TMEFF2 shedding is regulated by oxidative stress and mediated by ADAMs and transmembrane serine proteases implicated in prostate cancer

TMEFF2 is a type I transmembrane protein with two follistatin (FS) and one EGF‐like domain over‐expressed in prostate cancer; however its biological role in prostate cancer development and progression remains unclear, which may, at least in part, be explained by its proteolytic processing. The extracellular part of TMEFF2 (TMEFF2‐ECD) is cleaved by ADAM17 and the membrane‐retained fragment is further processed by the gamma‐secretase complex. TMEFF2 shedding is increased with cell crowding, a condition associated with the tumour microenvironment, which was mediated by oxidative stress signalling, requiring jun‐kinase (JNK) activation. Moreover, we have identified that TMEFF2 is also a novel substrate for other proteases implicated in prostate cancer, including two ADAMs (ADAM9 and ADAM12) and the type II transmembrane serine proteinases (TTSPs) matriptase‐1 and hepsin. Whereas cleavage by ADAM9 and ADAM12 generates previously identified TMEFF2‐ECD, proteolytic processing by matriptase‐1 and hepsin produced TMEFF2 fragments, composed of TMEFF2‐ECD or FS and/or EGF‐like domains as well as novel membrane retained fragments. Differential TMEFF2 processing from a single transmembrane protein may be a general mechanism to modulate transmembrane protein levels and domains, dependent on the repertoire of ADAMs or TTSPs expressed by the target cell.     

Phosphorylation-dependent interactions between ADAM15 cytoplasmic domain and Src family protein-tyrosine kinases.

The adamalysins (ADAMs) are transmembrane glycoproteins involved in cell adhesion and proteolytic ectodomain processing of cytokines and adhesion molecules. Many ADAM cytoplasmic domains are proline-rich and have potential phosphorylation sites. We show here that the cytoplasmic domain of ADAM15, metargidin, can interact specifically with Src family protein-tyrosine kinases (PTKs) and the adaptor protein Grb2 in hematopoietic cells (Jurkat, THP-1, U937, and K562 cell lines). Src homology 3 domains from several Src family PTKs including Lck, Fyn, Abl, and Src associate with ADAM15 in vitro. Dephosphorylation of cell extracts resulted in decreased association of ADAM15 with Src family PTK SH3 domains, indicating that phosphorylation influences ADAM15 interactions with its binding partners. This was confirmed in vitro for Hck, Lck, and Grb2, which showed enhanced association with tyrosine-phosphorylated glutathione S-transferase-ADAM15 cytoplasmic domain compared with unphosphorylated protein. In contrast, binding of MAD2 to ADAM15 was slightly reduced by phosphorylation of the ADAM. Immunoprecipitation of ADAM15 from Jurkat cells confirmed the association with Lck in vivo, and upon PMA stimulation, the phosphorylation level of ADAM15 was increased. Cotransfection of ADAM15 and Hck showed Hck-dependent phosphorylation of ADAM15 in vivo. Hck, and to a lesser extent Lck, phosphorylated the ADAM15 cytoplasmic domain in vitro in immune complex kinase assays. Binding of ADAM15 cytoplasmic domain to Hck and Lck was also shown by Far Western analysis. In contrast to Hck, Lck activity was not required for binding to ADAM15, as shown by treatment of cells with PP1. Deletion and point mutation analysis of the ADAM15 cytoplasmic domain confirmed the importance of the proline-rich motifs for Grb2 and Lck binding and indicated the regulatory nature of Tyr(715) and Tyr(735). These data demonstrate selective, phosphorylation-dependent interactions of ADAM15 with Src family PTKs and Grb2, which highlight the potential for integration of ADAM functions and cellular signaling.     

