PACSIN3 Binds ADAM12/Meltrin α and Up-regulates Ectodomain Shedding of Heparin-binding Epidermal Growth Factor-like Growth Factor

Abstract

A disintegrin and metalloprotease 12 (ADAM12/meltrin α) 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. A disintegrin and metalloprotease 12 (ADAM12/meltrin α) 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. The transactivation of epidermal growth factor receptor (EGFR) 1The abbreviations used are: EGFR, EGF receptor; EGF, epidermal growth factor; HB-EGF, heparin-binding EGF-like growth factor; GPCR, G-protein-coupled receptor; TPA, 12-O-tetradecanoylphorbol-13-acetate; SH3, Src homology 3; siRNA, small interfering RNA; HA, hemagglutinin; AP, alkaline phosphatase; GST, glutathione S-transferase; CBB, Coomassie Brilliant Blue; ADAM, a disintegrin and metalloprotease. by G-protein-coupled receptor (GPCR) agonists is a critical element in various responses of diverse cell types including fibroblasts, keratinocytes, astrocytes, and smooth muscle cells (1Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (588) Google Scholar, 2Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). A novel mechanistic concept of the EGFR transactivation-signaling pathway involves the proteolytic release of heparin-binding EGF-like growth factor (HB-EGF) at the surface of cells stimulated with GPCR agonists (3Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar). HB-EGF is a member of the EGF family that directly binds EGFR and thereby enhances its phosphorylation, resulting in cell growth and differentiation (4Higashiyama 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 proteolytically processed to become a bioactive soluble form, a process that is called ectodomain shedding. The ectodomain shedding of proHB-EGF is an important post-translational modification that converts a tethered insoluble juxtacrine growth factor into a soluble ligand leading to the autocrine or paracrine activation of EGFR. Studies of GPCR mitogenic signaling have proven that EGFR transactivation is dependent on HB-EGF in smooth muscle cells (2Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar), cardiac endothelial cells (5Fujiyama S. Matsubara H. Nozawa Y. Maruyama K. Mori Y. Tsutsumi Y. Masaki H. Uchiyama Y. Koyama Y. Nose A. Iba O. Tateishi E. Ogata N. Jyo N. Higashiyama S. Iwasaka T. Circ. Res. 2001; 88: 22-29Crossref PubMed Scopus (203) Google Scholar), and cardiomyocytes (6Asakura 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 (641) Google Scholar), as well as in various pathological processes such as cardiac hypertrophy (6Asakura 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 (641) Google Scholar), chronic active gastritis associated with Helicobacter pylori (7Keates S. Sougioultzis S. Keates A.C. Zhao D. Peek Jr., R.M. Shaw L.M. Kelly C.P. J. Biol. Chem. 2001; 276: 48127-48134Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), and cystic fibrosis (8Lemjabbar H. Basbaum C. Nat. Med. 2002; 8: 41-46Crossref PubMed Scopus (301) Google Scholar). Growing evidence points to a disintegrin and metalloproteases (ADAMs) as key enzymes of proHB-EGF shedding in EGFR transactivation signaling. All ADAMs have an extracellular portion with a metalloprotease domain, a transmembrane region and a cytoplasmic tail, and several ADAMs have metalloprotease activity. Lemjabbar and Basbaum (8Lemjabbar H. Basbaum C. Nat. Med. 2002; 8: 41-46Crossref PubMed Scopus (301) Google Scholar) described that stimulation of platelet-activating factor receptor transactivated EGFR through the shedding of proHB-EGF by ADAM10 in the human epithelial cell line HM3. Yan et al. (9Yan Y. Shirakabe K. Werb Z. J. Cell Biol. 2002; 158: 221-226Crossref PubMed Scopus (279) Google Scholar) demonstrated that stimulation with the bombesin receptor transactivated EGFRs via the ADAM10-dependent cleavage of proHB-EGF in COS-7 cells. We also identified ADAM12 as a specific enzyme that catalyzes proHB-EGF shedding in EGFR transactivation by GPCR agonists, such as phenylephrine, endothelin-1, and angiotensin II causing cardiac hypertrophy (6Asakura 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 (641) Google Scholar). Izumi et al. (10Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Crossref PubMed Scopus (475) Google Scholar) showed that ADAM9 is involved in TPA-induced shedding of HB-EGF in Vero-H cells when protein kinase Cδ is activated. However, Weskamp et al. (11Weskamp G. Cai H. Brodie T.A. Higashyama S. Manova K. Ludwig T. Blobel C.P. Mol. Cell Biol. 2002; 22: 1537-1544Crossref PubMed Scopus (175) Google Scholar) found that TPA-stimulated shedding of HB-EGF is unaffected in embryonic fibroblasts derived from mice lacking ADAM9. Moreover, Kurisaki et al. (12Kurisaki T. Masuda A. Sudo K. Sakagami J. Higashiyama S. Matsuda Y. Nagabukuro A. Tsuji A. Nabeshima Y. Asano M. Iwakura Y. Sehara-Fujisawa A. Mol. Cell Biol. 2003; 23: 55-61Crossref PubMed Scopus (126) Google Scholar) discovered that TPA-induced proHB-EGF shedding is completely abrogated in embryonic fibroblasts derived from mice lacking ADAM12, arguing against an essential role for ADAM9 in proHB-EGF shedding. These reports indicate that the ectodomain shedding of EGFR ligands, especially of proHB-EGF, is central to GPCR and EGFR communication. However, the underlying mechanisms of the ligand shedding-dependent EGFR transactivation pathway are largely unknown. Thus, elucidation of the regulatory mechanisms of ADAMs is essential to understand the ligand shedding-dependent EGFR transactivation pathway. The present study focuses on ADAM12, which is involved in TPA or angiotensin II induced HB-EGF shedding and EGFR transactivation. ADAM12 has several Src homology 3 (SH3) domain-binding motifs, (R/K)XXPXXP or PXXPX(R/K) (13Kay B.K. Williamson M.P. Sudol M. FASEB J. 2000; 14: 231-241Crossref PubMed Scopus (1050) Google Scholar) in its cytoplasmic tails, indicating that ADAM12 probably interacts with signaling molecules containing SH3 domains. Therefore, we performed a yeast two-hybrid screening to identify proteins that bind ADAM12 cytoplasmic tails. We found that a protein containing an SH3 domain called PACSIN3 is required for the proHB-EGF shedding induced by TPA and angiotensin II. Expression Vectors and Small Interfering RNA (siRNA)—The yeast expression plasmid encoding the GAL4 DNA-binding domain fused to the human ADAM12 cytoplasmic domain was constructed by inserting ADAM12 cytoplasmic domain complementary DNA (cDNA) into EcoRI and SalI sites in the multiple cloning sites of the pBTM116 vector (Clontech). We similarly constructed yeast expression plasmids encoding GAL4 DNA-binding domain fused to human ADAM9, ADAM10, ADAM15, ADAM17, and ADAM19 cytoplasmic domains. We prepared an adenovirus carrying a gene encoding FLAG epitope-tagged ADAM12 as described (6Asakura 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 (641) Google Scholar). ADAM12 full-length was cloned into the pEGFP-N1 vector (Clontech). The cytoplasmic domain of ADAM12 and its truncated regions were cloned into the pGEX4T-1 vector (Amersham Biosciences). PACSIN3 full-length and truncated (ΔSH3, 1-362 amino acids) cDNAs isolated from a human heart cDNA library by the polymerase chain reaction (PCR) were introduced below the hemagglutinin (HA) sequence into BamHI and NotI sites among the multiple cloning sites of the pcDNA3.1 mammalian expression vector (Invitrogen). PACSIN3 full-length cDNA was also cloned