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Carl P Blobel

Publications and source records attributed to Carl P Blobel.

At least 19 recordsLinked to original sources
PubMed2006

Ectodomain shedding of the EGF-receptor ligand epigen is mediated by ADAM17.

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are made as transmembrane precursors. Proteolytic processing by ADAMs (a disintegrin and metalloprotease) regulates the bioavailability of several EGFR-ligands, yet little is known about the enzyme responsible for processing the recently identified EGFR ligand, epigen. Here we show that ectodomain shedding of epigen requires ADAM17, which can be stimulated by phorbol esters, phosphatase inhibitors and calcium influx. These results suggest that ADAM17 might be a good target to block the release of bioactive epigen, a highly mitogenic ligand of the EGFR which has been implicated in wound healing and cancer.

ADAM Proteins↗
PubMed2006

Proteolytic processing of delta-like 1 by ADAM proteases.

Delta-like 1 (Dll1) is a mammalian ligand for Notch receptors. Interactions between Dll1 and Notch in trans activate the Notch pathway, whereas Dll1 binding to Notch in cis inhibits Notch signaling. Dll1 undergoes proteolytic processing in its extracellular domain by ADAM10. In this work we demonstrate that Dll1 represents a substrate for several other members of the ADAM family. In co-transfected cells, Dll1 is constitutively cleaved by ADAM12, and the N-terminal fragment of Dll1 is released to medium. ADAM12-mediated cleavage of Dll1 is cell density-dependent, takes place in cis orientation, and does not require the presence of the cytoplasmic domain of ADAM12. Full-length Dll1, but not its N- or C-terminal proteolytic fragment, co-immunoprecipitates with ADAM12. By using a Notch reporter construct, we show that Dll1 processing by ADAM12 increases Notch signaling in a cell-autonomous manner. Furthermore, ADAM9 and ADAM17 have the ability to process Dll1. In contrast, ADAM15 does not cleave Dll1, although the two proteins still co-immunoprecipitate with each other. Asn-353 present in the catalytic motif of ADAM12 and other Dll1-processing ADAMs, but absent in ADAM15, is necessary for Dll1 cleavage. Dll1 cleavage is reduced in ADAM9/12/15(-/-) mouse embryonic fibroblasts (MEFs), suggesting that the endogenous ADAM9 and/or ADAM12 present in wild type MEFs contribute to Dll1 processing. Finally, the endogenous Dll1 present in primary mouse myoblasts undergoes cleavage in confluent, differentiating myoblast cultures, and this cleavage is decreased by ADAM12 small interfering RNAs. Our findings expand the role of ADAM proteins in the regulation of Notch signaling.

ADAM Proteins↗
PubMed2006

Substrate selectivity of epidermal growth factor-receptor ligand sheddases and their regulation by phorbol esters and calcium influx.

Signaling via the epidermal growth factor receptor (EGFR), which has critical roles in development and diseases such as cancer, is regulated by proteolytic shedding of its membrane-tethered ligands. Sheddases for EGFR-ligands are therefore key signaling switches in the EGFR pathway. Here, we determined which ADAMs (a disintegrin and metalloprotease) can shed various EGFR-ligands, and we analyzed the regulation of EGFR-ligand shedding by two commonly used stimuli, phorbol esters and calcium influx. Phorbol esters predominantly activate ADAM17, thereby triggering a burst of shedding of EGFR-ligands from a late secretory pathway compartment. Calcium influx stimulates ADAM10, requiring its cytoplasmic domain. However, calcium influx-stimulated shedding of transforming growth factor alpha and amphiregulin does not require ADAM17, even though ADAM17 is essential for phorbol ester-stimulated shedding of these EGFR-ligands. This study provides new insight into the machinery responsible for EGFR-ligand release and thus EGFR signaling and demonstrates that dysregulated EGFR-ligand shedding may be caused by increased expression of constitutively active sheddases or activation of different sheddases by distinct stimuli.

ADAM Proteins↗
PubMed2006

ADAM10 is a principal 'sheddase' of the low-affinity immunoglobulin E receptor CD23.

