The shedding activity of ADAM17 is sequestered in lipid rafts

The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-disintegrin responsible for the cleavage of several biologically active transmembrane proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding activity are still poorly understood. Here, we report that during its transport through the Golgi apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts) where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of these substrates demonstrating the importance of lipid rafts in the control of this process. Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting that the entry of each of these substrates in these particular membrane microdomains is specifically regulated. Our data support the idea that one of the mechanisms regulating ADAM17 substrate cleavage involves protein partitioning in lipid rafts.     

The role of ADAM-mediated shedding in vascular biology

Within the vasculature the disintegrins and metalloproteinases (ADAMs) 8, 9, 10, 12, 15, 17, 19, 28 and 33 are expressed on endothelial cells, smooth muscle cells and on leukocytes. As surface-expressed proteases they mediate cleavage of vascular surface molecules at an extracellular site close to the membrane. This process is termed shedding and leads to the release of a soluble substrate ectodomain thereby critically modulating the biological function of the substrate. In the vasculature several surface molecules undergo ADAM-mediated shedding including tumour necrosis factor (TNF) α, interleukin (IL) 6 receptor α, L-selectin, vascular endothelial (VE)-cadherin, the transmembrane CX3C-chemokine ligand (CX3CL) 1, Notch, transforming growth factor (TGF) and heparin-binding epidermal growth factor (HB-EGF). These substrates play distinct roles in vascular biology by promoting inflammation, permeability changes, leukocyte recruitment, resolution of inflammation, regeneration and/or neovascularisation. Especially ADAM17 and ADAM10 are capable of cleaving many substrates with diverse function within the vasculature, whereas other ADAMs have a more restricted substrate range. Therefore, targeting ADAM17 or ADAM10 by pharmacologic inhibition or gene knockout not only attenuates the inflammatory response in animal models but also affects tissue regeneration and neovascularisation. Recent discoveries indicate that other ADAMs (e.g. ADAM8 and 9) also play important roles in vascular biology but appear to have more selective effects on vascular responses (e.g. on neovascularisation only). Although, targeting of ADAM17 and ADAM10 in inflammatory diseases is still a promising approach, temporal and spatial as well as substrate-specific inhibition approaches are required to minimise undesired side effects on vascular cells.     

HDLs activate ADAM17-dependent shedding

The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease that cleaves several transmembrane proteins, including TNF and its receptors (TNFR1 and TNFR2). We recently showed that the shedding activity of ADAM17 is sequestered in lipid rafts and that cholesterol depletion increased the shedding of ADAM17 substrates. These data suggested that ADAM17 activity could be regulated by cholesterol movements in the cell membrane. We investigated if the membrane cholesterol efflux induced by high-density lipoproteins (HDLs) was able to modify the shedding of ADAM17 substrates. HDLs added to different cell types, increased the ectodomain shedding of TNFR2, TNFR1, and TNF, an effect reduced by inhibitors active on ADAM17. The HDLs-stimulated TNF release occurred also on cell-free isolated plasma membranes. Purified apoA1 increased the shedding of TNF in an ABCA1-dependent manner, suggesting a role for the cholesterol efflux in this phenomenon. HDLs reduced the cholesterol and proteins (including ADAM17) content of lipid rafts and triggered the ADAM17-dependent cleavage of TNF in the non-raft region of the membrane. In conclusion, these data demonstrate that HDLs alter the lipid raft structure, which in turn activates the ADAM17-dependent processing of transmembrane substrates.     

Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening

In this paper we describe novel fluorescent substrates for the human ADAM family members ADAM17, ADAM10, ADAM8, and ADAM12 that have good specificity constants and are useful for high-throughput screening of inhibitors. The fluorescence resonance energy transfer substrates contain a 4-(4-dimethylaminophenylazo)benzoyl and 5-carboxyfluorescein (Dabcyl/Fam) pair and are based on known cleavage sequences in precursor tumor necrosis factor-alpha (TNF-alpha) and CD23. The precursor TNF-alpha-based substrate, Dabcyl-Leu-Ala-Gln-Ala-Homophe-Arg-Ser-Lys(Fam)-NH2, is a good substrate for all the ADAMs tested, including ADAM12 for which there is no reported fluorescent substrate. The CD23-based substrate, Dabcyl-His-Gly-Asp-Gln-Met-Ala-Gln-Lys-Ser-Lys(Fam)-NH2, is more selective, being hydrolyzed efficiently only by ADAM8 and ADAM10. The substrates were used to obtain inhibition constants for four inhibitors that are commonly used in shedding assays: TMI-1, GM6001, GW9471, and TAPI-2. The Wyeth Aerst compound, TMI-1, is a potent inhibitor against all of the ADAMs tested and is slow binding against ADAM17.     

Catalytic activity of ADAM8, ADAM15, and MDC-L (ADAM28) on synthetic peptide substrates and in ectodomain cleavage of CD23

The ADAM family of disintegrin metalloproteases plays important roles in "ectodomain shedding," the process by which biologically active, soluble forms of cytokines, growth factors, and their receptors are released from membrane-bound precursors. Whereas ADAM8, ADAM15, and MDC-L (ADAM28) are expressed in specific cell types and tissues, their in vivo functions and substrates are not known. By screening a library of synthetic peptides as potential substrates, we show that soluble recombinant forms of these enzymes have similar proteolytic substrate specificity, clearly distinct from that of ADAM17 (TNFalpha-converting enzyme). A number of tumor necrosis factor (TNF) family proteins and CD23 were screened as potential substrates for ectodomain cleavage. We found that ADAM8, ADAM15, and MDC-L, but not ADAM17, catalyzed ectodomain shedding of CD23, the low affinity IgE receptor. ADAM8-dependent, soluble CD23 release required proteolytically active ADAM8, and a physical association of ADAM8 was observed with the membrane-bound form of CD23. The ADAM8-dependent release of sCD23 and the endogenous release from B cell lines could be similarly inhibited by a hydroxamic acid, metalloprotease inhibitor compound. We conclude that ADAM8 could contribute to ectodomain shedding of CD23 and may thus be a potential target for therapeutic intervention in allergy and inflammation.     

MiR-152 reduces human umbilical vein endothelial cell proliferation and migration by targeting ADAM17

As a cleavage enzyme of precursor TNF-α, the high expression level of ADAM17 in endothelial cells is an important factor in atherosclerosis. In this study, we demonstrate that ADAM17 is the target of miR-152. We found that miR-152 could reduce TNF precursor cleavage and inhibit cell proliferation and migration by targeting ADAM17 in human umbilical vein endothelial cells (HUVECs). Furthermore, the expression pattern of miR-152 and corresponding target ADAM17 was opposite in HUVECs under hypoxic conditions. The levels of circulating miR-152 in AS patient sera were lower than those detected in the sera of normal individuals. Our results indicate that miR-152 may be involved in the development of human atherosclerosis and could be used as diagnostic biomarker or therapeutic target in atherosclerosis.     

ADAM17 orchestrates Interleukin-6, TNFα and EGF-R signaling in inflammation and cancer

It was realized in the 1990s that some membrane proteins such as TNFα, both TNF receptors, ligands of the EGF-R and the Interleukin-6 receptor are proteolytically cleaved and are shed from the cell membrane as soluble proteins. The major responsible protease is a metalloprotease named ADAM17. So far, close to 100 substrates, including cytokines, cytokine receptors, chemokines and adhesion molecules of ADAM17 are known. Therefore, ADAM17 orchestrates many different signaling pathways and is a central signaling hub in inflammation and carcinogenesis. ADAM17 plays an important role in the biology of Interleukin-6 (IL-6) since the generation of the soluble Interleukin-6 receptor (sIL-6R) is needed for trans-signaling, which has been identified as the pro-inflammatory activity of this cytokine. In contrast, Interleukin-6 signaling via the membrane-bound Interleukin-6 receptor is mostly regenerative and protective. Probably due to its broad substrate spectrum, ADAM17 is essential for life and most of the few human individuals identified with ADAM17 gene defects died at young age. Although the potential of ADAM17 as a therapeutic target has been recognized, specific blockade of ADAM17 is not trivial since the metalloprotease domain of ADAM17 shares high structural homology with other proteases, in particular matrix metalloproteases. Here, the critical functions of ADAM17 in IL-6, TNFα and EGF-R pathways and strategies of therapeutic interventions are discussed.     