The α-helical content of the transmembrane domain of the British dementia protein-2 (Bri2) determines its processing by signal peptide peptidase-like 2b (SPPL2b)

Regulated intramembrane proteolysis is a widely accepted concept describing the processing of various transmembrane proteins via ectodomain shedding followed by an intramembrane cleavage. The resulting cleavage products can be involved in reverse signaling. Presenilins, which constitute the active center of the γ-secretase complex, signal peptide peptidase (SPP), and its homologues, the SPP-like (SPPL) proteases are members of the family of intramembrane-cleaving aspartyl proteases of the GXGD-type. We recently demonstrated that Bri2 (itm2b) is a substrate for regulated intramembrane proteolysis by SPPL2a and SPPL2b. Intramembrane cleavage of Bri2 is triggered by an initial shedding event catalyzed by A Disintegrin and Metalloprotease 10 (ADAM10). Additionally primary sequence determinants within the intracellular domain, the transmembrane domain and the luminal juxtamembrane domain are required for efficient cleavage of Bri2 by SPPL2b. Using mutagenesis and circular dichroism spectroscopy we now demonstrate that a high α-helical content of the Bri2 transmembrane domain (TMD) reduces cleavage efficiency of Bri2 by SPPL2b, while the presence of a GXXXG dimerization motif influences the intramembrane cleavage only to a minor extent. Surprisingly, only one of the four conserved intramembrane glycine residues significantly affects the secondary structure of the Bri2 TMD and thereby its intramembrane cleavage. Other glycine residues do not influence the α-helical content of the transmembrane domain nor its intramembrane processing.     

Evidence for disulfide involvement in the regulation of intramolecular autolytic processing by human adamalysin19/ADAM19

Human adamalysin 19 (a disintegrin and metalloproteinase 19, hADAM19) is activated by furin-mediated cleavage of the prodomain followed by an autolytic processing within the cysteine-rich domain at Glu586-Ser587, which occurs intramolecularly, producing an NH2 terminal fragment (N-fragment) associated with its COOH-terminal fragment (C-fragment), most likely through disulfide bonds. When stable Madin-Darby canine kidney (MDCK) transfectants overexpressing soluble hADAM19 were treated with dithiothreitol (DTT) or with media at pH 6.5, 7.5, or 8.5, the secretion and folding of the enzyme were not affected. Autolytic processing was blocked by DTT and pH 6.5 media, which favor disulfide reduction, but was increased by pH 8.5 media, which promotes disulfide formation. Cys605, Cys633, Cys639, and Cys643 of the C-fragment appear to be partially responsible for the covalent association between the C-fragment and the N-fragment. A new autolytic processing site at Lys543-Val544 was identified in soluble mutants when these cysteine residues were individually mutated to serine residues. Shed fragments were also detectable in the media from MDCK cells stably expressing the full-length Cys633Ser mutant. Ilomastat/GM6001 inhibited hADAM19 with an IC50 of 447 nM, but scarcely affected the shedding process. The cysteine-rich domain likely forms disulfide bonds to regulate the autolytic processing and shedding of hADAM19.     

Sequential processing of the transmembrane chemokines CX3CL1 and CXCL16 by alpha- and gamma-secretases

The chemokines CX3CL1/Fractalkine and CXCL16 are expressed as transmembrane molecules and can mediate cell-cell-adhesion. By proteolytic processing, CX3CL1 and CXCL16 are released from the cell surface by proteolytic shedding resulting in the generation of soluble chemoattractants. This ectodomain release is mediated by the alpha-secretase-like activity of the two disintegrins and metalloproteinases ADAM10 and ADAM17. Using CX3CL1 and CXCL16 constructs C-terminally fused to two Z-domains of Protein A (2Z-tag) we detect C-terminal fragments (CTFs) of both chemokines resulting from ADAM10-mediated cleavages at multiple sites as examined by inhibitor studies. Furthermore, inhibitor studies as well as genetic studies using presenilin 1/2-deficient cell lines suggest the involvement of gamma-secretase-but not beta-secretase-like activity in the processing of transmembrane chemokines. The combination of alpha- and gamma-secretase and proteasomal inhibitors points towards a sequential processing of transmembrane chemokines by first ADAM10 and then gamma-secretases and possible further degradation. This proteolytic processing cascade of transmembrane chemokines is similar to that described for Notch and E-cadherin where CTFs generated by gamma-secretase serve as intracellular signal transmitters.     

