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1.
A disintegrin and metalloprotease 12 (ADAM12/meltrin alpha) is a key enzyme implicated in the ectodomain shedding of membrane-anchored heparin-binding epidermal growth factor (EGF)-like growth factor (proHB-EGF)-dependent epidermal growth factor receptor (EGFR) transactivation. However, the activation mechanisms of ADAM12 are obscure. To determine how ADAM12 is activated, we screened proteins that bind to the cytoplasmic domain of ADAM12 using a yeast two-hybrid system and identified a protein called PACSIN3 that contains a Src homology 3 domain. An analysis of interactions between ADAM12 and PACSIN3 using glutathione S-transferase fusion protein revealed that a proline-rich region (amino acid residues 829-840) of ADAM12 was required to bind PACSIN3. Furthermore, co-immunoprecipitation and co-localization analyses of ADAM12 and PACSIN3 proteins also revealed their interaction in mammalian cells expressing both of them. The overexpression of PACSIN3 in HT1080 cells enhanced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced proHB-EGF shedding. Furthermore, knockdown of endogenous PACSIN3 by small interfering RNA in HT1080 cells significantly attenuated the shedding of proHB-EGF induced by TPA and angiotensin II. Our data indicate that PACSIN3 has a novel function as an up-regulator in the signaling of proHB-EGF shedding induced by TPA and angiotensin II.  相似文献   

2.
Meltrin beta (ADAM19) is a metalloprotease-disintegrin expressed in the peripheral nervous system and other organs during embryogenesis. We report here an alternatively spliced isoform, meltrin beta mini, that lacks the prodomain, metalloprotease and disintegrin domains. A comparison of the cDNA and genomic sequences suggested the existence of a new exon. This isoform was detected in murine dorsal root ganglion and neuronal cell lines by RT-PCR. Overexpression of meltrin beta mini but not meltrin beta induced neurite outgrowth in neuronal cells. These studies suggest that the novel meltrin beta isoform has a distinct function related to neurogenesis.  相似文献   

3.
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.  相似文献   

4.
ADAM12 has been implicated in cell-cell interactions in myogenesis and cancer, but the structure of the mature form of ADAM12 is not known, and its localization on the cell surface has been questioned. In this report, we show that full-length ADAM12 is N-glycosylated in the endoplasmic reticulum (ER) and proteolytically processed in the trans-Golgi network to an approximately 90-kDa form. The approximately 90-kDa form, which lacks the prodomain, was the predominant form present at the cell surface. Replacement of Leu(73) in the putative alpha-helical region in the prodomain with proline resulted in retention of ADAM12 in the ER and a complete lack of its processing. However, deletion of the entire pro- and metalloprotease domains did not affect the processing and trafficking of ADAM12. In contrast, replacement of the cytoplasmic domain of ADAM12 with that of ADAM9 or adding a c-Myc tag at the C terminus led to a significant increase in transport of the protein to the cell surface. These results suggest that the cytoplasmic domain of ADAM12 plays an important role in regulating ADAM12 exit from the ER. We conclude that properly folded mouse ADAM12, after passing a rate-limiting step of exit from the ER, is processed in the secretory pathway and reaches the cell surface, where it can mediate adhesion-mediated signaling.  相似文献   

5.
Roles of Meltrin beta /ADAM19 in the processing of neuregulin   总被引:7,自引:0,他引:7  
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.  相似文献   