into the pGEX4T-1 vector. The siRNA duplexes were chemically synthesized and purified by Dharmacon Research Inc. The PACSIN3 target sequence 5′-AAGAGGCTGAAGGAGGTTGAG-3′ was selected for PACSIN3 knockdown. This sequence was not substantially similar to any other sequence in the NCBI data base. Scramble siRNA directed against 5′-GCGCGCUUUGUAGGAUUCG-3′ was the negative control. No mammalian mRNAs contained this sequence in the NCBI data base. Yeast Two-hybrid Screening—Yeast strain L40 containing pBTM116-ADAM12-ΔCyto was selected on synthetic complete medium lacking tryptophan. A human heart cDNA library in pACT2 was introduced into the transformant, and then yeast cells were plated onto synthetic complete medium lacking tryptophan, leucine, and histidine in the presence of 3.5 mm 3-aminotriazole. We assayed the β-galactosidase activity of transformants grown on the dropout plates at 30 °C for 3-7 days. Library plasmid DNA was recovered by transformation into Escherichia coli HB101 cells and sequenced. Cell Culture and Transfection—HT1080 cells were cultured in Eagle's minimum essential medium supplemented with 0.1 mm non-essential amino acids and 10% fetal bovine serum at 37 °C in 5% CO2. HT1080 cells stably expressing one of HA-tagged PACSIN3 (HA-PACSIN3), HA-tagged PACSIN3-ΔSH3 (HA-PACSIN3-ΔSH3), alkaline phosphatase (AP)-tagged proHB-EGF (proHB-EGF-AP) (14Tokumaru S. Higashiyama S. Endo T. Nakagawa T. Miyagawa J.I. Yamamori K. Hanakawa Y. Ohmoto H. Yoshino K. Shirakata Y. Matsuzawa Y. Hashimoto K. Taniguchi N. J. Cell Biol. 2000; 151: 209-220Crossref PubMed Scopus (264) Google Scholar), or both proHB-EGF-AP and angiotensin type I (AT1) receptor were also maintained under the same conditions except for the presence of 200 μg/ml hygromycin B. Cells at 80-90% confluence were transfected with mammalian expression vectors using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions or infected with adenovirus at 50 multiplicity of infection and then incubated for 40 h. Preparation of Cell Extracts—Cells were washed with phosphate-buffered saline and lysed in 50 mm Tris-HCl (pH 7.4) containing 120 mm NaCl, 0.5% Nonidet P-40, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 mm leupeptin, 1.5 mm pepstatin, 1 mm aprotinin, and 50 mm sodium fluoride (lysis buffer). After centrifugation for 15 min at 12,000 rpm, the supernatant was collected as cell extract. Pull-down Assay with Immobilized Glutathione S-Transferase (GST) Fusion Proteins—We separated GST and GST-fusion proteins using SDS-PAGE and stained them with Coomassie Brilliant Blue. The proteins were immobilized onto glutathione-Sepharose and exposed to pull-down FLAG-tagged ADAM12 (FLAG-ADAM12) from cell lysates. Cell lysates were incubated with either GST or GST fusion proteins on the resin in 500 μl of the lysis buffer described above with gentle agitation at 4 °C for 4 h. After extensive washing with lysis buffer, bound proteins were released by boiling in SDS-sample buffer, separated by SDS-PAGE, and then immunoblotted. Immunoprecipitation—The HT1080/HA-PACSIN3 cell lysates were incubated for 2 h with an anti-HA polyclonal antibody (Y11) (Santa Cruz Biotechnology). Protein G-Sepharose (Amersham Biosciences) was added, and the mixture was incubated for 4 h. The immunoprecipitates were collected by centrifugation, washed five times with lysis buffer, resolved by SDS-PAGE, and immunoblotted. Immunoblot Analysis—Resolved proteins were transferred onto an Immobilon-P membrane (Millipore) and immunoblotted against the following primary antibodies: anti-HA monoclonal antibody (12CA5) (Roche Applied Science), anti-FLAG monoclonal antibody (M2) (Sigma), or anti-PACSIN3 polyclonal antibody raised against GST fusion