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Microfibrillar Proteins MAGP-1 and MAGP-2 Induce Notch1 Extracellular Domain Dissociation and Receptor Activation

2006; Elsevier BV; Volume: 281; Issue: 15 Linguagem: Inglês

10.1074/jbc.m600298200

1083-351X

Alison Miyamoto, Rhiana L. Lau, Patrick W. Hein, J. Michael Shipley, Gerry Weinmaster,

Cell Adhesion Molecules Research

Unlike most receptors, Notch serves as both the receiver and direct transducer of signaling events. Activation can be mediated by one of five membrane-bound ligands of either the Delta-like (-1, -2, -4) or Jagged/Serrate (-1, -2) families. Alternatively, dissociation of the Notch heterodimer with consequent activation can also be mediated experimentally by calcium chelators or by mutations that destabilize the Notch1 heterodimer, such as in the human disease T cell acute lymphoblastic leukemia. Here we show that MAGP-2, a protein present on microfibrils, can also interact with the EGF-like repeats of Notch1. Co-expression of MAGP-2 with Notch1 leads to both cell surface release of the Notch1 extracellular domain and subsequent activation of Notch signaling. Moreover, we demonstrate that the C-terminal domain of MAGP-2 is required for binding and activation of Notch1. Based on the high level of homology, we predicted and further showed that MAGP-1 can also bind to Notch1, cause the release of the extracellular domain, and activate signaling. Notch1 extracellular domain release induced by MAGP-2 is dependent on formation of the Notch1 heterodimer by a furin-like cleavage, but does not require the subsequent ADAM metalloprotease cleavage necessary for production of the Notch signaling fragment. Together these results demonstrate for the first time that the microfibrillar proteins MAGP-1 and MAGP-2 can function outside of their role in elastic fibers to activate a cellular signaling pathway. Unlike most receptors, Notch serves as both the receiver and direct transducer of signaling events. Activation can be mediated by one of five membrane-bound ligands of either the Delta-like (-1, -2, -4) or Jagged/Serrate (-1, -2) families. Alternatively, dissociation of the Notch heterodimer with consequent activation can also be mediated experimentally by calcium chelators or by mutations that destabilize the Notch1 heterodimer, such as in the human disease T cell acute lymphoblastic leukemia. Here we show that MAGP-2, a protein present on microfibrils, can also interact with the EGF-like repeats of Notch1. Co-expression of MAGP-2 with Notch1 leads to both cell surface release of the Notch1 extracellular domain and subsequent activation of Notch signaling. Moreover, we demonstrate that the C-terminal domain of MAGP-2 is required for binding and activation of Notch1. Based on the high level of homology, we predicted and further showed that MAGP-1 can also bind to Notch1, cause the release of the extracellular domain, and activate signaling. Notch1 extracellular domain release induced by MAGP-2 is dependent on formation of the Notch1 heterodimer by a furin-like cleavage, but does not require the subsequent ADAM metalloprotease cleavage necessary for production of the Notch signaling fragment. Together these results demonstrate for the first time that the microfibrillar proteins MAGP-1 and MAGP-2 can function outside of their role in elastic fibers to activate a cellular signaling pathway. Notch signaling is best known for its role in cell fate determination and is critical for regulating multiple cellular processes in many different tissues, including those of the nervous, hematopoietic, and vascular systems (1Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4874) Google Scholar). Controlled by membrane-tethered ligands on apposing cells, ligand binding initiates canonical Notch signaling and leads to the proteolytic release of the Notch intracellular domain (NICD). 