6.
The ADAMs (a disintegrin and metalloprotease) comprise a family of multidomain proteins with metalloprotease, cell adhesion, and signaling activities. Human ADAM12, which is implicated in diseases such as cancer, is expressed in two splice forms, the transmembrane ADAM12-L and the shorter and soluble ADAM12-S. ADAM12 is synthesized as a zymogen with the prodomain keeping the metalloprotease inactive through a cysteine-switch mechanism. Maturation and activation of the protease involves the cleavage of the prodomain in the trans-Golgi or possibly at the cell surface by a furin-peptidase. The aim of the present study was to determine the fate of the prodomain following furin cleavage. Here we demonstrate that, following cleavage of the human ADAM12-S prodomain in the trans-Golgi by a furin-peptidase, the prodomain remains non-covalently associated with the mature molecule. Accordingly, both the 68-kDa mature form of ADAM12-S and the 25-kDa prodomain could be detected using domain-specific antisera in immunoprecipitation and Western blot analyses of human serum ADAM12 and purified recombinant human ADAM12. Using electron microscopy after negative staining we have furthermore obtained the first visualization of a full-length ADAM molecule, human ADAM12-S, and report that it appears to be a compact clover composed of four globular domains, one of which is the prodomain. Finally, our data demonstrate that the presence of the metalloprotease domain appears to be sufficient for the prodomain to remain associated with the mature ADAM12-S. Thus, we conclude that the prodomain of human ADAM12-S is an integral domain of the mature molecule and as such might have specific biological functions in the extracellular space.  相似文献   

7.
Sperm–egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell–cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm–egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (α and β subunits). Fertilin α and β are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin β functions in sperm–egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin α, fertilin β, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin β, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm–egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80–90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin β active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin β also strongly inhibited (80–90%) both sperm–egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin β showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin β in sperm–egg plasma membrane adhesion leading to fusion.  相似文献   

8.
Fertilin alpha (also known as ADAM1) is a member of the ADAM (A disintegrin and A metalloprotease domain) family of proteins. In this study, we examine the mechanism of mouse fertilin alpha's in adhesion of sperm to the egg plasma membrane during fertilization. We find that recombinant forms of fertilin alpha corresponding to either the disintegrin-like domain or the cysteine-rich domain and the EGF-like repeat can perturb sperm-egg binding, suggesting that both of these domains can participate in fertilin alpha-mediated adhesion events. In further examination of the fertilin alpha disintegrin-like domain, we find that a subdomain of disintegrin-like domain with the sequence DLEECDCG outside the putative disintegrin loop but with homology to the fertilin beta disintegrin loop can inhibit the binding of both sperm and recombinant fertilin alpha to eggs, suggesting that this is an adhesion-mediating motif of the fertilin alpha disintegrin-like domain. This sequence also inhibits the binding of recombinant fertilin beta to eggs and thus is the first peptide sequence found to block two different sperm ligands. Finally, a monoclonal antibody to the tetraspanin protein CD9, KMC.8, inhibited the binding of recombinant fertilin alpha to eggs in one type of binding assay, suggesting that, under certain conditions, fertilin alpha may interact with a KMC.8-sensitive binding site on the egg plasma membrane.  相似文献   

9.
Fertilin is reported to be a heterodimeric protein composed of A Disintegrin And Metalloprotease 1 (ADAM1, fertilin alpha) and ADAM2 (fertilin beta) located on the sperm surface. In the process of clarifying the molecular basis of mouse ADAM1, we have identified two intron-less mouse genes encoding different isoforms of ADAM1, termed ADAM1a and ADAM1b. The amino acid sequences of ADAM1a and ADAM1b deduced from the DNA sequences were homologous to each other (99% identity) in the pro- and metalloprotease domains, whereas the C-terminal half region of ADAM1a, including the disintegrin and Cys-rich domains, shared only a low degree of identity (37%) with that of ADAM1b. These two genes were both localized on mouse chromosome 5 as a single copy gene, and were expressed specifically in the testis. These data demonstrate the presence of the ADAM1a (Adam1a) and ADAM1b (Adam1b) genes in mouse, instead of the ADAM1 gene, and may imply different roles of ADAM1a and ADAM1b in spermatogenesis, sperm maturation, and/or fertilization.  相似文献   