CD23, the low-affinity immunoglobulin E receptor, is an important modulator of the allergic response and of diseases such as rheumatoid arthritis. The proteolytic release of CD23 from cells is considered a key event in the allergic response. Here we used loss-of-function and gain-of-function experiments with cells lacking or overexpressing candidate CD23-releasing enzymes (ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM33), ADAM-knockout mice and a selective inhibitor to identify ADAM10 as the main CD23-releasing enzyme in vivo. Our findings provide a likely target for the treatment of allergic reactions and set the stage for further studies of the involvement of ADAM10 in CD23-dependent pathologies.

ADAM Proteins↗
PubMed2006

A sensitive method to monitor ectodomain shedding of ligands of the epidermal growth factor receptor.

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.

Alkaline Phosphatase↗
PubMed2005

Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans.

The Eph family of receptor tyrosine kinases and their ephrin ligands are mediators of cell-cell communication. Cleavage of ephrin-A2 by the ADAM10 membrane metalloprotease enables contact repulsion between Eph- and ephrin-expressing cells. How ADAM10 interacts with ephrins in a regulated manner to cleave only Eph bound ephrin molecules remains unclear. The structure of ADAM10 disintegrin and cysteine-rich domains and the functional studies presented here define an essential substrate-recognition module for functional interaction of ADAM10 with the ephrin-A5/EphA3 complex. While ADAM10 constitutively associates with EphA3, the formation of a functional EphA3/ephrin-A5 complex creates a new molecular recognition motif for the ADAM10 cysteine-rich domain that positions the proteinase domain for effective ephrin-A5 cleavage. Surprisingly, the cleavage occurs in trans, with ADAM10 and its substrate being on the membranes of opposing cells. Our data suggest a simple mechanism for regulating ADAM10-mediated ephrin proteolysis, which ensures that only Eph bound ephrins are recognized and cleaved.

ADAM Proteins↗
PubMed2005

Critical function for ADAM9 in mouse prostate cancer.

ADAM9 is a membrane-anchored metalloprotease that is markedly up-regulated in several human carcinomas. Here, we show that ADAM9 is similarly up-regulated in mouse models for prostate, breast, and intestinal carcinoma. To assess whether ADAM9 is critical for the pathogenesis of prostate carcinoma, one of the most common cancers in men, we evaluated how loss of ADAM9 affects tumorigenesis in W(10) mice, a mouse model for this disease. In the absence of ADAM9, most tumors in 50-week-old W(10) mice were well differentiated, whereas littermate controls expressing wild-type ADAM9 had predominantly poorly differentiated, and in some cases significantly larger, tumors. Moreover, gain-of-function experiments in which ADAM9 was overexpressed in mouse prostate epithelium resulted in significant abnormalities, including epithelial hyperplasia at 4 to 6 months of age, and prostatic intraepithelial neoplasia after 1 year. A potential underlying mechanism for the role of ADAM9 in prostate cancer emerged from cell-based assays: ADAM9 can cleave and release epidermal growth factor and FGFR2iiib from cells, both of which have pivotal functions in the pathogenesis of this disease. Taken together, these results suggest that ADAM9 contributes to the pathogenesis of prostate cancer and potentially also other carcinomas, raising the possibility that ADAM9 might be a good target for antitumor drugs.

ADAM Proteins↗
PubMed2005

Evaluation of the contributions of ADAMs 9, 12, 15, 17, and 19 to heart development and ectodomain shedding of neuregulins beta1 and beta2.

Defects in heart development are the most common congenital abnormalities in humans, providing a strong incentive to learn more about the underlying causes. Previous studies have implicated the metalloprotease-disintegrins ADAMs (a disintegrin and metalloprotease) 17 and 19 as well as heparin binding EGF-like growth factor (HB-EGF) and neuregulins in heart development in mice. Here, we show that mice lacking both ADAMs 17 and 19 have exacerbated defects in heart development compared to mice lacking either ADAM, providing the first evidence for redundant or compensatory functions of ADAMs in development. Moreover, we identified additional compensatory or redundant roles of ADAMs 9 and 19 in morphogenesis of the mitral valve and cardiac outflow tract. Cell biological studies designed to address the functions of these ADAMs in shedding of HB-EGF uncovered a contribution of ADAM19 to this process, but this was only evident in the absence of the major HB-EGF sheddase, ADAM17. In addition, ADAM17 emerged as the major sheddase for neuregulins beta1 and beta2 in mouse embryonic fibroblasts. These results raise the possibility that ADAMs 9, 17, and 19 contribute to heart development in humans and have implications for understanding the mechanisms underlying congenital heart disease.