2The abbreviations used are: NICD, Notch intracellular domain; ADAM, a disintegrin and metalloprotease; ECD, extracellular domain; EGF, epidermal growth factor; HA, hemagglutinin; Hes, Hairy/Enhancer of split; MAGP, microfibril-associated glycoprotein; N1, Notch1; T-ALL, T-cell acute lymphoblastic leukemia; TM, transmembrane; DAPT, N-[N-(3,5-difluorophenacetyl-l-alanyl]-S-phenylglycine tert-butyl ester; TGF, transforming growth factor. 2The abbreviations used are: NICD, Notch intracellular domain; ADAM, a disintegrin and metalloprotease; ECD, extracellular domain; EGF, epidermal growth factor; HA, hemagglutinin; Hes, Hairy/Enhancer of split; MAGP, microfibril-associated glycoprotein; N1, Notch1; T-ALL, T-cell acute lymphoblastic leukemia; TM, transmembrane; DAPT, N-[N-(3,5-difluorophenacetyl-l-alanyl]-S-phenylglycine tert-butyl ester; TGF, transforming growth factor. The NICD fragment travels to the nucleus, interacts with a DNA-binding protein CSL (CBF1/Su(H)/LAG-1), and activates expression of target genes such as HES1. At least three proteolytic events are required for Notch activation through CSL. The first cleavage is ligand-independent and occurs during maturation of the co-linear Notch protein. Either during trafficking or at the cell surface, Notch is cleaved by a furin-like convertase into two fragments, the extracellular domain (NEC) and the transmembrane-anchored intracellular domain (NTM), that remain associated through non-covalent interactions (2Logeat F. Bessia C. Brou C. LeBail O. Jarriault S. Seiday N. Israel A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8108-8112Crossref PubMed Scopus (573) Google Scholar, 3Rand M.D. Grimm L.M. Artavanis-Tsakonas S. Patriub V. Blacklow S.C. Sklar J. Aster J.C. Mol. Cell. Biol. 2000; 20: 1825-1835Crossref PubMed Scopus (325) Google Scholar, 4Bush G. diSibio G. Miyamoto A. Denault J.B. Leduc R. Weinmaster G. Dev. Biol. 2001; 229: 494-502Crossref PubMed Scopus (117) Google Scholar). This "heterodimer" is the predominant form of Notch on the plasma membrane and is required for ligand-induced CSL-dependent Notch signaling (4Bush G. diSibio G. Miyamoto A. Denault J.B. Leduc R. Weinmaster G. Dev. Biol. 2001; 229: 494-502Crossref PubMed Scopus (117) Google Scholar). Accordingly, ligand engagement by the heterodimeric Notch receptor leads to sequential ADAM and γ-secretase cleavage events that facilitate the release of NICD from its membrane tether to activate signaling (5Mumm J.S. Kopan R. Dev. Biol. 2000; 228: 151-165Crossref PubMed Scopus (838) Google Scholar, 6Weinmaster G. Curr. Opin. Genet. Dev. 2000; 10: 363-369Crossref PubMed Scopus (177) Google Scholar). Underscoring the importance of Notch signaling is the finding that more than 50% of T-ALL patient samples tested so far carry activating mutations of Notch1 (N1) (7Weng A.P. Ferrando A.A. Lee W. Morris J.P.t. Silverman L.B. Sanchez-Irizarry C. Blacklow S.C. Look A.T. Aster J.C. Science. 2004; 306: 269-271Crossref PubMed Scopus (2222) Google Scholar). Interestingly, one of the mutation "hot spots" marks the area around the furin-processing site designated the heterodimerization domain (8Sanchez-Irizarry C. Carpenter A.C. Weng A.P. Pear W.S. Aster J.C. Blacklow S.C. Mol. Cell. Biol. 2004; 24: 9265-9273Crossref PubMed Scopus (159) Google Scholar). At least some of the heterodimerization domain mutations potentiate the dissociation of an engineered soluble form of the heterodimer, mimicking the biological effects of ligand-induced Notch signaling, and constructs encoding just the NTM sequences are constitutively active (8Sanchez-Irizarry C. Carpenter A.C. Weng A.P. Pear