The good, the bad and the ugly substrates for ADAM10 and ADAM17 in brain pathology, inflammation and cancer

Various surface molecules undergo regulated cleavage by the disintegrin and metalloproteinases ADAM10 and ADAM17. The list of substrates includes molecules involved in brain pathology, inflammation and cancer. In the brain both proteases mediate neuroprotective cleavage events such as inactivation of amyloid precursor protein. In inflammatory settings signaling of cytokines including TNFalpha and IL-6 is triggered by proteolytic release of soluble agonists and leukocyte recruitment is controlled by the cleavage of adhesion molecules. Moreover, in tumors, ADAM10- and ADAM17-mediated shedding events trigger proliferative signaling via activation of growth factors including ErbB family members. Concepts of either increasing ADAM10- or ADAM17-activity to limit neurodegeneration or suppressing their activity to block inflammation or tumor growth have to be carefully scrutinized for their potential side effects in various tissues and pathologies.     

Distinct Intracellular Domain Substrate Modifications Selectively Regulate Ectodomain Cleavage of NRG1 or CD44

Ectodomain cleavage by A-disintegrin and -metalloproteases (ADAMs) releases many important biologically active substrates and is therefore tightly controlled. Part of the regulation occurs on the level of the enzymes and affects their cell surface abundance and catalytic activity. ADAM-dependent proteolysis occurs outside the plasma membrane but is mostly controlled by intracellular signals. However, the intracellular domains (ICDs) of ADAM10 and -17 can be removed without consequences for induced cleavage, and so far it is unclear how intracellular signals address cleavage. We therefore explored whether substrates themselves could be chosen for proteolysis via ICD modification. We report here that CD44 (ADAM10 substrate), a receptor tyrosine kinase (RTK) coreceptor required for cellular migration, and pro-NRG1 (ADAM17 substrate), which releases the epidermal growth factor (EGF) ligand neuregulin required for axonal outgrowth and myelination, are indeed posttranslationally modified at their ICDs. Tetradecanoyl phorbol acetate (TPA)-induced CD44 cleavage requires dephosphorylation of ICD serine 291, while induced neuregulin release depends on the phosphorylation of several NRG1-ICD serines, in part mediated by protein kinase Cδ (PKCδ). Downregulation of PKCδ inhibits neuregulin release and reduces ex vivo neurite outgrowth and myelination of trigeminal ganglion explants. Our results suggest that specific selection among numerous substrates of a given ADAM is determined by ICD modification of the substrate.     

Soluble RAGE but not endogenous secretory RAGE is associated with albuminuria in patients with type 2 diabetes

Aims

Type 2 diabetes is characterised by increased plasma concentrations of pro-inflammatory cytokines [such as tumour necrosis factor – alpha; TNF-α] and soluble forms of adhesion molecules involved in leukocyte – endothelial interactions. These molecules are synthesised as transmembrane proteins and the plasma soluble forms are generated by ectodomain cleavage from the cell surface by members of the ADAM [a disintegrin and metalloproteinase] proteinase family. We hypothesised that plasma low density lipoprotein [LDL] from subjects with Type 2 diabetes would influence in vitro monocytic ADAM and matrix metalloproteinase [MMP] gene expression differently compared to control LDL.

Methods

We examined relative mRNA expression by real time PCR in a monocytic cell line [THP-1] cultured for 4, 8 and 24 hrs with human plasma LDL derived from subjects with [n = 5] or without [n = 4] Type 2 diabetes. Gene expression for MMP-1 and 9, and ADAM – 8, 15, 17 and 28 was studied.