protein. The secondary antibody was either horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (Promega). The immunoreactive proteins were visualized using the ECL detection system (Amersham Biosciences). Confocal Microscopy—HT1080/HA-PACSIN3 and HT1080/HA-PACSIN3-ΔSH3 cells were transiently transfected with pEGFP-ADAM12. Thereafter, the cells were fixed with 4% paraformaldehyde, permeabilized using 0.1% Nonidet P-40, and stained with anti-HA monoclonal antibody followed by goat Cy3-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Fluorescent images were acquired at the resolution of 512 × 512 using a Bio-Rad Radiance 2000 confocal device fitted on a Nikon Eclipse E-600 microscope with a 100 x/1.4 PlanAPO oil-immersion objective. Red and green signals were collected sequentially to avoid bleed through. Assay of ProHB-EGF-AP Shedding—Cells at 80-90% confluence were transfected with 0.8 μg of siRNA (per well of 24-well plates) and incubated for 40 h. The cells were then incubated for 60 min at 37 °C with 50 nm TPA or 100 nm angiotensin II. Aliquots (100 μl) of conditioned media were transferred to 96-well plates, and AP activity was measured as described previously (14Tokumaru S. Higashiyama S. Endo T. Nakagawa T. Miyagawa J.I. Yamamori K. Hanakawa Y. Ohmoto H. Yoshino K. Shirakata Y. Matsuzawa Y. Hashimoto K. Taniguchi N. J. Cell Biol. 2000; 151: 209-220Crossref PubMed Scopus (264) Google Scholar). Identification of Binding Proteins That Interact with ADAM12 by Yeast Two-hybrid Screening—To identify proteins that interact with the cytoplasmic domain of ADAM12, 3.0 × 106 clones of a human heart cDNA library were screened using the yeast two-hybrid system and the ADAM12 cytoplasmic domain as bait. Thirteen positive clones were obtained and sequenced. A homology search in GenBank™ cDNA data bases using the BLAST program revealed that two of the isolated clones overlap with the carboxyl-terminal region of PACSIN3 cDNA. PACSIN3 full-length cDNA was cloned by PCR from a human heart cDNA library, and interaction between PACSIN3 and the cytoplasmic domain of ADAM12 in yeast cells was confirmed by growth under nutritional selection and by β-galactosidase production (Fig. 1A). The domain structure of PACSIN3 has a Fes/CIP4 homology domain in the amino-terminal region, followed by a domain and a SH3 domain at the region (Fig. R. N. M. 2001; PubMed Scopus Google Scholar). PACSIN3 with ADAM12 in and in performed in to that PACSIN3 and ADAM12 bound to fusion protein not to GST in from HT1080 cells expressing (Fig. To that two proteins in mammalian was transiently in HT1080/HA-PACSIN3 cells. that anti-HA antibody from cell lysates of HT1080 cells transfected with not from cell lysates and which was by the anti-FLAG antibody expression was by anti-FLAG antibody in the lysates of HT1080 cells transfected with not in lysates from HT1080 cells of of ADAM12 for PACSIN3 the of ADAM12 for we assayed in using a of truncated of the cytoplasmic domain of ADAM12 (Fig. A and bound to and and to and to a in from HT1080 cells expressing (Fig. not bind to and (Fig. These that (amino acid residues and (amino acid residues 829-840) are required for to PACSIN3. of ADAM12 and determine the of ADAM12 and ADAM12 was transiently in HT1080/HA-PACSIN3 or HT1080/HA-PACSIN3-ΔSH3 cells. revealed that and at and at the leading of the cell (Fig. the other the of and (Fig. not of PACSIN3 on of by TPA and the of PACSIN3 on the ectodomain shedding of proHB-EGF induced by TPA, PACSIN3 or PACSIN3-ΔSH3 was in cells. The AP activity in the conditioned medium of was then measured a with 50 nm The overexpression of PACSIN3 enhanced TPA-induced proHB-EGF-AP shedding with the the of PACSIN3-ΔSH3 overexpression was (Fig. However, PACSIN3 overexpression not proHB-EGF-AP shedding induced by angiotensin II to a (Fig. To that PACSIN3 as an up-regulator of proHB-EGF ectodomain we the using siRNA To the of on we the of endogenous PACSIN3 protein by We endogenous PACSIN3 protein in the from HT1080 cells transfected with in transfected with negative siRNA (Fig. This confirmed that endogenous PACSIN3 protein We then the of on the ectodomain shedding of proHB-EGF induced by cells were transfected with or with and AP activity was measured in the same as described of PACSIN3 by HT1080 cell lysates were immunoblotted using anti-PACSIN3 and of PACSIN3 knockdown on shedding of proHB-EGF induced by 50 nm TPA and 100 nm angiotensin II AP activity in conditioned medium of or cells transfected with siRNA was measured with 50 nm TPA or 100 nm angiotensin II for 60 min at 37 for siRNA with TPA or angiotensin proHB-EGF-AP shedding induced by angiotensin II was assayed using cells. PACSIN3 knockdown by attenuated 50 and of the of and angiotensin proHB-EGF-AP (Fig. and of PACSIN3 Binding to the of ADAMs for PACSIN3 the of the cytoplasmic of various proteins for PACSIN3 by the yeast two-hybrid The cytoplasmic of ADAMs and with PACSIN3 not of both in yeast growth (Fig. and β-galactosidase (Fig. The study the regulatory mechanisms of proHB-EGF shedding induced by TPA or GPCR We then on ADAM12 that has identified as a shedding enzyme involved in the cytoplasmic of human ADAM12 has I (R/K)XXPXXP and II PXXPX(R/K) SH3 domain-binding into proline-rich regions (Fig. ADAM12 probably interacts with several signaling molecules containing SH3 domains. Yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as in the of PACSIN3 by Fes/CIP4 homology and SH3 in the and carboxyl-terminal PACSIN3 is to a cytoplasmic involved in PACSIN3 binds proteins such as and in and PACSIN3 overexpression J. B. B. M. M. J. Cell 2000; PubMed Google Scholar). ADAM12 interacts with signaling molecules containing an SH3 domain such as Src and the regulatory of and the sites for proteins are in and in or of the ADAM12 cytoplasmic tail, A. N. T. Y. Izumi T. T. H. Endo T. 2000; PubMed Scopus Google Scholar, Y. A. J. 2000; PubMed Scopus Google Scholar, Y. A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Binding using a of truncated the sites to and in ADAM12 cytoplasmic These that the sites are by several proteins containing SH3 the of proteins to ADAM12 cytoplasmic is unknown. revealed that was the and in the membrane endogenous ADAM12 also the other was in a with other reports indicating that ADAM12 is in the and in the membrane N. A. Y. Izumi T. Endo T. J. 2000; PubMed Scopus Google Scholar, S. F. R. R. Res. 2000; PubMed Scopus Google Scholar). with in the that PACSIN3 with ADAM12 in the Our are also by the that PACSIN3-ΔSH3 not with type ADAM12, and previously binds to and with H. A. C. T. D. Biol. 2000; PubMed Scopus Google Scholar). We demonstrated that PACSIN3 knockdown by siRNA in HT1080 cells attenuated the shedding of proHB-EGF induced by TPA and angiotensin II. Thus, PACSIN3 is to the signaling pathway of the ectodomain shedding of HB-EGF induced by both TPA and by angiotensin II. molecules involved in the ectodomain shedding of proHB-EGF induced by TPA and angiotensin II. We also PACSIN3 the ectodomain shedding of proHB-EGF induced by other PACSIN3 interact with the cytoplasmic of ADAM9, ADAM10, ADAM15, and ADAM19 as well as with of ADAM12 (Fig. that PACSIN3 post-translational The of interactions between PACSIN3 and obscure. We that PACSIN3 a role as a of ADAM12 in the signaling pathway of proHB-EGF shedding induced by GPCR analyses the signaling pathway from to the of proHB-EGF leading to EGFR transactivation. We R. and M. for