W.S. Aster J.C. Blacklow S.C. Mol. Cell. Biol. 2004; 24: 9265-9273Crossref PubMed Scopus (159) Google Scholar, 9Aster J.C. Robertson E.S. Hasserjian R.P. Turner J.R. Kieff E. Sklar J. J. Biol. Chem. 1997; 272: 11336-11343Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Together, these findings imply that heterodimer dissociation leads to ligand-independent, constitutive cleavage of NTM to produce active NICD. Further evidence that preservation of the heterodimer is important for maintaining Notch in an inactive state is the finding that treatment with calcium chelators, such as EDTA, disrupts the non-covalent interactions that hold the heterodimer together, leading to both receptor dissociation and activation of downstream signaling events (3Rand M.D. Grimm L.M. Artavanis-Tsakonas S. Patriub V. Blacklow S.C. Sklar J. Aster J.C. Mol. Cell. Biol. 2000; 20: 1825-1835Crossref PubMed Scopus (325) Google Scholar). Notch receptors and ligands of the DSL (Delta/Serrate/LAG-2) class have similarly structured extracellular domains; the bulk of which is comprised of tandem EGF-like repeats. We have recently shown that MAGP-2 interacts with the EGF-like repeats of DSL ligands Jagged1, Jagged2, and Delta1, and specifically potentiates Jagged1 shedding by ADAM sheddases (10Nehring L.C. Miyamoto A. Hein P.W. Weinmaster G. Shipley J.M. J. Biol. Chem. 2005; 280: 20349-20355Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore, given the presence of similar tandem EGF-like repeats in the N1 receptor we asked whether MAGP-2 could interact with N1 and whether this interaction had any functional consequences on N1 activity. MAGPs are best characterized as components of microfibrils, which are important structural components of elastic tissues such as the lung, skin, and vasculature but are also present in non-elastic tissues such as the ciliary zonule of the eye (11Kielty C.M. Sherratt M.J. Shuttleworth C.A. J. Cell Sci. 2002; 115: 2817-2828Crossref PubMed Google Scholar). Biochemical dissection of microfibrils has identified the major component to be fibrillin, a large modular protein that contains 47 EGF-like repeats among other motifs (12Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Crossref PubMed Scopus (899) Google Scholar). Under reducing conditions a number of small molecular weight proteins are also released from microfibrils, including MAGP-1 and MAGP-2 (13Gibson M.A. Hughes J.L. Fanning J.C. Cleary E.G. J. Biol. Chem. 1986; 261: 11429-11436Abstract Full Text PDF PubMed Google Scholar, 14Gibson M.A. Hatzinikolas G. Kumaratilake J.S. Sandberg L.B. Nicholl J.K. Sutherland G.R. Cleary E.G. J. Biol. Chem. 1996; 271: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The function of the small microfibril-associated proteins has been inferred from protein-protein interactions with both extracellular matrix and cell-associated proteins. MAGP-1 and MAGP-2 share a C-terminal domain with conserved cysteine spacing that defines their gene family (15Segade F. Trask B.C. Broekelmann T.J. Pierce R.A. Mecham R.P. J. Biol. Chem. 2002; 277: 11050-11057Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). This domain has been shown to interact with different regions of the fibrillin molecule (16Hanssen E. Hew F.H. Moore E. Gibson M.A. J. Biol. Chem. 2004; 279: 29185-29194Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 17Penner A.S. Rock M.J. Kielty C.M. Shipley J.M. J. Biol. Chem. 2002; 277: 35044-35049Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). MAGP-1 has also been shown to interact with a number of elastic fiber components beyond fibrillin, including tropoelastin and decorin, and therefore is thought to be an integral component of the elastic fiber (18Brown-Augsburger P. Broekelmann T. Mecham L. Mercer