Roles of Meltrin beta /ADAM19 in the Processing of Neuregulin*

Kyoko ShirakabeDagger, Shuji Wakatsuki, Tomohiro Kurisaki, and Atsuko Fujisawa-Sehara§

From the Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan and the § Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan

Received for publication, August 29, 2000, and in revised form, November 17, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta 1 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-beta 1. Furthermore, expression of protease-deficient mutants of meltrin beta  exerted dominant negative effects on the basal processing of NRG-beta 1. These results indicate that meltrin beta  participates in the processing of NRG-beta 1. Since meltrin beta  affected the processing of NRG-beta 4 but not that of NRG-alpha 2, 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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Various intracellular signaling and adhesion molecules govern the cell-cell interactions during the development of multicellular organisms. The actions of these molecules are regulated not only by transcriptional and translational controls but also by post-translational modifications such as phosphorylations and proteolytic processings. Numerous membrane-anchored signaling molecules are subjected to proteolytic processing to release their extracellular domains. Such modifications may cause qualitative and irreversible changes in the functions of these molecules.

ADAMs1 (a disintegrin and metalloproteases; also known as MDC proteins, metalloprotease/disintegrin/cysteine-rich proteins) are a family of membrane-anchored glycoproteins (1, 2) which play important roles in sperm-egg binding and fusion (3, 4), muscle cell fusion (5), neurogenesis (6), and development of various epithelial tissues (7). At present, more than 30 ADAM cDNAs have been cloned from various species. Since more than half of these have a catalytic site consensus sequence for metalloproteases (HEXGHXXGXXHD), they are predicted to be catalytically active proteases. Genetic and biochemical evidence indicate that some ADAMs participate in the processing of the extracellular domain of membrane-anchored proteins. TACE (tumor necrosis factor-alpha converting enzyme)/ADAM17 was initially identified as the protease responsible for the processing of pro-tumor necrosis factor-alpha (8, 9). Furthermore, studies on the disruption of the mouse TACE gene demonstrated that TACE is involved in the processing of extracellular domains of several membrane-anchored proteins including tumor necrosis factor p75 receptor, the adhesion molecule L-selectin, amyloid precursor protein, and transforming growth factor-alpha (7, 10). Kuzbanian/ADAM10 is involved in the neurogenesis of Drosophila (6), and processes and releases a soluble form of Delta, a Notch ligand (11). Recently, it has been reported that meltrin gamma /ADAM9 is involved in the processing of heparin-binding EGF-like growth factor (12). Mouse Kuzbanian and meltrin gamma  also cleave amyloid precursor protein (13, 14). These findings strongly suggest potential roles of ADAM metalloproteases in the proteolytic processing of various membrane-anchored proteins.

Previously, our group and C. P. Blobel and his colleagues (15, 19) cloned an ADAM with a conserved active site of a metalloprotease, meltrin beta /ADAM19. Meltrin beta  consists of multiple domains including prodomain, metalloprotease domain, disintegrin domain, cysteine-rich domain, epidermal growth factor (EGF)-like domain, transmembrane domain, and cytoplasmic tail. During mouse embryogenesis, meltrin beta  mRNA is markedly expressed in craniofacial and dorsal root ganglia (DRG) and ventral horns of the spinal cord, where peripheral neuronal cell lineages differentiate (16). Heart, lung, skeletal muscle, and intestine also express meltrin beta  mRNA transiently (16). These expression patterns of meltrin beta  coincide well with that of neuregulin-1 (NRG) (17, 18).

NRGs (also known as acetylcholine receptor inducing activity, glial growth factor, heregulin or neu differentiation factor) are a group of growth factors that are members of the EGF family. NRGs mediate an array of biological effects, including the synthesis of acetylcholine receptors in skeletal muscle (19) and the stimulation of Schwann cell growth (17). These biological effects of NRGs are mediated by the ErbB family of tyrosine kinase receptors (20, 21). Gene disruption studies indicate that NRGs are essential for early heart and central nervous system development (22). A variety of different protein isoforms are produced from the single NRG gene via alternative splicing mechanisms. All isoforms contain an EGF-like domain sufficient for biological activity. Although alternatively spliced transcripts also generate some secreted isoforms (17), most soluble NRGs are derived from membrane-anchored precursor proteins via proteolytic cleavage of the extracellular region including EGF-like domain. It has been reported that this processing occurs in intracellular organellas (23). However, the nature of the processing enzyme remains elusive.