Processing of the human protocadherin Fat1 and translocation of its cytoplasmic domain to the nucleus

The giant protein hFat1, a member of the cadherin superfamily, has been proposed to play roles in cerebral development, glomerular slit formation, and also to act as a tumor suppressor, but its mechanisms of action have not been elucidated. To examine functions of the transmembrane and cytoplasmic domains, they were expressed in HEK293 and HeLa cells as chimeric proteins in fusion with EGFP and extracellular domains derived from E-cadherin. Proteins comprising the transmembrane domain localized to the membrane fraction. Deletion of this domain resulted in a predominantly nuclear localization of the cytoplasmic segment of hFat1. Nuclear localization was largely reduced by deletion of a presumed juxta-membrane NLS. Fusion proteins located in the plasma membrane underwent proteolytic processing. In a first proteolytic step, only the extracellular domain was cleaved off. In another step, the cleavage product was released to the cytosol and was also found in a low speed pellet fraction, in accordance with the nuclear localization of the cytoplasmic domain of hFat1.     

Roles of Meltrin beta /ADAM19 in the processing of neuregulin

Meltrin beta/ADAM19 is a member of ADAMs (a disintegrin and metalloproteases), which are a family of membrane-anchored glycoproteins that play important roles in fertilization, myoblast fusion, neurogenesis, and proteolytic processing of several membrane-anchored proteins. The expression pattern of meltrin beta during mouse development coincided well with that of neuregulin-1 (NRG), a member of the epidermal growth factor family. Then we examined whether meltrin beta participates in the proteolytic processing of membrane-anchored NRGs. When NRG-beta1 was expressed in mouse L929 cells, its extracellular domain was constitutively processed and released into the culture medium. This basal processing activity was remarkably potentiated by overexpression of wild-type meltrin beta, which lead to the significant decrease in the cell surface exposure of extracellular domains of NRG-beta1. Furthermore, expression of protease-deficient mutants of meltrin beta exerted dominant negative effects on the basal processing of NRG-beta1. These results indicate that meltrin beta participates in the processing of NRG-beta1. Since meltrin beta affected the processing of NRG-beta4 but not that of NRG-alpha2, meltrin beta was considered to have a preference for beta-type NRGs as substrate. Furthermore, the effects of the secretory pathway inhibitors suggested that meltrin beta participates in the intracellular processing of NRGs rather than the cleavage on the cell surface.     

Regulation of human ADAM 12 protease by the prodomain. Evidence for a functional cysteine switch

The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins that are believed to play key roles in cell-cell and cell-matrix interactions. We have shown recently that human ADAM 12-S (meltrin alpha) is an active metalloprotease. It is synthesized as a zymogen, with the prodomain maintaining the protease in a latent form. We now provide evidence that the latency mechanism of ADAM 12 can be explained by the cysteine switch model, in which coordination of Zn2+ in the active site of the catalytic domain by a cysteine residue in the prodomain is critical for inhibition of the protease. Replacing Cys179 with other amino acids results in an ADAM 12 proform that is proteolytically active, but latency can be restored by placing cysteine at other positions in the propeptide. None of the amino acids adjacent to the crucial cysteine residue is essential for blocking activity of the protease domain. In addition to its latency function, the prodomain is required for exit of ADAM 12 protease from the endoplasmic reticulum. Tissue inhibitor of metalloprotease-1, -2, and -3 were not found to block proteolytic activity of ADAM 12, hence a physiological inhibitor of ADAM 12 protease in the extracellular environment remains to be identified.     

Multimerisation of A disintegrin and metalloprotease protein-17 (ADAM17) is mediated by its EGF-like domain

A disintegrin and metalloprotease protein 17 (ADAM17) is a transmembrane zinc dependent metalloprotease. The catalytic activity of the enzyme results in the shedding of a broad range of membrane proteins. The release of the corresponding ectodomains induces a switch in various physiological and pathophysiological processes. So far there is not much information about the molecular mechanism of ADAM17 activation available. As for other transmembrane proteases, multimerisation may play a critical role in the activation and function of ADAM17. The present work demonstrates that ADAM17 indeed exists as a multimer in the cell membrane and that this multimerisation is mediated by its EGF-like domain.     