R. Gibson M.A. Cleary E.G. Abrams W.R. Rosenbloom J. Mecham R.P. J. Biol. Chem. 1994; 269: 28443-28449Abstract Full Text PDF PubMed Google Scholar, 19Trask B.C. Trask T.M. Broekelmann T. Mecham R.P. Mol. Biol. Cell. 2000; 11: 1499-1507Crossref PubMed Scopus (119) Google Scholar). MAGP-2, on the other hand, contains a RGD sequence that can mediate interactions with integrins and has a more restricted expression pattern than MAGP-1, leading to the notion that MAGP-2 may be involved in cell signaling events (20Gibson M.A. Leavesley D.I. Ashman L.K. J. Biol. Chem. 1999; 274: 13060-13065Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 21Gibson M.A. Finnis M.L. Kumaratilake J.S. Cleary E.G. J. Histochem. Cytochem. 1998; 46: 871-886Crossref PubMed Scopus (79) Google Scholar). Experimental evidence that induction of MAGP-2 expression increased collagen deposition in fibroblast culture is supportive of this notion, although in this system, no direct effect of MAGP-2 on signaling was identified (22Lemaire R. Korn J.H. Shipley J.M. Lafyatis R. Arthritis Rheum. 2005; 52: 1812-1823Crossref PubMed Scopus (28) Google Scholar). We now show that MAGP-2 can directly participate in a cell signaling pathway via an interaction with the Notch receptor that induces heterodimer dissociation and activation of signaling. DNA Constructs—The N-terminal MAGP-2 construct was generated via PCR to delete amino acids 84–162 in the pCAGGS vector. The C-terminal MAGP-2 construct was made via standard molecular biology techniques to replace the sequences between HincII and PstI with six tandem myc epitopes. The Dll3 ECDHA construct was generated via PCR to fuse the N-terminal 488 amino acids of Dll3 to three tandem HA epitope tags. The N1c/s construct was generated via a PCR overlap strategy to change the two conserved cysteines at positions 1675 and 1682 in the rat N1 sequence to serines. All constructs were sequenced to verify that no PCR-induced mutations were generated. The N1Δmyc series of constructs (23Yang L.T. Nichols J.T. Yao C. Manilay J.O. Robey E.A. Weinmaster G. Mol. Biol. Cell. 2005; 16: 927-942Crossref PubMed Scopus (168) Google Scholar), pBosHA-N1 and pBosDll3HA (24Ladi E. Nichols J.T. Ge W. Miyamoto A. Yao C. Yang L.T. Boulter J. Sun Y.E. Kintner C. Weinmaster G. J. Cell Biol. 2005; 170: 983-992Crossref PubMed Scopus (216) Google Scholar), pBosOEDN1 (25Tan-Pertel H.T. Walker L. Browning D. Miyamoto A. Weinmaster G. Gasson J.C. J. Immunol. 2000; 165: 4428-4436Crossref PubMed Scopus (45) Google Scholar), pBosΔEDN1 and pBosZEDN1 (26Shawber C. Nofziger D. Hsieh J.J.-D. Lindsell C. Bogler O. Hayward D. Weinmaster G. Development (Camb.). 1996; 122: 3765-3773Crossref PubMed Google Scholar), AT-EK1 (4Bush G. diSibio G. Miyamoto A. Denault J.B. Leduc R. Weinmaster G. Dev. Biol. 2001; 229: 494-502Crossref PubMed Scopus (117) Google Scholar), mutant, and wild-type CSL reporter (JH26 and JH28, respectively) (27Hsieh J.J. Henkel T. Salmon P. Robey E. Peterson M.G. Hayward S.D. Mol. Cell. Biol. 1996; 16: 952-959Crossref PubMed Scopus (395) Google Scholar) constructs have been previously published. A number of constructs were kind gifts, and include the N1-Fc construct from A. Chitnis, the Hes5-luciferase reporter from R. Kageyama, and the optimized reporter construct pGL3PJH26 from M. Hancock and A. Orth. Reporter Assays—COS7 cells were transfected via Lipofectamine (Invitrogen) using a total of 800 ng of DNA and 2 μl of Lipofectamine/well of a 6-well dish and incubation on cells in serum-free medium for 5 h. Usually 100 ng of Notch receptor, 100 ng of CSL-reporter, and 5 ng of CMV-Renilla luciferase (Promega) were transfected with 100 or 200 ng of MAGP-2. After serum was added back to the cultures, cells were collected at 48 h