In this study, we examined whether meltrin beta  participates in the processing of membrane-anchored NRGs. First, both meltrin beta  and NRG proteins were expressed in DRG neurons at the same stages of mouse embryogenesis. Next, overexpression of wild-type meltrin beta  significantly increased the release of soluble NRGs in culture medium and decreased the cell surface expression of the extracellular domains of NRG-beta 1. Furthermore, the processing of NRGs was abrogated by expression of protease-deficient mutants of meltrin beta . Finally, the enhanced processing of NRGs by meltrin beta  was blocked by the treatment with brefeldin A but not by monensin, which suggested the action of meltrin beta  in the Golgi apparatus. Taken together, we concluded that meltrin beta  (or similar ADAM proteases) participates in the cleavage of membrane-anchored NRGs.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Immunohistochemistry-- Anti-meltrin beta  antiserum used in this study was raised in rabbits against a keyhole limpet hemocyanin-coupled peptide (PEYRSQRVGAIISSKI) corresponding to the extreme C-terminal sequence of meltrin beta . E12.5 mouse embryos were dissected, rinsed with phosphate-buffered saline (PBS), incubated in PBS containing 20% sucrose at 4 °C overnight, and embedded in OCT compound (Tissue-Tek, Miles Inc.) on a dry ice block. Cryosections (6 µm in thickness) on 3-aminopropyltriethoxysilane-coated glass slides were prepared and fixed for 5 min in acetone at -20 °C. Antibodies were applied overnight at 4 °C in a humidified chamber in PBS containing 10% heat-inactivated normal goat serum at the following dilutions: anti-meltrin beta  antiserum, 1:300; anti-NRG Ab-3 (NeoMarker), 1:300, and anti-neurofilament 160 (NF160, Sigma), 1:500. The slides were washed three times for 10 min each in PBS containing 0.05% Tween 20 (PBST), then incubated in secondary antibodies in PBS containing 10% heat-inactivated normal goat serum for 1 h at room temperature. The slides were then washed three times in PBST and mounted with PERMAFLOUR (Immunotech). The anti-meltrin beta  and anti-NRG immunoreactivities were amplified using biotinylated anti-rabbit IgG (1:500 or 1:1000 dilution, Vector Laboratories) and streptavidine-Cy3 (1:500 or 1:1000 dilution, Jackson ImmunoResearch). Fluorescein isothiocyanate-conjugated anti-mouse IgG (1:500 dilution, Jackson ImmunoResearch) was used for the anti-NF160 immunoreactivity. Imaging was carried out using a Leica DM IRBE inverted confocal microscope using ×10 and ×40 objectives (Leica) and TCS-NT software (Leica).

Cell Culture-- P19 rat embryonic carcinoma cells were cultured in minimum essential medium alpha  medium supplemented with 10% fetal bovine serum. Mouse L929 fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. All cultures were maintained at 37 °C in the presence of 5% CO2. To induce differentiation, P19 cells were cultured on bacterial grade dishes to form aggregates for 4 days in the presence of 1 µM retinoic acid (Sigma) and then replated on tissue culture grade dishes in growing medium.

RT-PCR-- mRNA was extracted from P19 cells using Micro-FastTrack 2.0 kit (Invitrogen). Two ng of mRNA was subjected to one RT-PCR reaction. Reverse transcription was carried out using SuperScript II reverse transcriptase (Life Technologies, Inc.). PCR reactions were performed using an annealing temperature of 50 °C for 20 cycles (NRG and meltrin beta ) or 55 °C for 15 cycles (glyceraldehyde-3-phosphate dehydrogenase). Signals are roughly proportional to the amount of cDNA under these conditions. The following primer pairs were used: 5'-ACATCAACATCCACGACTGGGACCAGCCATCT-3' and 5'-GCAGTAGGCCACCACACACATGATGCC-3' (NRG), 5'-GCGGAATTCGGAGGCCGGAGGTGTGGCAAC-3' and 5'-GGCGTCGACGGTGCCATCCATCTGATAATA-3' (meltrin beta ), 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3' (glyceraldehyde-3-phosphate dehydrogenase).