Cell‐surface Metalloprotease ADAM12 is Internalized by a Clathrin‐ and Grb2‐dependent Mechanism

ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell‐adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell‐surface are possibly crucial in these contexts. We here investigated internalization and subsequent recycling or degradation of ADAM12 as a potentially important regulatory mechanism. Our results show that ADAM12 is constitutively internalized primarily via the clathrin‐dependent pathway and is subsequently detected in both early and recycling endosomes. The protease activity of ADAM12 does not influence this internalization mechanism. Analysis of essential elements for internalization established that proline‐rich regions in the cytoplasmic domain of ADAM12, previously shown to interact with Src‐homology 3 domains, were necessary for proper internalization. These sites in the ADAM12 cytoplasmic domain interacted with the adaptor protein growth factor receptor‐bound protein 2 (Grb2) and knockdown of Grb2 markedly reduced ADAM12 internalization. These studies establish that internalization is indeed a mechanism that regulates ADAM cell surface levels and show that ADAM12 internalization involves the clathrin‐dependent pathway and Grb2 .     

C-terminal processing of the toxoplasma protein MIC2 is essential for invasion into host cells

Host cell invasion by apicomplexan parasites is accompanied by the rapid, polarized secretion of parasite proteins that are involved in cell attachment. The Toxoplasma gondii micronemal protein MIC2 contains several extracellular adhesive domains, a transmembrane domain, and a short cytoplasmic tail. Following apical secretion, MIC2 is transiently present on the parasite surface before being translocated backward and released by proteolytic cleavage. Mutations in the extracellular domain of MIC2, directly upstream of the transmembrane domain, prevented processing and release of the soluble protein into the supernatant. A conserved basic residue in MIC2 was essential for cleavage, and basic residues are similarly positioned in other microneme proteins. Following the induction of secretion, MIC2 processing mutants were stably expressed on the surface of the parasite. Surface MIC2-expressing mutants showed increased adhesion to host cells, yet were impaired in their capacity to invade. These data demonstrate that proteolysis is essential for releasing cell surface adhesins prior to cell entry by apicomplexan parasites.     

Identification of SH3 Domain Proteins Interacting with the Cytoplasmic Tail of the A Disintegrin and Metalloprotease 10 (ADAM10)

The a disintegrin and metalloproteases (ADAMs) play a pivotal role in the control of development, adhesion, migration, inflammation and cancer. Although numerous substrates of ADAM10 have been identified, the regulation of its surface expression and proteolytic activity is still poorly defined. One current hypothesis is that both processes are in part modulated by protein-protein interactions mediated by the intracellular portion of the protease. For related proteases, especially proline-rich regions serving as docking sites for Src homology domain 3 (SH3) domain-containing proteins proved to be important for mediating regulatory interactions. In order to identify ADAM10-binding SH3 domain proteins, we screened the All SH3 Domain Phager library comprising 305 human SH3 domains using a GST fusion protein with the intracellular region of human ADAM10 as a bait for selection. Of a total of 291 analyzed phage clones, we found 38 SH3 domains that were precipitated with the ADAM10-derived fusion protein but not with GST. We verified the binding to the cytosolic portion of ADAM10 for several candidates by co-immunoprecipitation and/or pull down analyses. Intriguingly, several of the identified proteins have been implicated in regulating surface appearance and/or proteolytic activity of related ADAMs. Thus, it seems likely that they also play a role in ADAM10 biology.     