post-transfection for luciferase assays that were performed using the dual-luciferase kit (Promega) following the manufacturer's instructions on a Turner Designs Luminometer (TD-20/20). Co-culture reporter assays using Delta1- or Jagged1-expresssing L fibroblasts to activate 3T3 fibroblasts transiently expressing N1 and increasing amounts of MAGP-2 were performed as described in Ref. 24Ladi E. Nichols J.T. Ge W. Miyamoto A. Yao C. Yang L.T. Boulter J. Sun Y.E. Kintner C. Weinmaster G. J. Cell Biol. 2005; 170: 983-992Crossref PubMed Scopus (216) Google Scholar. Biotinylation of Cell Surface Proteins—Cell surface labeling and isolation of biotinylated proteins were performed as described in Ref. 24Ladi E. Nichols J.T. Ge W. Miyamoto A. Yao C. Yang L.T. Boulter J. Sun Y.E. Kintner C. Weinmaster G. J. Cell Biol. 2005; 170: 983-992Crossref PubMed Scopus (216) Google Scholar with the following modifications. 400 ng of N1 plasmid DNA was transfected into COS7 cells with 0, 400, or 800 ng of MAGP-2 plasmid DNA, using pCAGGS as filler plasmid for MAGP-2. Cells were harvested in lysis buffer as described below for immunoprecipitations. Lysates were incubated overnight at 4 °C with streptavidin-agarose (Pierce) on a rotator and then collected and washed three times with the wash buffer described below before SDS-PAGE analysis. Immunoprecipitations of the Soluble N1 Extracellular Domain—293T cells were transfected via standard calcium phosphate precipitation with 1 μg of HAN1 and 2 μg of MAGP2 in a total of 5 μg of DNA/transfection into a 60-mm dish. Approximately 24 h post-transfection, Dulbecco's modified Eagle's medium was placed on cells and collected 2 days later. Where BB94 (British Biotech) or DAPT (Calbiochem) was used, each was added with the Dulbecco's modified Eagle's medium and reapplied after 1 day in culture. Conditioned medium was collected, centrifuged at 1500 × g, then the supernatant was removed and spun again at 10,000 × g. Cleared supernatants were then immunoprecipitated with a 1/100 dilution of 12CA5 myeloma supernatant, and immune complexes were collected either on Protein G-Sepharose (Amersham Biosciences) or on Protein A-agarose (Invitrogen) after an additional incubation with rabbit anti-mouse antiserum. Beads were washed twice in phosphate-buffered saline and once in wash buffer (10 mm Tris, pH 7.4, 0.5 m NaCl, 0.5% IGEPAL, 1% deoxycholate, 1 mm EDTA) before elution and SDS-PAGE. Cell lysates were also collected at the same time as conditioned medium in lysis buffer (10 mm Tris, pH 8.5, 14 mm NaCl, 1 mm MgCl2, 0.5% deoxycholate, 1% IGEPAL, 0.1% SDS) supplemented with protease inhibitors aprotinin, leupeptin, and phenylmethylsulfonyl fluoride. Co-immunoprecipitations of N1 with MAGP-2 or MAGP-1—293T or COS7 cells were transfected either via calcium phosphate precipitation for 293T or via Lipofectamine for COS7. For co-immunoprecipitation studies, N1 constructs and MAGP-2 constructs were used at a 1:1 ratio, and cell lysates were collected 48 h post-transfection. Cell extracts were generated in lysis buffer as above and used for immunoprecipitation with either anti-N1 antiserum (PCR12), anti-MAGP2 antiserum, or anti-myc antibodies (9E10, from Santa Cruz Biochemicals). Beads were washed three times in wash buffer before immune complexes were eluted from the agarose beads. For NICD immunoprecipitations, at 2 days post-transfection cells were treated for 5 h with 10 μm MG132 (BIOMOL) to block proteasomal degradation prior to collection in lysis buffer. Western Blotting—Val1744 antiserum (Cell Signaling Technologies) and 9E10 (Santa Cruz Biochemicals) were used as per manufacturer's instructions. Antisera to intracellular and extracellular N1 (93-4 and 93-2, respectively) and MAGP-2 have been described