Expression Plasmids and Transfection-- The full-length mouse NRG cDNAs were isolated using the primers corresponding to the nucleotide sequences (5'-GGCTCTAGACATGTCTGAGCGCAAAGAAGGCAG-3' and 5'-GGCTCTAGATTATACAGCAATAGGGTCTTGGTTAGC-3') from murine neonatal muscle and E12.5 mouse embryo trunk cDNAs. The mouse NRG cDNAs were fused with a synthetic DNA cassette coding for the hemagglutinin (HA)-epitope tag (MYPYDVPDYA) and subcloned into pEF-BOS, which has the promoter region of the human EF-1alpha chromosomal gene (24), to obtain pEF-BOS-HA-NRGs. A couple of protease-deficient (E347Q and H346A,H350A) meltrin beta  cDNAs and a metalloprotease domain-deleted (Delta MP) meltrin beta  cDNA were constructed by mutagenesis based on a PCR technique using mutated primers. In E347Q mutant meltrin beta , glutamine is substituted for the conserved glutamic acid at position 347. In H346A,H350A mutant meltrin beta , alanines are substituted for the conserved histidines at positions 346 and 350. In Delta MP meltrin beta , amino acid residues 208-430 are deleted. The nucleotide sequences of the mutants were confirmed by direct sequencing. The cDNAs of wild-type and mutant meltrin beta  were subcloned into pEF-BOS. Wild-type meltrin gamma  was also subcloned into pEF-BOS (12). pBIE plasmid was generated by deletion of the human cytomegalovirus promoter region of pIRES2-EGFP (CLONTECH) and replaced by the promoter region of pEF-BOS. Wild-type and Delta MP meltrin beta  cDNAs were subcloned into pBIE to generate pBIE-meltrin beta  and pBIE-Delta MP meltrin beta , respectively. These plasmids were transfected by the LipofectAMINE PLUS method according to the manufacturer's instructions (Life Technologies Inc.).

Western Blot Analysis-- Before harvesting the conditioned medium, the cells were incubated in Opti-MEM (Life Technologies, Inc.) containing CaCl2 (110 µg/ml) for 12 h. The conditioned medium was initially filtered through a sterile filter unit (pore size: 0.2 µm, Millipore) and then concentrated up to 100-fold by centrifugation using a Centriplus 10 (Amicon) concentrator. Cells were extracted in extraction buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Nonidet P-40, 0.1% sodium deoxycholate) containing CompleteTM protease inhibitor mixture (Roche Molecular Biochemicals). Extracts were clarified by centrifugation at 15,000 × g for 10 min. Protein concentration was determined using the Bradford method (Bio-Rad). Approximately 20 µg of protein was loaded into each well. After SDS-PAGE, proteins were electroblotted onto Immobilon (Millipore). Anti-HA mouse monoclonal antibody (1:40 dilution; 12CA5, Roche Molecular Biochemicals), anti-meltrin beta  rabbit antiserum (1:500 dilution), and anti-C terminus of NRG rabbit polyclonal antibody (1:100 dilution; sc-348, Santa Cruz) were used as primary antibodies. After incubation with primary antibody, the blots were incubated with biotinylated anti-mouse or anti-rabbit IgG (1:2500 dilution, Jackson ImmunoResearch) and then with horseradish peroxidase-conjugated streptavidin (1:5000 dilution, Amersham Pharmacia Biotech). The blots were developed using the ECL plus system (Amersham Pharmacia Biotech). Prestained protein molecular weight marker was from Bio-Rad.

Cell Staining-- The cells transfected with pEF-BOS-HA-NRG-beta 1 together with pBIE, pBIE-meltrin beta , or pBIE-Delta MP meltrin beta  were incubated with anti-HA mouse monoclonal antibody (1:200 dilution; 16B12, Babco) at 4 °C for 30 min, and then washed four times with ice-cold PBS. After fixation with 4% paraformaldehyde in PBS for 15 min, the cells were incubated with Cy3-conjugated goat antibody to mouse IgG (1:400 dilution, Jackson ImmunoResearch) at 25 °C for 30 min. Imaging was carried out using a Leica DM IRBE inverted confocal microscope using a ×63 oil objective (Leica) and TCS-NT software (Leica).

