding Translocated to the Nucleoplasm and Bound to RNA Binding Proteins

Mitochondrial ATP synthase (F₁Fo-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, cDNA coding the human homolog of the inhibitor protein was isolated and sequenced. The deduced protein sequence shows that the protein was composed of 106 amino acids and had a molecular weight of 12248. The structural features of the protein show that the cDNA isolated in this study codes the human ATPase inhibitor.     

Lipid rafts identified as locations of ectodomain shedding mediated by Meltrin beta/ADAM19

Meltrin beta (Mel beta, also called ADAM19) is a member of the ADAM (adisintegrin and metalloprotease) family, which are membrane-anchored glycoproteins that play crucial roles in various biological processes. Many intercellular signaling molecules are membrane-anchored proteins, which are proteolytically processed after becoming membrane-bound, to liberate their extracellular domains (ectodomain shedding). Genetic and biochemical studies have shown that some ADAMs participate in these events. We found previously that Mel beta can cleave the extracellular region of the membrane-anchored beta-exon-containing Neuregulin-1 (NRG beta1) protein, which is one of the main ligands for the neural ErbB receptor. Mel beta-deficient mice showed developmental defects in the nervous system. These observations raise the possibility that the NRG ectodomain shedding mediated by Mel beta is closely related to the neural development. Here we show that Mel beta-mediated ectodomain shedding of NRG beta1 takes place in the lipid rafts of neurons. The lipid rafts localization of Mel beta requires its membrane-anchoring region, and NRG beta1 ectodomain shedding is not enhanced if Mel beta cannot reach the lipid rafts. These results indicate that localization of Mel beta in lipid rafts is critical for its ectodomain shedding.     

Characterization of nectin processing mediated by presenilin-dependent γ-secretase

Nectins play an important role in forming various intercellular junctions including synapses. This role is regulated by several secretases present at intercellular junctions. We have investigated presenilin (PS)-dependent secretase-mediated processing of nectins in PS1 KO cells and primary hippocampal neurons. The loss of PS1/γ-secretase activity delayed the processing of nectin-1 and caused the accumulation of its full-length and C-terminal fragments. Over-expression of PS2 in PS1 KO cells compensated for the loss of PS1, suggesting that PS2 also has the ability to regulate nectin-1 processing. In mouse brain slices, a pronounced increase in levels of 30 and 24 kDa C-terminal fragments in response to chemical long-term potentiation was observed. The mouse brain synaptosomal fractionation study indicated that nectin-1 localized to post-synaptic and preferentially pre-synaptic membranes and that shedding occurs in both compartments. These data suggest that nectin-1 shedding and PS-dependent intramembrane cleavage occur at synapses, and is a regulated event during conditions of synaptic plasticity in the brain. Point mutation analysis identified several residues within the transmembrane domain that play a critical role in the positioning of cleavage sites by ectodomain sheddases. Nectin-3, which forms hetero-trans-dimers with nectin-1, also undergoes intramembrane cleavage mediated by PS1/γ-secretase, suggesting that PS1/γ-secreatse activity regulates synapse formation and remodeling by nectin processing.     

Activity-dependent shedding of heparin-binding EGF-like growth factor in brain neurons

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is initially produced as a membrane-anchored precursor (pro-HB-EGF) and subsequently liberated from the cell membrane through ectodomain shedding. Here, we characterized the molecular regulation of pro-HB-EGF shedding in the central nervous system. Cultured neocortical or hippocampal neurons were transfected with the alkaline-phosphatase-tagged pro-HB-EGF gene and stimulated with various neurotransmitters. Both kainate and N-methyl-D-aspartate, but not agonists for metabotropic glutamate receptors, promoted pro-HB-EGF shedding and HB-EGF release, which were attenuated by an exocytosis blocker and metalloproteinase inhibitors. In the brain of transgenic mice over-expressing human pro-HB-EGF, kainate-induced seizure activity decreased content of pro-HB-EGF-like immunoreactivity and conversely increased levels of soluble HB-EGF. There was concomitant phosphorylation of EGF receptors (ErbB1) following seizures, suggesting that seizure activities liberated HB-EGF and activated neighboring ErbB1 receptors. Therefore, we propose that glutamatergic neurotransmission in the central nervous system plays a crucial role in regulating ectodomain shedding of pro-HB-EGF.