previously (4Bush G. diSibio G. Miyamoto A. Denault J.B. Leduc R. Weinmaster G. Dev. Biol. 2001; 229: 494-502Crossref PubMed Scopus (117) Google Scholar, 10Nehring L.C. Miyamoto A. Hein P.W. Weinmaster G. Shipley J.M. J. Biol. Chem. 2005; 280: 20349-20355Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Co-immunoprecipitation of Metabolically Labeled A7R5 Cells—The rat aortic smooth muscle cell line A7R5 were grown in 100-mm dishes until they reached confluence. Each dish was washed three times with phosphate-buffered saline and then incubated for 1 h with Dulbecco's modified Eagle's medium (-Cys, -Met) (Cellgro). After the initial starvation period, fresh Dulbecco's modified Eagle's medium containing 50 μCi/ml 35S translabel (MP Biomedicals) was added to the cells and incubated overnight. The next day cell lysates were generated in a lysis buffer containing 50 mm Tris, pH 8.0, 150 mm NaCl, 0.5% Nonidet P-40, and 1 mm CaCl2. Lysates were precleared with normal rabbit serum and then used for immunoprecipitation with anti-N1 extracellular domain antiserum (αN1-e, 93-2), preimmune serum for 93-2, anti-MAGP-2 antiserum, or anti-Fibrillin1 antiserum (exons 36–44) (28Ritty T.M. Broekelmann T.J. Werneck C.C. Mecham R.P. Biochem. J. 2003; 375: 425-432Crossref PubMed Scopus (67) Google Scholar). After collection on Protein A-agarose beads, three washes were performed with the lysis buffer before the immune complexes were eluted from the agarose beads and run on SDS-PAGE. Gels were fixed in a solution of 20% methanol and 10% acetic acid, dried, and exposed to PhosphorImager screens (Amersham Biosciences). Scanning was done on the Typhoon 9410 and visualized using ImageQuant software. Notch1 and MAGP-2 Interact via the Notch EGF-like Repeats—Having previously characterized Jagged1 interactions with MAGP-2 via co-immunoprecipitation (10Nehring L.C. Miyamoto A. Hein P.W. Weinmaster G. Shipley J.M. J. Biol. Chem. 2005; 280: 20349-20355Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), we first asked whether N1 and MAGP-2 would also interact in co-immunoprecipitation experiments. Given the likelihood that the EGF-like repeats of N1 would mediate this interaction, we made use of a panel of deletion constructs that encoded various amount of the extracellular domain of N1 (Fig. 1A). Either full-length Notch1 (N1) or constructs encoding 4 EGF-like repeats (ΔEDN1), 1.5 EGF-like repeats (OEDN1), or no EGF-like repeats (ZEDN1) were expressed with MAGP-2 in COS7 cells, and immunoprecipitation with N1 antiserum was performed on cell lysates. MAGP-2 was found in immunoprecipitations from lysates containing N1 or ΔEDN1 (Fig. 1B, lanes 2 and 3) but not OEDN1 or ZEDN1 (Fig. 1B, lanes 4 and 5). This interaction was specific, because MAGP-2 was not recognized on its own by the N1 antiserum (Fig. 1B, lane 1) and required that MAGP-2 and N1 be in the same cell, because MAGP-2 did not interact with N1 in mixed lysate controls (Fig. 1D, lower panels). Together this suggests that the EGF-like repeats of N1 mediate an interaction with MAGP-2 and that more than one N1 EGF-like repeat is required for the interaction. Because the LIN-12/Notch repeats are also deleted in the two constructs that do not interact with MAGP-2, it remained possible that it was this domain that mediated the interaction. Therefore, we also co-expressed MAGP-2 with an Fc-tagged construct encoding only N1 EGF-like repeats 1–12 and found that the two proteins formed a complex in the conditioned medium (Fig. 1C). Together, these data indicate that the EGF-like repeats of N1 are sufficient to mediate an interaction between MAGP-2 and N1. Endogenous MAGP-2 and Notch1 Interact—To confirm the possibility of a biological role of MAGP-2 in Notch signaling, we asked