Animals↗
PubMed2005

Homeostatic effects of the metalloproteinase disintegrin ADAM15 in degenerative cartilage remodeling.

OBJECTIVE: The membrane-anchored metalloproteinase disintegrin ADAM15 is up-regulated in osteoarthritis and has been implicated in proteolysis and cell-matrix interactions. To address its role in cartilage metabolism, we performed an analysis of joint morphology in aging mice with a targeted inactivation of the ADAM15 gene (ADAM15(-/-)). In addition, a human chondrocyte cell line overexpressing ADAM15 was used to investigate the role of ADAM15 in an in vitro model of chondrocyte-matrix interactions. METHODS: Knee joint sections from 3-, 6-, and 12-14-month-old ADAM15(-/-) and wild-type (WT) 129/SvJ mice were examined for synovial hyperplasia, cartilage degradation, and osteophyte formation. Stable transfection of the human T/C28a4 chondrocyte cell line with full-length human ADAM15 complementary DNA led to the establishment of ADAM15-overexpressing chondrocytes that were further analyzed for their capability to adhere to and to survive on cartilage matrix molecules (fibronectin and types II and VI collagen) under conditions of serum starvation. ADAM15 expression was investigated by reverse transcription-polymerase chain reaction and Western blotting. RESULTS: Aging ADAM15(-/-) mice exhibited accelerated development of osteoarthritic lesions compared with WT mice, and the difference was statistically significant at age 12 months. The osteoarthritic changes preferentially affected male ADAM15(-/-) mice. ADAM15 overexpression in T/C28a4 cells led to the specific reinforcement of chondrocyte adhesion to cartilage types II and VI collagen, and this was associated with enhanced cell viability under conditions of serum starvation. CONCLUSION: The accelerated development of murine osteoarthritis in ADAM15 deficiency as well as the proadhesive and cell survival-promoting in vitro effect of ADAM15 overexpression suggest a homeostatic rather than a destructive role of ADAM15 in cartilage remodeling.

ADAM Proteins↗
PubMed2005

Metalloprotease-disintegrin ADAM8: expression analysis and targeted deletion in mice.

ADAM8 (a disintegrin and metalloprotease 8, also referred to as MS2/CD156a) is a membrane-anchored metalloprotease that was first identified in a macrophage cell line and has been implicated in neurodegenerative diseases. Here, we evaluated the expression of ADAM8 during mouse development and generated mice lacking ADAM8 (Adam8-/- mice). During early mouse development, ADAM8 is expressed by maternal cells in the decidua and by trophoblast derivatives of the embryo but not in the derivatives of the inner cell mass. At later stages, prominent expression of ADAM8 is seen in the embryo proper, in the gonadal ridge, thymus, developing cartilage and bone, brain and spinal cord, and in the mesenchyme in close proximity to the branch point between the jugular vein and developing lymphatic vessels. Examination of Adam8-/- mice, however, revealed no major defects in these or other structures during development or in adult tissues and no evident pathological phenotypes.

ADAM Proteins↗
PubMed2005

ADAMs: key components in EGFR signalling and development.

ADAM (a disintegrin and metalloprotease) proteins are membrane-anchored metalloproteases that process and shed the ectodomains of membrane-anchored growth factors, cytokines and receptors. ADAMs also have essential roles in fertilization, angiogenesis, neurogenesis, heart development and cancer. Research on ADAMs and their role in protein ectodomain shedding is emerging as a fertile ground for gathering new insights into the functional regulation of membrane proteins.

Amino Acid Sequence↗
PubMed2004

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.

ADAM Proteins↗
PubMed2004

Essential role for ADAM19 in cardiovascular morphogenesis.

Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19(-/-) animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects.

ADAM Proteins↗
PubMed2003

Evidence for a critical role of the tumor necrosis factor alpha convertase (TACE) in ectodomain shedding of the p75 neurotrophin receptor (p75NTR).

