ADAM Binding Protein Eve-1 Is Required for Ectodomain Shedding of Epidermal Growth Factor Receptor Ligands

Abstract

A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-α, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding. A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-α, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding. Transactivation of the epidermal growth factor receptor (EGFR) 1The abbreviations used are: EGFR, epidermal growth factor receptor; GPCR, G-protein-coupled receptor; HB-EGF, heparin-binding epidermal growth factor-like growth factor; EGF, epidermal growth factor; proHB-EGF, membrane-anchored form of HB-EGF; TPA, 12-O-tetradecanoylphorbol-13-acetate; ADAM, a disintegrin and metalloprotease; SH3, Src homology 3; TGF, transforming growth factor; AP, alkaline phosphatase; siRNA, small interfering RNA; RT, reverse transcriptase; GST, glutathione S-transferase.1The abbreviations used are: EGFR, epidermal growth factor receptor; GPCR, G-protein-coupled receptor; HB-EGF, heparin-binding epidermal growth factor-like growth factor; EGF, epidermal growth factor; proHB-EGF, membrane-anchored form of HB-EGF; TPA, 12-O-tetradecanoylphorbol-13-acetate; ADAM, a disintegrin and metalloprotease; SH3, Src homology 3; TGF, transforming growth factor; AP, alkaline phosphatase; siRNA, small interfering RNA; RT, reverse transcriptase; GST, glutathione S-transferase. has been shown to play a crucial role in the signaling by G-protein-coupled receptors (GPCRs), cytokine receptors, receptor tyrosine kinases, and integrins to a variety of cellular responses (1Hackel P.O. Zwick E. Prenzel N. Ullrich A. Curr. Opin. Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (545) Google Scholar, 2Moghal N. Sternberg P.W. Curr. Opin. Cell Biol. 1999; 11: 190-198Crossref PubMed Scopus (287) Google Scholar). The transactivation of EGFR is mediated, in some cases, by EGFR ligand shedding. In particular, the shedding of heparin-binding EGF-like growth factor (HB-EGF) (3Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1493) Google Scholar, 4Nanba D. Higashiyama S. Cytokine Growth Factor Rev. 2004; 15: 13-19Crossref PubMed Scopus (51) Google Scholar) is known to have a prominent role in regulating the function of this growth factor. HB-EGF plays a physiological role by binding to and stimulating phosphorylation of the EGFR (5Higashiyama S. Abraham J.A. Fiddes J.C. Klagsbrun M. Science. 1991; 251: 936-939Crossref PubMed Scopus (1042) Google Scholar, 6Higashiyama S. Lau K. Besner G.E. Abraham J.A. Klagsbrun M. J. Biol. Chem. 1992; 267: 6205-6212Abstract Full Text PDF PubMed Google Scholar). Like other members of the EGF family, HB-EGF is synthesized as a membrane-anchored form (proHB-EGF) and then processed to a bioactive soluble form (5Higashiyama S. Abraham J.A. Fiddes J.C. Klagsbrun M. Science. 1991; 251: 936-939Crossref PubMed Scopus (1042) Google Scholar, 6Higashiyama S. Lau K. Besner G.E. Abraham J.A. Klagsbrun M. J. Biol. Chem. 1992; 267: 6205-6212Abstract Full Text PDF PubMed Google Scholar). This process, called ectodomain shedding of proHB-EGF, is an important post-translational modification that converts a tethered insoluble juxtacrine form into a soluble growth factor with autocrine/paracrine activity (7Goishi K. Higashiyama S. Klagsbrun M. Nakano N. Umata T. Ishikawa M. Mekada E. Taniguchi N. Mol. Biol. Cell. 1995; 6: 967-980Crossref PubMed Scopus (252) Google Scholar). The ectodomain shedding of proHB-EGF is induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) (7Goishi K. Higashiyama S. Klagsbrun M. Nakano N. Umata T. Ishikawa M. Mekada E. Taniguchi N. Mol. Biol. Cell. 1995; 6: 967-980Crossref PubMed Scopus (252) Google Scholar) and mitogen-activated protein kinase signaling pathway also contributes to this proteolytic processing (8Gechtman Z. Alonson J.L. Raab G. Ingber D.E. Klagsbrun M. J. Biol. Chem. 1999; 274: 28828-28835Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 9Umata T. Hirata M. Takahashi T. Ryu F. Shida S. Takahashi Y. Tsuneoka M. Miura Y. Masuda M. Horiguchi Y. Mekada E. J. Biol. Chem. 2001; 276: 30475-30482Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In addition, GPCR agonists induce the ectodomain shedding of proHB-EGF, resulting in EGFR transactivation, which is a critical element in GPCR-induced mitogenic signaling (3Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1493) Google