Results

Type 2 diabetes LDL significantly increased gene expression of MMP – 1 [p < 0.01] MMP – 9 [p < 0.001], and ADAM 17 [p < 0.05], – 28 [p < 0.01] and – 15 [p < 0.01] compared to control LDL. Type 2 diabetes LDL had disparate effects on inhibitors of MMP.

Conclusion

These data suggest that Type 2 diabetes LDL could lead to increased adhesion molecule and TNF alpha cell surface shedding, and vascular plaque instability, by promoting increased expression of ADAM and MMP genes.     

Substrate specificity and inducibility of TACE (tumour necrosis factor alpha-converting enzyme) revisited: the Ala-Val preference, and induced intrinsic activity

Tumour necrosis factor alpha (TNF alpha)-converting enzyme (TACE/ADAM-17, where ADAM stands for a disintegrin and metalloproteinase) releases from the cell surface the extracellular domains of TNF and several other proteins. Previous studies have found that, while purified TACE preferentially cleaves peptides representing the processing sites in TNF and transforming growth factor alpha, the cellular enzyme nonetheless also sheds proteins with divergent cleavage sites very efficiently. More recent work, identifying the cleavage site in the p75 TNF receptor, quantifying the susceptibility of additional peptides to cleavage by TACE and identifying additional protein substrates, underlines the complexity of TACE-substrate interactions. In addition to substrate specificity, the mechanism underlying the increased rate of shedding caused by agents that activate cells remains poorly understood. Recent work in this area, utilizing a peptide substrate as a probe for cellular TACE activity, indicates that the intrinsic activity of the enzyme is somehow increased.     

Differential Surface Expression of ADAM10 and ADAM17 on Human T Lymphocytes and Tumor Cells

A disintegrin and metalloproteases (ADAMs) have been implicated in many processes controlling organismic development and integrity. Important substrates of ADAM proteases include growth factors, cytokines and their receptors and adhesion proteins. The inducible but irreversible cleavage of their substrates alters cell-cell communication and signaling. The crucial role of ADAM proteases (e.g. ADAM10 and 17) for mammalian development became evident from respective knockout mice, that displayed pre- or perinatal lethality with severe defects in many organs and tissues. Although many substrates for these two ADAM proteases were identified over the last decade, the regulation of their surface appearance, their enzymatic activity and their substrate specificity are still not well understood. We therefore analyzed the constitutive and inducible surface expression of ADAM10 and ADAM17 on a variety of human T cell and tumor cell lines. We demonstrate that ADAM10 is constitutively present at comparably high levels on the majority of the tested cell types. Stimulation with phorbol ester and calcium ionophore does not significantly alter the amount of surface ADAM10, except for a slight down-regulation from T cell blasts. Using FasL shedding as a readout for ADAM10 activity, we show that PKC activation and calcium mobilization are both prerequisite for activation of ADAM10 resulting in a production of soluble FasL. In contrast to ADAM10, the close relative ADAM17 is detected at only low levels on unstimulated cells. ADAM17 surface expression on T cell blasts is rapidly induced by stimulation. Since this inducible mobilization of ADAM17 is sensitive to inhibitors of actin filament formation, we propose that ADAM17 but not ADAM10 is prestored in a subcellular compartment that is transported to the cell surface in an activation- and actin-dependent manner.     

ADAM15 is an adherens junction molecule whose surface expression can be driven by VE-cadherin

ADAM15 belongs to the family of proteins containing disintegrin and metalloprotease domains (ADAM) that have been implicated in cell adhesion via integrin binding and shedding of cell surface molecules. Here we provide the first report on the localization of an ADAM in adherens junctions. We show that ADAM15 colocalizes with a cell adhesion molecule, vascular endothelial (VE)-cadherin, which mediates endothelial cell adherens junction formation. In contrast, the distribution of ADAM15 correlates poorly with the localization in cell contacts of one of its proposed ligands, the beta1-integrin. Furthermore, ADAM15 accumulation in cell-cell contacts is preceded by VE-cadherin-mediated adherens junction formation. To investigate the dependence of ADAM15 surface expression on adherens junction formation, we coexpressed VE-cadherin with ADAM15 and an ADAM15 green fluorescence protein (GFP) fusion protein in Chinese hamster ovary cells. VE-cadherin coexpression results in the translocation of ADAM15-GFP to the cell periphery. Analysis of cell surface levels of ADAM15 and ADAM15-GFP, with or without VE-cadherin coexpression, clearly demonstrates that VE-cadherin can drive surface expression of ADAM15. Our data suggest that ADAM15 may be a novel component of adherens junctions and thus could play a role in endothelial functions that are mediated by these cell contacts.     