Protein ectodomain shedding, the proteolytic release of the extracellullar domain of membrane-tethered proteins, can dramatically affect the function of cell surface receptors, growth factors, cytokines, and other proteins. In this study, we evaluated the activities involved in ectodomain shedding of p75NTR, a neurotrophin receptor with critical roles in neuronal differentiation and survival. p75NTR is shed in a variety of cell types, including dorsal root ganglia cells and PC12 cells. In Chinese hamster ovary cells, inhibitors of the MEK/ERK and p38 MAP kinase pathways uncovered distinct signaling pathways required for the constitutive and stimulated shedding of p75NTR. Stimulated p75NTR shedding is abrogated in M2 mutant Chinese hamster ovary cells that lack functional tumor necrosis factor-alpha converting enzyme (TACE, also referred to as ADAM17) and in cells isolated from adam17-/- mice, but not in cells from adam9/12/15-/- or adam10-/- mice. Stimulated p75(NTR) shedding is strongly reduced by deletion of 15 amino acid residues in its extracellular membrane-proximal stalk domain. However, similar to other shed proteins, point mutations and overlapping shorter deletions within this region have little or no effect on shedding. Because ectodomain shedding of p75NTR releases a soluble ectodomain and could also be a prerequisite for its regulated intramembrane proteolysis, these findings may have important implications for the functional regulation of p75NTR.

ADAM Proteins↗
PubMed2003

Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-alpha-converting enzyme (ADAM 17).

A variety of cell surface adhesion molecules can exist as both transmembrane proteins and soluble circulating forms. Increases in the levels of soluble adhesion molecules have been correlated with a variety of inflammatory diseases, suggesting a pathological role. Although soluble forms are thought to result from proteolytic cleavage from the cell surface, relatively little is known about the proteases responsible for their release. In this report we demonstrate that under normal culture conditions, cells expressing vascular cell adhesion molecule 1 (VCAM-1) release a soluble form of the extracellular domain that is generated by metalloproteinase-mediated cleavage. VCAM-1 release can be rapidly simulated by phorbol 12-myristate 13-acetate (PMA), and this induced VCAM-1 shedding is mediated by metalloproteinase cleavage of VCAM-1 near the transmembrane domain. PMA-induced VCAM-1 shedding occurs as the result of activation of a specific pathway, as the generation of soluble forms of three other adhesion molecules, E-selectin, platelet-endothelial cell adhesion molecule 1, and intercellular adhesion molecule 1, are not altered by PMA stimulation. Using cells derived from genetically deficient mice, we identify tumor necrosis factor-alpha-converting enzyme (TACE or ADAM 17) as the protease responsible for PMA-induced VCAM-1 release, including shedding of endogenously expressed VCAM-1 by murine endothelial cells. Therefore, TACE-mediated shedding of VCAM-1 may be important for the regulation of VCAM-1 function at the cell surface.

ADAM Proteins↗
PubMed2003

Impaired trafficking and activation of tumor necrosis factor-alpha-converting enzyme in cell mutants defective in protein ectodomain shedding.

Protein ectodomain shedding is a specialized type of regulated proteolysis that releases the extracellular domain of transmembrane proteins. The metalloprotease disintegrin tumor necrosis factor-alpha-converting enzyme (TACE) has been convincingly shown to play a central role in ectodomain shedding, but despite its broad interest, very little is known about the mechanisms that regulate its activity. An analysis of the biosynthesis of TACE in mutant cell lines that have a gross defect in ectodomain shedding (M1 and M2) shows a defective removal of the prodomain that keeps TACE in an inactive form. Using LoVo, a cell line that lacks of active furin, and alpha1-Antitrypsin Portland, a protein inhibitor of proprotein convertases, we show that TACE is normally processed by furin and other proprotein convertases. The defect in M1 and M2 cells is due to a blockade of the exit of TACE from the endoplasmic reticulum. The processing of other zinc-dependent metalloproteases, previously suggested to participate in activated ectodomain shedding is normal in the mutant cells, indicating that the component mutated is highly specific for TACE. In summary, the characterization of shedding-defective somatic cell mutants unveils the existence of a specific mechanism that directs the proteolytic activation of TACE through the control of its exit from the ER.

ADAM Proteins↗
PubMed2003

Catalytic properties of ADAM19.

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.

ADAM Proteins↗