Scholar). We identified a disintegrin and metalloprotease 12 (ADAM12) as a specific enzyme that catalyzes proHB-EGF shedding in EGFR transactivation by GPCR agonists, such as phenylephrine, endothelin-1, and angiotensin II, and results in cardiac hypertrophy (10Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (640) Google Scholar). Further, ADAM9, ADAM10, ADAM17, matrix metalloproteases 3 and 7 as well as ADAM12 have been reported to be involved in the ectodomain shedding of proHB-EGF (4Nanba D. Higashiyama S. Cytokine Growth Factor Rev. 2004; 15: 13-19Crossref PubMed Scopus (51) Google Scholar) These reports suggest that the ectodomain shedding of EGFR ligands, especially HB-EGF, is key to the molecular mechanism of EGFR transactivation, which would be an important network of GPCR signaling. However, the pathway by which GPCR signaling links to the activation of ADAM enzymes is largely unknown. Elucidation of the regulatory mechanisms of ADAM activation could clarify the signaling pathway of EGFR transactivation by GPCRs. ADAM12 is a single membrane-spanning protein with a metalloprotease domain in its extracellular portion, and several Src homology 3 (SH3) binding motifs in the cytoplasmic tail. Therefore, it has been anticipated to interact with SH3 domain-containing proteins. Indeed, it has been demonstrated that ADAM12 binds to p85α (11Kang Q. Cao Y. Zolkiewska A. J. Biol. Chem. 2001; 276: 24466-24472Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), Src (12Kang Q. Cao Y. Zolkiewska A. Biochem. J. 2000; 352: 883-892Crossref PubMed Scopus (69) Google Scholar, 13Suzuki A. Kadota N. Hara T. Nakagami Y. Izumi T. Takenawa T. Sabe H. Endo T. Oncogene. 2000; 19: 5842-5850Crossref PubMed Scopus (51) Google Scholar), Grb2 (13Suzuki A. Kadota N. Hara T. Nakagami Y. Izumi T. Takenawa T. Sabe H. Endo T. Oncogene. 2000; 19: 5842-5850Crossref PubMed Scopus (51) Google Scholar), and Fish (14Abram C.L. Seals D.F. Pass I. Salinsky D. Maurer L. Roth T.M. Courtneidge S.A. J. Biol. Chem. 2003; 278: 16844-16851Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Moreover, the cytoplasmic domain of ADAM12 has been shown to interact with actin cytoskeleton via α-actinin-2 (15Galliano M.F. Huet C. Frygelius J. Polgren A. Wewer U.M. Engvall E. J. Biol. Chem. 2000; 275: 13933-13939Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). However, it has not been demonstrated that these molecules could affect ADAM12 metalloprotease activity. In this study, we performed yeast two-hybrid screening to identify ADAM12-binding proteins and discovered a protein containing SH3 domains that we designated Eve-1. We showed that Eve-1 binds not only ADAM12 but also ADAM9, ADAM10, ADAM15, and ADAM 17 and is involved in ADAM-mediated ectodomain shedding of several EGFR ligands, including proHB-EGF, proTGF-α, proamphiregulin and proepiregulin. Antibodies—Mouse anti-myc monoclonal antibody (9E10) and anti-FLAG monoclonal antibody (M2) were purchased from Santa Cruz Biotechnology and Sigma-Aldrich, respectively. Rabbit polyclonal antibodies against human ADAM10 and ADAM17 were from BIOMOL Research Laboratories Inc. and R&D Systems Inc., respectively. Rabbit polyclonal antibody against the cysteine-rich region of human ADAM12 was a kind gift from Dr. A. Sehara (Kyoto University, Kyoto, Japan). Rabbit polyclonal antibody against the cytoplasmic region of pro-HB-EGF (H1) has been described previously (16Miyagawa J. Higashiyama S. Kawata S. Inui Y. Tamura S. Yamamoto K. Nishida M. Nakamura T. Yamashita S. Matsuzawa Y. Taniguchi N. J. Clin. Investig. 1995; 95: 404-411Crossref PubMed Google Scholar). Rabbit anti-Eve-1 polyclonal antibody was raised against recombinant Eve-1c. Goat alkaline phosphatase (AP) or horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from Promega. Goat rhodamine-conjugated anti-mouse antibody was from Chemicon. Expression Vectors and Small Interfering RNA (siRNA)—We constructed a yeast expression plasmid encoding GAL4 DNA-binding domain fused with the ADAM12 cytoplasmic domain by inserting ADAM12 cytoplasmic domain cDNA into the EcoRI and SalI multiple cloning sites of the pBTM116 vector (BD Biosciences Clontech). ADAM12 full-length cDNA was cloned into the SacI and SalI sites of the pEYFP-N1 vector (BD Biosciences Clontech). We prepared an adenovirus carrying a gene encoding FLAG epitope-tagged ADAM12 (ADAM12-FLAG) as described previously (10Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (640) Google Scholar). The cytoplasmic domain of ADAM12 and its truncated regions were cloned into the EcoRI and XhoI sites of the pGEX6P-1 vector (Amersham Biosciences). Eve-1a and Eve-1b full-length cDNAs isolated from a human heart cDNA library by PCR were introduced into the SalI and NotI multiple cloning sites of the modified pME18S vector (pME18S-myc) that possesses an SRα promoter (17Tanabe Y. Seiki M. Fujisawa J. Hoy P. Yokota K. Arai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar). Eve-1c and Eve-1d cDNAs were amplified using vectors with Eve-1a and Eve-1b inserted as templates, respectively, and cloned into the BglII and NotI sites of the pME18S-myc vector. A series of truncated mutants of the SH3 domains region of Eve-1b were cloned into the EcoRI and NotI sites of the pME18S-myc vector. The pME18S-myc vector was provided by Dr. K. Touhara (University of Tokyo, Tokyo, Japan). Dharmacon Research Inc. chemically synthesized and purified siRNA duplexes. The siRNA sequence targeting Eve-1 corresponded to the coding region 155–175 (5′-AACCUUCUGUAGCUCCCAAAC-3′) relative to the first nucleotide of the start codon of Eve-1a (Eve-1b). This sequence had no substantial similarity to any other sequence in the NCBI data base. Scramble-siRNA directed against 5′-GCGCGCUUUGUAGGAUUCG-3′ was the negative control. No this sequence in the NCBI data base. containing was A human heart cDNA library in was introduced into the and yeast cells were and in the of the at were activity. plasmid was by into cells and Northern with human RNA Northern Biosciences was with a Eve-1 full-length were in with and using the Tokyo, Japan). To the of the were performed and human was the Cell and cells and that proHB-EGF proHB-EGF and angiotensin receptor proamphiregulin or proepiregulin were in with and at in was performed using to the HT1080 cells at in were with of the expression vectors or with and then HT1080 cells in were with of and of analysis or with of siRNA reverse and then and cells were with of siRNA cell or with of siRNA well of shedding and then of Cell cells were with and in containing and at the was as cell cell was with anti-myc anti-FLAG antibody or antibody Japan). (Amersham was and the was The were by with by and with and proteins (Amersham were The cell was with or proteins the with at with proteins were by in by and with or to proteins were and against the anti-myc anti-FLAG or The antibody was or horseradish peroxidase-conjugated anti-mouse or anti-rabbit The proteins were using and as or the (Amersham horseradish Cell cells were with and with of in containing was by with The cell was with as described using antibody or and was performed as described (7Goishi K. Higashiyama S. Klagsbrun M. Nakano N. Umata T. Ishikawa M. Mekada E. Taniguchi N. Mol. Biol. Cell. 1995; 6: 967-980Crossref PubMed Scopus (252) Google Scholar). as described the cells were with using and with anti-myc antibody by rhodamine-conjugated anti-mouse were using a The of was as described RNA was from the cells using and cDNA was synthesized using II PCR using a specific Eve-1 and or cells were in and then at with angiotensin II or of were to and activity was as described previously S. Higashiyama S. Endo T. T. J. K. Y. Ohmoto H. Yoshino K. Y. Matsuzawa Y. K. Taniguchi N. J. Cell Biol. 2000; PubMed Scopus Google Scholar). by of with identify proteins that interact with the cytoplasmic domain of of a human heart cDNA library were with the of the yeast two-hybrid and the ADAM12 cytoplasmic domain as of the isolated a encoding by four SH3 the cDNA sequence that it was to the region of a protein with an of We designated the full-length protein Eve-1 it was the binding of Two full-length cDNAs that were cloned by PCR from a human heart cDNA proteins of and and with a respectively. We designated these proteins Eve-1a and Eve-1b, respectively. analysis revealed that Eve-1a and Eve-1b have four and five tandem SH3 domains in the carboxyl-terminal region, respectively. proline-rich SH3 domain binding S. H. J.A. Science. Scopus Google Scholar, S. A. 1995; PubMed Scopus Google Scholar), were also in the amino-terminal region A and The short forms translated at Met-371 of Eve-1a and Eve-1b are human counterparts of mouse which was identified as a protein Y. N. K. M. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). We designated Eve-1c and Eve-1d A and analysis of the Eve-1 gene revealed that Eve-1a and Eve-1b and are alternatively spliced isoforms from a single gene Expression of analysis the expression of Eve-1 in human with an Eve-1 a single in with expression in skeletal and and expression in and To Eve-1 human cancer cell cancer cancer and and HT1080 were to and Western Eve-1 gene expression was in and HT1080 cells but not in Western showed a cell and HT1080 that with recombinant Eve-1c of recombinant Eve-1c into HT1080 cell in the of the to Eve-1c of the and was no of any However, this was not in The to Eve-1b, and Eve-1d were in cell The in and cells by an be or a These data suggest that Eve-1c is in the cancer cell lines. of Eve-1c and Eve-1d with ADAM12 in Eve-1b, or Eve-1d was expressed in HT1080 and showed the of Eve-1 To that Eve-1 with ADAM12 in Eve-1 and ADAM12 were in HT1080 that anti-myc antibody and from cell and corresponded to an form that was by antibody However, to that Eve-1c and Eve-1d with was expressed in HT1080 that was from cell using anti-FLAG antibody and Eve-1c was by anti-Eve-1 antibody of the molecular to Eve-1b, or Eve-1d was by anti-Eve-1 of the binding of ADAM12 Eve-1 and we performed using a series of truncated mutants of cytoplasmic domain of ADAM12 or a series of truncated mutants of SH3 domains region of Eve-1d to and In not to or The results were using not These results suggest that region of ADAM12 is the binding to Eve-1c or of ADAM12 binding sites in of SH3 domain region mutants of Eve-1. ADAM12 binding to the prepared from HT1080 cells Eve-1 mutants were with or cytoplasmic domain glutathione Eve-1 mutants were by using anti-myc the other and In not and In the of to the of is of These results suggest that the region containing and domains of Eve-1d is binding to of ADAM12 and ADAM12 and we and in HT1080 revealed that with at the of the cell of Eve-1 of the of Eve-1 ectodomain shedding of proHB-EGF, using to the of Eve-1 and of and respectively. Eve-1 cDNA was amplified by using RNA isolated from HT1080 cells with negative but not with we the of Eve-1c protein in the from HT1080 cells with in with These that Eve-1 We the of the ectodomain shedding of cells were with Scramble-siRNA or with and then we activity in the of a with angiotensin shedding induced by was using the of expression was Eve-1 by and of angiotensin and we reported previously that binds ADAM12 cytoplasmic domain and ectodomain shedding of proHB-EGF S. M. D. E. Ishiguro H. Higashiyama S. N. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), we the of Eve-1 and the ectodomain shedding of of Eve-1 and by using not showed the of proHB-EGF shedding induced by or angiotensin II with the of the suggesting that Eve-1 and are the proHB-EGF shedding. Eve-1c and Eve-1d with ADAM9, ADAM10, ADAM15, and ADAM17 in ADAMs proline-rich regions in cytoplasmic suggesting with Eve-1. To Eve-1 with the other ADAM members such as ADAM9, ADAM10, ADAM15, and ADAM17 implicated in the ectodomain shedding of Eve-1b, or Eve-1d was expressed in HT1080 cells and using anti-myc shown in ADAM10 and ADAM17 were with and and were also with and However, any or ADAM of Eve-1 of proTGF-α, and the of the ectodomain shedding of proTGF-α, and proepiregulin. shown in of and shedding and of expression the of siRNA and RNA were in We to of the regulatory mechanisms of ADAM-mediated proHB-EGF and the other EGFR-ligand shedding induced by GPCR agonists or We which was identified as a shedding enzyme of two-hybrid screening using the cytoplasmic domain of ADAM12 as of a protein containing SH3 domains that we called Eve-1. The Eve-1 gene at alternatively spliced Eve-1a and Eve-1a has an SH3 domain by four tandem SH3 the other Eve-1b has to the SH3 domain of resulting in the production of SH3 Eve-1b five SH3 suggesting that these isoforms are involved in ADAM with PCR showed that the cloning of Eve-1a and Eve-1b was suggesting that Eve-1a is Eve-1b in HT1080 is a protein Y. N. K. M. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) that to a mouse of Northern revealed a single of Eve-1, and siRNA 155–175 coding region of Eve-1a reduced Eve-1c protein suggesting that Eve-1a and Eve-1c are translated from a single Western revealed that the expression of Eve-1a not Eve-1c and that the Eve-1c was in human cancer cell the dominant of as an codon of Eve-1 of the expression of the Eve-1 isoforms in are to the mechanism of in the of the to the of the a but of and cellular the of the called P. 2001; 15: PubMed Scopus Google Scholar, S. S. 2001; PubMed Scopus Google Scholar, S. C. L. S. 2003; PubMed Scopus Google Scholar). and are translated at the first and the in of the E. S. G. G. T. L. J. PubMed Scopus Google Scholar), and growth factor has five isoforms alternatively translated from a single S. A. F. F. H. Mol. Cell. Biol. 1995; 15: PubMed Scopus Google Scholar). has been reported that the growth factor is is and RNA and a S. C. L. S. H. S. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). the region of the Eve-1 has no it is to be especially in and by using M. 2003; PubMed Scopus Google Scholar) not suggesting the of an Eve-1 the the of of growth factor is in and cells S. C. S. F. F. H. J. Cell Biol. PubMed Scopus Google Scholar), the of Eve-1a and Eve-1c be also in and of Eve-1a and Eve-1c and of proteins are the other analysis of the NCBI data revealed that a of not mouse counterparts of Eve-1b of a codon of its coding region. The from the data that Eve-1a and Eve-1b have in Eve-1 has multiple SH3 we that Eve-1 with the proline-rich SH3 in the cytoplasmic of A binding Eve-1 was in the region of the ADAM12 cytoplasmic and domains of Eve-1d were shown to be binding to the cytoplasmic domain of ADAM12 it is that a of SH3 domains 3 and of Eve-1 and of ADAM12 are ADAM12 also with p85α and and the binding sites these proteins are in or and of the ADAM12 cytoplasmic (11Kang Q. Cao Y. Zolkiewska A. J. Biol. Chem. 2001; 276: 24466-24472Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, Q. Cao Y. Zolkiewska A. Biochem. J. 2000; 352: 883-892Crossref PubMed Scopus (69) Google Scholar). Therefore, it is that several proteins containing SH3 domains the members of the ADAM family, such as ADAM9, ADAM10, ADAM15, and ADAM17 as well as ADAM12 have such regions in cytoplasmic D.F. Courtneidge S.A. 2003; PubMed Scopus Google Scholar). Indeed, some of these ADAM proteins interact with signaling molecules containing an SH3 domain via cytoplasmic domains (11Kang Q. Cao Y. Zolkiewska A. J. Biol. Chem. 2001; 276: 24466-24472Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, Q. Cao Y. Zolkiewska A. Biochem. J. 2000; 352: 883-892Crossref PubMed Scopus (69) Google Scholar, 13Suzuki A. Kadota N. Hara T. Nakagami Y. Izumi T. Takenawa T. Sabe H. Endo T. Oncogene. 2000; 19: 5842-5850Crossref PubMed Scopus (51) Google Scholar, C.L. Seals D.F. Pass I. Salinsky D. Maurer L. Roth T.M. Courtneidge S.A. J. Biol. Chem. 2003; 278: 16844-16851Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, L. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, Z. A. G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (87) Google Scholar). several proteins containing SH3 domains are to form in the cytoplasmic domains of and form with and L. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). expressed also ADAM9, ADAM10, ADAM15, and ADAM17 as well as ADAM12 ADAM9, ADAM10, and ADAM17 have been shown to be involved in the shedding of (4Nanba D. Higashiyama S. Cytokine Growth Factor Rev. 2004; 15: 13-19Crossref PubMed Scopus (51) Google Scholar, G. H. Higashiyama S. J. D. P. J. Cell Biol. 2004; PubMed Scopus Google Scholar), it is that Eve-1 is involved in the of ectodomain shedding of We demonstrated that the of Eve-1 by siRNA in HT1080 cells the shedding of proHB-EGF induced by angiotensin II and as well as the shedding of proTGF-α, and proepiregulin induced by suggesting that Eve-1 post-translational processing of EGFR Eve-1 be to the signaling of angiotensin II EGFR-ligand shedding. To the physiological of Eve-1 and these are ADAM-mediated ectodomain shedding of other proteins and the other ADAM such as cell We have identified SH3 domain-containing which to ADAM12 and proHB-EGF shedding activity induced by angiotensin II and S. M. D. E. Ishiguro H. Higashiyama S. N. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). it has not been reported proteins such as and α-actinin-2 could affect ADAM metalloprotease Eve-1 and are the first molecules that have been as of metalloprotease activity of Eve-1, which was identified as an ADAM12-binding is to play a physiological role as a binding of ADAMs in the signaling pathway of EGFR transactivation by GPCR clarify the signaling pathway from to the shedding of proHB-EGF and the other EGFR ligands to EGFR transactivation.