Glycosylation of a disintegrin and metalloprotease 17 affects its activity and inhibition

ADAM17 (a disintegrin and metalloprotease 17) is believed to be a tractable target in various diseases, including cancer and rheumatoid arthritis; however, it is not known whether glycosylation of ADAM17 expressed in healthy cells differs from that found in diseased tissue and, if so, whether glycosylation affects inhibitor binding. We expressed human ADAM17 in mammalian and insect cells and compared their glycosylation, substrate kinetics, and inhibition profiles. We found that ADAM17 expressed in mammalian cells was more heavily glycosylated than its insect-expressed analog. To determine whether differential glycosylation modulates enzymatic activity, we performed kinetic studies with both ADAM17 analogs and various TNFα-based substrates. The mammalian form of ADAM17 exhibited 10- to 30-fold lower k_cat values than the insect analog, while the K_M was unaffected, suggesting that glycosylation of ADAM17 can potentially play a role in regulating enzyme activity in vivo. Finally, we tested ADAM17 forms for inhibition by several well-characterized inhibitors. Active-site zinc-binding small molecules did not exhibit differences between the two ADAM17 analogs, while a non-zinc-binding exosite inhibitor of ADAM17 showed significantly lower potency toward the mammalian-expressed analog. These results suggest that glycosylation of ADAM17 can affect cell signaling in disease and might provide opportunities for therapeutic intervention using exosite inhibitors.     

TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions: EVIDENCE FOR DISTINCT BINDING MECHANISMS FOR DIFFERENT TspanC8 PROTEINS*

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunoprecipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipitation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunoprecipitation with Tspan14 and other TspanC8s. This TspanC8-interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reducing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conformations in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study identifies regions of TspanC8s and ADAM10 for potential interaction-disrupting therapeutic targeting.     

Different signaling pathways stimulate a disintegrin and metalloprotease-17 (ADAM17) in neutrophils during apoptosis and activation

ADAM17 is a membrane-associated metalloprotease that cleaves proteins from the surface of neutrophils and modulates the density of various receptors and adhesion molecules. The protease activity of ADAM17 is highly inducible and occurs upon neutrophil activation as well as apoptosis. At this time, little is known about the signal transduction pathway that promotes ADAM17 activity in neutrophils upon the induction of apoptosis. We show that caspase-8 activation, Bid cleavage, and the release of mitochondrial reactive oxygen species are sequential transduction components of the Fas signaling cascade that induces ADAM17. This is different from ADAM17 stimulation upon overt neutrophil activation, which requires MAPK p38 or ERK, but not caspases and reactive oxygen species. ADAM17 activity in apoptotic neutrophils may serve to inactivate select effector molecules that promote the pro-inflammatory activity of recruited neutrophils. For instance, TNFα receptors TNF-RI and TNF-RII are substrates of ADAM17, and we show that they are shed during apoptosis, decreasing neutrophil sensitivity to TNFα. Altogether, our findings provide significant new insights into the signal transduction pathway that stimulates ADAM17 during induced neutrophil apoptosis. ADAM17 induction during apoptosis may rapidly diminish neutrophil sensitivity to the inflammatory environment, complementing other anti-inflammatory activities by these cells during inflammation resolution.     