Proteolytic processing of the protein tyrosine phosphatase α extracellular domain is mediated by ADAM17/TACE

The receptor protein tyrosine phosphatase alpha (PTPα) is involved in the regulation of tyrosine kinases like the Src kinase and the insulin receptor. As with other PTPs, its function is determined by alternative splicing, dimerisation, phosphorylation and proteolytical processing. PTPα is cleaved by calpain in its intracellular domain, which decreases its potential to dephosphorylate Src kinase. Here, we demonstrate that PTPα is also processed in the extracellular domain. Extracellular processing was exclusively found for a splice variant containing an extra nine amino acid insert three residues amino-terminal from the transmembrane domain. Processing was sensitive to the metalloprotease-inhibitor Batimastat, and CHO-M2 cells lacking a disintegrin and metalloproteinase 17 (ADAM17; tumor-necrosis-factor α converting enzyme) activity were not able to cleave PTPα. After transient overexpression of ADAM17 and PTPα in these cells, processing was restored, proving that ADAM17 is involved in this process. Further characterization of the consequences of processing revealed that dephosphorylation of the insulin receptor or activation of Src was not affected but focus formation was reduced. We conclude that extracellular proteolytic processing is a novel mechanism for PTPα regulation.     

Requirements for presenilin-dependent cleavage of notch and other transmembrane proteins

Ligand binding to receptors of the LIN-12/Notch family causes at least two proteolytic cleavages: one between the extracellular and transmembrane domains, and the other within the transmembrane domain. The transmembrane cleavage depends on Presenilin, a protein also required for transmembrane cleavage of beta-APP. Here, we have assayed the substrate requirements for Presenilin-dependent processing of Notch and other type I transmembrane proteins in vivo. We find that the Presenilin-dependent cleavage does not depend critically on the recognition of particular sequences in these proteins but rather on the size of the extracellular domain: the smaller the size, the greater the efficiency of cleavage. Hence, Notch, beta-APP, and perhaps other proteins may be targeted for Presenilin-mediated transmembrane cleavage by upstream processing events that sever the extracellular domain from the rest of the protein.     

The metalloprotease disintegrin ADAM8. Processing by autocatalysis is required for proteolytic activity and cell adhesion

ADAMs (a disintegrin and metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu(330) to Gln exchange (EQ-A8) in the Zn(2+) binding motif (HE(330)LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substrate-bound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC(50) approximately 50 nm) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion.     

The ADAM-integrin-tetraspanin complex in fetal and postnatal testicular cords

New insights have emerged about the expression, during testicular cord formation, of the ADAM (a disintegrin and metalloprotease) domain family of proteins that combines both cell surface adhesion and proteolytic activity; this family includes integrins alpha3beta1 and alpha6beta1 and tetraspanins, a distinct family of proteins containing four transmembrane domains, a small and a large extracellular loop, and short cytoplasmic tails. ADAM3 (cyritestin), ADAM5, ADAM6, and ADAM15 are expressed in fetal rat testes. In contrast, the expression of the ADAM1/ADAM2 pair (fertilin alpha/fertilin beta, respectively) is not detected in fetal testis. Yet the expression of ADAM1 starts immediately after birth, and is followed within 24 hr by the expression of ADAM2. Therefore, the ADAM1/ADAM2 heterodimer is visualized far in advance of the meiotic and spermiogenic phase of spermatogenesis. A similar expression pattern was observed for integrin subunits alpha3, alpha6, and beta1, as well as for tetraspanins CD9, CD81, and CD98; the latter is a single-pass integrin subunit beta1-binding protein. ADAM2, integrin subunits alpha3, alpha6, and beta1, and tetraspanin CD9 and CD81 immunoreactive sites are observed in prespermatogonia (also known as primordial germ cells or gonocytes). A model is proposed in which the ADAM-integrin-tetraspanin complex, known to constitute a network of membrane microdomains called the tetraspanin web, may be involved in the migration of prespermatogonia from the center to the periphery of the testicular cords and in the reinitiation of mitotic activity during the initial wave of spermatogenesis. A complementary model consists in the rearrangement of the tetraspanin web in prespermatogonia/spermatogonia undergoing spontaneous or Fas-induced apoptosis upon coculturing with Sertoli cells. In this model, the cellular site involved in the formation of preapoptotic bodies is devoid of tetraspanin-integrin clusters, in contrast with nonapoptotic cells, which display a diffuse circumferential distribution. In apoptotic prespermatogonia, immunoreactive clusters are restricted to sites where the attachment of prespermatogonia/spermatogonia to Sertoli cell surfaces is still preserved.     