whether the endogenous proteins also interact. N1 and MAGP-2 are both expressed in the A7R5 rat aortic smooth muscle cell line, facilitating detection of interactions using co-immunoprecipitation of metabolically labeled cells. For these experiments we used antiserum to the extracellular domain of N1 (αN1-E), which we determined did not cross-react with MAGP-2 in overexpression studies (Fig. 2A). The αN1-E antiserum, but not preimmune serum, could co-immunoprecipitate a protein similar in apparent molecular weight as MAGP-2 immunoprecipitated with anti-MAGP-2 antiserum (Fig. 2B, lanes 1–3). Furthermore, we also tested antiserum against fibrillin-1, one of the best characterized MAGP-2-interacting proteins, and detected a protein similar in size to MAGP-2 in the fibrillin-1 immunoprecipitates (Fig. 2B, lane 4). Finding the previously characterized fibrillin-1·MAGP-2 complex under our co-immunoprecipitation conditions verified that these conditions were suitable for characterizing other relevant MAGP-2 interacting proteins. MAGP-2 Generates a Soluble Form of the Extracellular Domain of Heterodimeric Notch1—We next asked what consequence MAGP-2 binding had on the N1 receptor, specifically if MAGP-2 induced release of the N1 extracellular domain (ECD), much like what we reported for Jagged1. For these experiments, we used an engineered form of rat N1 containing three HA-epitope tags just downstream of the signal peptide to facilitate detection of the N1 ECD (HA-N1). 293T cells were transfected with HA-N1 and either vector or MAGP-2 and conditioned medium collected and immunoprecipitated with anti-HA monoclonal antibodies (12CA5). The anti-HA precipitates from the conditioned medium were run on Western blots and probed with 12CA5 for HA-N1. Anti-HA Western blotting revealed a protein of ∼200 kDa in the conditioned medium that was specific to samples containing MAGP-2 (Fig. 3A, top left panel). The protein was approximately the same size as the furin-cleaved HA-NEC present in whole cell lysates and did not run similarly to full-length HA-N1 (HA-N1FL) (Fig. 3A, compare right and top left panels). This is an important consideration, because it indicates that we did not simply induce cell lysis or "blebbing" that would allow detection of full-length N1 molecules in the medium. Moreover, when the same blots were reprobed with antiserum to the N1 intracellular domain that would recognize both full-length HA-N1 and the NTM fragment, neither of these proteins were detected in the anti-HA precipitates (data not shown). Importantly, MAGP-2 was detected in the anti-HA precipitates from the conditioned medium (Fig. 3A, bottom left panel), indicating that MAGP-2 and the HA-N1 ECD remained stably associated following release from the cell surface. Because N1 exists as both an uncleaved co-linear protein and a heterodimer on the cell surface, we asked whether the MAGP-2-mediated generation of soluble HA-N1 ECD was dependent on furin-like cleavage and heterodimer formation. Cells were co-transfected with a competitive substrate for furin-like convertases, AT-EK1, that we have previously shown to prevent N1 furin-like cleavage and heterodimerization (4Bush G. diSibio G. Miyamoto A. Denault J.B. Leduc R. Weinmaster G. Dev. Biol. 2001; 229: 494-502Crossref PubMed Scopus (117) Google Scholar), along with HA-N1 and MAGP-2 and conditioned medium was assayed for HA-N1 ECD via 12CA5 immunoprecipitation. In the presence of AT-EK1, the MAGP-2-induced generation of soluble HA-N1 ECD was greatly reduced (Fig. 3B, top panel), suggesting that the furin-like cleavage of N1 into a heterodimeric receptor is necessary for MAGP-2-induced release of N1 ECD from cells. Notch1 ECD Release Facilitated by MAGP-2 Does Not Require ADAM Cleavage—We initially