10.
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.  相似文献   

11.
We have studied the subcellular distribution of the alpha 1 and alpha 2 subunits of the skeletal muscle dihydropyridine (DHP) receptor with immunofluorescence labeling of normal and dysgenic (mdg) muscle in culture. In normal myotubes both alpha subunits were localized in clusters associated with the T-tubule membranes of longitudinally as well as transversely oriented T-tubules. The DHP receptor-rich domains may represent the sites where triad junctions with the sarcoplasmic reticulum are being formed. In cultures from dysgenic muscle the alpha 1 subunit was undetectable and the distribution patterns of the alpha 2 subunit were abnormal. The alpha subunit did not form clusters nor was it discretely localized in the T-tubule system. Instead, alpha 2 was found diffusely distributed in parts of the T-system, in structures in the perinuclear region and in the plasma membrane. These results suggest that an interaction between the two alpha subunits is required for the normal distribution of the alpha 2 subunit in the T-tubule membranes. Spontaneous fusion of normal non-muscle cells with dysgenic myotubes resulted in a regional expression of the alpha 1 polypeptide near the foreign nuclei, thus defining the nuclear domain of a T-tubule membrane protein in multi-nucleated muscle cells. Furthermore, the normal intracellular distribution of the alpha 2 polypeptide was restored in domains containing a foreign "rescue" nucleus; this supports the idea that direct interactions between the DHP receptor alpha 1 and alpha 2 subunits are involved in the organization of the junctional T-tubule membranes.  相似文献   

12.
ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the beta-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.  相似文献   

13.
During epididymal transit, mammalian sperm acquire selected proteins secreted by the epididymis. We previously showed that a disintegrin and metalloprotease (ADAM) 7 is expressed specifically in the epididymis and transferred to the sperm surface during epididymal transit. Here, we show that mouse ADAM7 secreted to the epididymal lumen is associated with membranous vesicles known as epididymosomes. Furthermore, we found that ADAM7 can be transferred directly from epididymal vesicles to sperm and that it is an integral plasma membrane protein in sperm. Thus, our study provides new information regarding the unique mode of secretion and interaction of ADAM7 during the epididymis-to-sperm transfer process.  相似文献   

14.
The first step of assembly of the nicotinic acetylcholine receptor (AChR) of adult skeletal muscle is the specific association of the alpha subunit with either delta or epsilon subunits to form a heterodimer with a ligand-binding site. Previous experiments have suggested that het erodimer formation in the ER arises from interaction between the luminal, NH2-terminal domains of the subunits. When expressed in COS cells with the delta subunit, however, the truncated NH2-terminal domain of the subunit folded correctly but did not form a heterodimer. Association with the delta subunit occurred only when the NH2-terminal domain was retained in the ER and was tethered to the membrane by its own M1 transmembrane domain, by the transmembrane domain of another protein, or by a glycolipid link. In each case, the ligand-binding sites of the resulting heterodimers were indistinguishable from that formed when the full-length alpha subunit was used. Attachment to the membrane may promote interaction by concentrating or orienting the subunit; alternatively, a membrane-bound factor may facilitate subunit association.  相似文献   

15.
The Na-K-ATPase, which maintains the Na(+) and K(+) gradients across the plasma membrane, can play a major role in modulation of skeletal muscle contractility. Although both alpha(1)- and alpha(2)-isoforms of the Na-K-ATPase are expressed in skeletal muscle, the physiological significance of these isoforms in contractility is not known. Evaluation of the contractile parameters of mouse extensor digitorum longus (EDL) was carried out using gene-targeted mice lacking one copy of either the alpha(1)- or alpha(2)-isoform gene of the Na-K-ATPase. The EDL muscles from heterozygous mice contain approximately one-half of the alpha(1)- or alpha(2)-isoform, respectively, which permits differentiation of the functional roles of these isoforms. EDL from the alpha(1)(+/-) mouse shows lower force compared with wild type, whereas that from the alpha(2)(+/-) mouse shows greater force. The different functional roles of these two isoforms are further demonstrated because inhibition of the alpha(2)-isoform with ouabain increases contractility of alpha(1)(+/-) EDL. These results demonstrate that the Na-K-ATPase alpha(1)- and alpha(2)-isoforms may play different roles in skeletal muscle contraction.  相似文献   