Cysteine array matrix metalloproteinase (CA-MMP)/MMP-23 is a type II transmembrane matrix metalloproteinase regulated by a single cleavage for both secretion and activation

Matrix metalloproteinases characterized so far are either secreted or membrane anchored via a type I transmembrane domain or a glycosylphosphatidylinositol linkage. Lacking either membrane-anchoring mechanism, the newly discovered CA-MMP/MMP-23 was reported to be expressed as a cell-associated protein. In this report, we present evidence that CA-MMP is expressed as an integral membrane zymogen with an N-terminal signal anchor, and secreted as a fully processed mature enzyme. We further demonstrate that L(20)GAALSGLCLLSALALL(36) is required for this unique membrane localization as a signal anchor and its secretion is regulated by a proprotein convertase motif RRRR(79) sandwiched between its pro- and catalytic domains. Thus, CA-MMP is a type II transmembrane MMP that can be regulated by a single proteolytic cleavage for both activation and secretion, establishing a novel paradigm for protein trafficking and processing within the secretory pathway.     

The transmembrane homotrimer of ADAM 1 in model lipid bilayers

Fertilin is a transmembrane protein heterodimer formed by the two subunits fertilin alpha and fertilin beta that plays an important role in sperm-egg fusion. Fertilin alpha and beta are members of the ADAM family, and contain each one transmembrane alpha-helix, and are termed ADAM 1 and ADAM 2, respectively. ADAM 1 is the subunit that contains a putative fusion peptide, and we have explored the possibility that the transmembrane alpha-helical domain of ADAM 1 forms homotrimers, in common with other viral fusion proteins. Although this peptide was found to form various homooligomers in SDS, the infrared dichroic data obtained with the isotopically labeled peptide at specific positions is consistent with the presence of only one species in DMPC or POPC lipid bilayers. Comparison of the experimental orientational data with molecular dynamics simulations performed with sequence homologues of ADAM 1 show that the species present in lipid bilayers is only consistent with an evolutionarily conserved homotrimeric model for which we provide a backbone structure. These results support a model where ADAM 1 forms homotrimers as a step to create a fusion active intermediate.     

Notch-induced proteolysis and nuclear localization of the Delta ligand

The Delta protein is a single-pass transmembrane ligand for the Notch family of receptors. Delta binding to Notch invokes regulated intramembrane proteolysis and nuclear translocation of the Notch intracellular domain. Delta is proteolytically processed at two sites, Ala(581) and Ala(593) in the juxtamembrane and transmembrane domains, respectively (Mishra-Gorur, K., Rand, M. D., Perez-Villamil, B., and Artavanis-Tsakonas, S. (2002) J. Cell Biol. 159, 313-324). Controversy over the role of Delta processing in propagating Notch signals has stemmed from conflicting reports on the activity or inactivity of soluble extracellular domain products of Delta. We have examined Delta proteolysis in greater detail and report that Delta undergoes three proteolytic cleavages in the region of the juxtamembrane and transmembrane domains. Only one of these cleavages, analogous to cleavage at Ala(581), is dependent on the Kuzbanian ADAM metalloprotease. The two additional cleavages correspond to the previously described cleavage at Ala(593) and a novel unidentified site within or close to the transmembrane domain. Delta processing is up-regulated in co-cultures with Notch-expressing cells and is similarly induced by p-aminophenylmercuric acetate, a well documented activator of metalloproteases. Furthermore, expression of a truncated intracellular isoform of Delta shows prominent nuclear localization. Altogether, these data demonstrate a role for Notch in inducing Delta proteolysis and implicate a nuclear function for Delta, consistent with a model of bi-directional signaling through Notch-Delta interactions.     

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.