predicted that the MAGP-2-induced shedding of HA-N1 ECD would be mediated by ADAM cleavage. This seemed likely because ADAM cleavage is a necessary step in Notch receptor activation and because MAGP-2 can induce an ADAM-like cleavage of the Notch ligand Jagged1. To test this theory we evaluated whether MAGP-2-induced production of soluble HA-N1 ECD was sensitive to ADAM inhibitors. For these studies we expressed HA-N1 and either vector or MAGP-2 in 293T cells and prepared conditioned medium in the presence of BB94, a hydroxamate-based metalloprotease inhibitor, or Me2SO control. Both in the presence and absence of BB94, HA-N1 ECD could be detected (Fig. 4A, compare lanes 2 and 4), indicating that HA-N1 ECD release by MAGP-2 is ADAM-independent. Parallel proteolysis experiments described in the next section verified that BB94 and DAPT blocked ADAM and γ-secretase activity, respectively (Fig. 4B). Therefore, this suggests that in contrast to ADAM shedding of Jagged1, MAGP-2 induces a dissociative event of the N1 heterodimer independent of ADAM cleavage. However, it remains possible that MAGP-2 induces an alternative cleavage event that is not blocked by the pan-metalloprotease inhibitor BB94. MAGP-2 Leads to Increased Production of NICD and Notch-dependent Reporter Activity—In other systems it has been shown that the presence of the extracellular domain of N1 acts to negatively regulate receptor activation (29Lieber T. Kidd S. Alcamo E. Corbin V. Young M.W. Genes Dev. 1993; 7: 1949-1965Crossref PubMed Scopus (361) Google Scholar, 30Kopan R. Schroeter E.H. Weintraub H. Nye J.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1683-1688Crossref PubMed Scopus (424) Google Scholar). Furthermore, destabilizing mutations found in the heterodimerization domain in T-ALL patient samples or treatment of N1-expressing cells with calcium chelators such as EDTA can lead to N1 heterodimer dissociation and subsequent activation of signaling (3Rand M.D. Grimm L.M. Artavanis-Tsakonas S. Patriub V. Blacklow S.C. Sklar J. Aster J.C. Mol. Cell. Biol. 2000; 20: 1825-1835Crossref PubMed Scopus (325) Google Scholar, 8Sanchez-Irizarry C. Carpenter A.C. Weng A.P. Pear W.S. Aster J.C. Blacklow S.C. Mol. Cell. Biol. 2004; 24: 9265-9273Crossref PubMed Scopus (159) Google Scholar). Because MAGP-2 induced dissociation of the HA-N1 heterodimer, we asked what happened to the NTM cell-associated portion that remained after MAGP-2-induced loss of the ECD. For these experiments, we employed a form of N1 (N1Δmyc) optimized to detect NICD in which the C terminus is replaced with six myc epitope tags (23Yang L.T. Nichols J.T. Yao C. Manilay J.O. Robey E.A. Weinmaster G. Mol. Biol. Cell. 2005; 16: 927-942Crossref PubMed Scopus (168) Google Scholar). This truncated protein facilitates detection of N1Δmyc cleavage products that differ by only a small number of amino acids. Transfection of 293T cells with this construct and either vector or MAGP-2 was followed by immunoprecipitation with anti-myc antibodies (9E10) and Western blotting with the same antibody. As seen in Fig. 4B, MAGP-2 increased production of a NICD-like fragment from N1Δmyc (lanes 7 and 8, asterisk). Production of NICD by MAGP-2 was suppressed by both ADAM and γ-secretase inhibitors (BB94 and DAPT, respectively), indicating that the MAGP-2-induced NICD fragment requires the same cleavage events as ligand-induced Notch signaling (Fig. 4B, lanes 8, 10, and 12). Furthermore, the same precursor-product relationships described for NICD generated through Notch ligand activation (23Yang L.T. Nichols J.T. Yao C. Manilay J.O. Robey E.A. Weinmaster G. Mol. Biol. Cell. 2005; 16: 927-942Crossref PubMed Scopus (168) Google Scholar) are also found for MAGP-2 treatment; BB94 blocks the ADAM eve