16.
17.
Al-Khalili L  Yu M  Chibalin AV 《FEBS letters》2003,536(1-3):198-202
We determined insulin-stimulated Na(+),K(+)-ATPase isoform-specific translocation to the skeletal muscle plasma membrane. When rat muscle plasma membrane fractions were isolated by discontinuous sucrose gradients, insulin-stimulated translocation of alpha(2)- but not alpha(1)-subunits was detected. However, using cell surface biotinylation techniques, an insulin-induced membrane translocation of both alpha(1) and alpha(2)-subunits in rat epitrochlearis muscle and cultured human skeletal muscle cells was noted. Na(+),K(+)-ATPase alpha-subunit translocation was abolished by the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, as well as by the protein kinase C inhibitor GF109203X. Thus, insulin mediates Na(+),K(+)-ATPase alpha(1)- and alpha(2)-subunit translocation to the skeletal muscle plasma membrane via a PI 3-kinase-dependent mechanism.  相似文献   

18.
The transmembrane and multidomain neural cell adhesion molecule (NCAM) plays important functional roles in the developing and adult nervous system. NCAM is proteolytically processed and appears in soluble forms in the cerebrospinal fluid and in serum under normal and pathological conditions. In this report, we present evidence that the metalloprotease a disintegrin and a metalloprotease (ADAM)17/tumour necrosis factor alpha converting enzyme (TACE) cleaves the polysialylated as well as the non-polysialylated transmembrane isoforms of NCAM, whereas the glycophosphatidylinositol-linked isoform of NCAM is not proteolytically cleaved. A truncated, enzymatically inactive mutant of TACE did not result in release of the NCAM110 cleavage product. Proteolytic cleavage was enhanced by a calmodulin-specific inhibitor and the actin-destabilizing agents cytochalasin D and latrunculin B. In contrast, the microtubule-stabilizing agent colchicine or microtubule-destabilizing agent paclitaxel did not affect the release of the 110-kDa fragment of NCAM. Neurite outgrowth from cerebellar microexplants was inhibited in the presence of the metalloprotease inhibitor GM 6001 on substrate-coated NCAM, but not on poly-l-lysine. Upon transfection of hippocampal neurones with an enzymatically inactive mutant of TACE, NCAM-stimulated neurite outgrowth was inhibited without affecting neurite outgrowth on poly-l-lysine, showing that proteolytic processing of NCAM by the metalloprotease TACE is involved in NCAM-mediated neurite outgrowth.  相似文献   

19.
ADAM13 is a member of the disintegrin and metalloprotease protein family that is expressed on cranial neural crest cells surface and is essential for their migration. ADAM13 is an active protease that can cleave fibronectin in vitro and remodel a fibronectin substrate in vivo. Using a recombinant secreted protein containing both disintegrin and cysteine-rich domains of ADAM13, we show that this "adhesive" region of the protein binds directly to fibronectin. Fibronectin fusion proteins corresponding to the various functional domains were used to define the second heparin-binding domain as the ADAM13 binding site. Mutation of the syndecan-binding site (PPRR --> PPTM) within this domain abolishes binding of the recombinant disintegrin and cysteine-rich domains of ADAM13. We further show that the adhesive disintegrin and cysteine-rich domain of ADAM13 can promote cell adhesion via beta(1) integrins. This adhesion requires integrin activation and can be prevented by antibodies to the cysteine-rich domain of ADAM13 and beta(1) integrin. Finally, wild type, but not the E/A mutant of ADAM13 metalloprotease domain, can be shed from the cell surface, releasing the metalloprotease domain associated with the disintegrin and cysteine-rich domains. This suggests that ADAM13 shedding may involve its own metalloprotease activity and that the released protease may interact with both integrins and extracellular matrix proteins.  相似文献   

20.
We describe a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Integrin alpha7beta1 extracted from human embryonic kidney 293 cells transfected with alpha7 cDNA was retained on an affinity column containing immobilized DC domain of ADAM12. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells, and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for Mn2+ than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase (FAK) at Tyr397. We also show that adhesion of L6 myoblasts to DC is mediated in part by the endogenous integrin alpha7beta1 expressed in these cells. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulate that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. We also conclude that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses.  相似文献   

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