Short term exposure to cis unsaturated free fatty acids inhibits degranulation of cytotoxic T lymphocytes

Degranulation of CTL stimulated by alloantigen-bearing target cells is shown to be inhibited by short term exposure to low concentrations of long chain cis unsaturated free fatty acids (FFA), whereas saturated FFA have no effect. The Ag-specific (TCR mediated) stimulation of cloned murine CTL was monitored by changes in intracellular calcium concentrations [( Ca2+]i) using the fluorescence indicator acetoxymethylester of fura-2 and by degranulation as measured by the release of BLT-esterase. Treatment of the CTL cells with any of the physiologically important FFA; oleic (18:1), linoleic (18:2), linolenic (18:3), or arachidonic (20:4) acid, at concentrations between 1 and 10 microM inhibits the target cell-mediated rise in [Ca2+]i which occurs within seconds of stimulation and the release of BLT-esterase, which occurs over a period of 1 to 3 h. These inhibitory effects are observed within seconds to minutes of FFA addition. Inhibition can be reversed by treating cells with fatty acid free BSA and, in agreement with our previous studies, indicates that the effects of FFA are due to physical perturbations of cellular components. To determine the locus of this perturbation, the effect of FFA on the lipid order of CTL plasma membrane was determined using fluorescence polarization of the membrane impermeable probe trimethylammoniumdiphenylhexatriene. Cis unsaturated FFA were found to disorder the lipid acyl chains and the degree of disorder was found to increase with the degree of cis unsaturation. These results, together with the previous studies, suggest that inhibition results from a physical perturbation of plasma membrane lipid order. Moreover, because degranulation requires elevated levels of [Ca2+]i, it is likely that inhibition of degranulation results from a FFA-induced decrease in Ca2+ permeability through the membrane.     

The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

The Coxsackievirus and Adenovirus Receptor (CAR) is a cell adhesion molecule originally characterized as a virus receptor but subsequently shown to be involved in physiological processes such as neuronal and heart development, epithelial tight junction integrity, and tumour suppression. Proteolysis of cell adhesion molecules and a wide variety of other cell surface proteins serves as a mechanism for protein turnover and, in some cases, cell signaling. Metalloproteases such as A Disintegrin and Metalloprotease (ADAM) family members cleave cell surface receptors to release their substrates’ ectodomains, while the presenilin/ɣ-secretase complex mediates regulated intramembrane proteolysis (RIP), releasing intracellular domain fragments from the plasma membrane. In the case of some substrates such as Notch and amyloid precursor protein (APP), the released intracellular domains enter the nucleus to modulate gene expression. We report that CAR ectodomain is constitutively shed from glioma cells and developing neurons, and is also shed when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. We identified ADAM10 as a sheddase of CAR using assays involving shRNA knockdown and rescue, overexpression of wild-type ADAM10 and inhibition of ADAM10 activity by addition of its prodomain. In vitro peptide cleavage, mass spectrometry and mutagenesis revealed the amino acids M224 to L227 of CAR as the site of ADAM10-mediated ectodomain cleavage. CAR also undergoes RIP by the presenilin/γ-secretase complex, and the intracellular domain of CAR enters the nucleus. Ectodomain shedding is a prerequisite for RIP of CAR. Thus, CAR belongs to the increasing list of cell surface molecules that undergo ectodomain shedding and that are substrates for ɣ-secretase-mediated RIP.     

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.     

The role of A Disintegrin and Metalloproteinase (ADAM)-10 in T helper cell biology

A Disintegrin and Metalloproteinases (ADAM)-10 is a member of a family of membrane-anchored proteinases that regulate a broad range of cellular functions with central roles within the immune system. This has spurred the interest to modulate ADAM activity therapeutically in immunological diseases. CD4 T helper (Th) cells are the key regulators of adaptive immune responses. Their development and function is strongly dependent on Notch, a key ADAM-10 substrate. However, Th cells rely on a variety of additional ADAM-10 substrates regulating their functional activity at multiple levels. The complexity of both, the ADAM substrate expression as well as the functional consequences of ADAM-mediated cleavage of the various substrates complicates the analysis of cell type specific effects.Here we provide an overview on the major ADAM-10 substrates relevant for CD4 T cell biology and discuss the potential effects of ADAM-mediated cleavage exemplified for a selection of important substrates.