Metabolic Labeling of Cells-- Cells were starved in medium lacking methionine and cysteine (ICN) for 1 h and pulse-labeled with [35S]methionine and -cysteine (EASYTAG express protein labeling mixture, PerkinElmer Life Sciences) at 0.1 mCi/ml. After a 1-h pulse, cells were either extracted in extraction buffer containing CompleteTM protease inhibitor mixture or chased with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Where indicated, the secretory pathway inhibitors, brefeldin A (10 µg/ml, Wako) and monensin (2 µM, Wako), were added during the chase period. After a 5-h chase, cells were extracted in extraction buffer. Cell extracts were clarified by centrifugation at 15,000 × g for 20 min. The supernatants were incubated with anti-HA monoclonal antibody (16B12, Babco) for 30 min on ice. After the addition of protein G-Sepharose beads, the extracts were incubated for 1 h on ice. Immunoprecipitates were washed six times with extraction buffer and subjected to SDS-PAGE.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Coincidental Expression of Meltrin beta  and NRGs-- We previously reported the expression pattern of meltrin beta  mRNA during mouse embryogenesis (16). Meltrin beta  mRNA is markedly expressed in the regions where peripheral neuronal cell lineages differentiate including craniofacial and DRG and ventral horns of the spinal cord. In addition, heart, lung, skeletal muscle, and intestine express meltrin beta  mRNA transiently. This expression pattern of meltrin beta  coincides well with that of an EGF family growth factor, NRGs (17, 18). In this study, we further investigated the precise expression sites of NRGs and meltrin beta  proteins in the developing mouse nervous system. Adjacent transverse sections through mouse E12.5 embryo were coimmunostained with antibodies against neuronal marker, neurofilament 160 (NF160), and C-terminal domain of meltrin beta  (Fig. 1, A-C), or EGF-like domain of NRGs (Fig. 1, D-F). As reported previously, high levels of NRG protein were expressed in DRG that give rise to sensory neurons (Ref. 17, Fig. 1E) and the ventral horns of the spinal cord that produce motor neurons (data not shown). Similarly, strong and specific immunoreactivity for meltrin beta  was observed in the DRG (Fig. 1B) and the ventral horns of the spinal cord (data not shown). This result indicates that meltrin beta  protein is expressed in the regions where peripheral neuronal cell lineages differentiate during embryogenesis. In addition, immunostainings of both meltrin beta  and NRGs were detected in most NF160-positive cells in the DRG region (Fig. 1, C and F). These results clearly demonstrate that the majority of NF160-positive neuronal cell lineages in DRG express both meltrin beta  and NRGs simultaneously.


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Fig. 1.   Expression of meltrin beta  and NRGs proteins in dorsal root ganglia during mouse embryogenesis. Adjacent transverse sections through forelimb of E12.5 mouse embryos were costained with antibodies against neurofilament 160 (NF160) and meltrin beta  (A-C) or NRGs (D-F). Asterisks and arrows indicate the neural tube and the DRG, respectively. A and D, anti-NF160 staining (green). B, anti-meltrin beta  staining (red). E, anti-NRGs staining (red). C, overlay of NF160 and meltrin beta  immunostaining. F, overlay of NF160 and NRGs immunostaining. Bar, 50 µm.

The mouse embryonic carcinoma P19 cell is a multipotential stem cell, which differentiates into a variety of cell types including neuron and glia. Since this cell line is used as an in vitro model for differentiation of the nervous system, we examined the expression of meltrin beta  and NRGs in this cell line during differentiation. Differentiation was induced by aggregating P19 cells in the presence of 1 µM retinoic acid for 4 days and then the cells were dissociated and plated in the absence of retinoic acid. Cells were harvested at the indicated times, and the cell extracts were subjected to Western blotting using antibodies against the markers of neurons (microtubule-associated protein-2), glial cells (glial fibrillary acidic protein), and smooth muscle cells (smooth muscle actin). The expression of microtubule-associated protein-2 was increased at day 6 and then decreased gradually (data not shown). On the other hand, the expression of glial fibrillary acidic protein was increased during the differentiation period (data not shown). To examine the expression level of NRGs and meltrin beta  mRNAs, total mRNAs were prepared from cells at the indicated times, and subjected to RT-PCR (Fig. 2). NRGs and meltrin beta  mRNAs were both expressed at very low levels at day 0. The transcripts of these genes began to appear at day 6, then reached a maximum level at day 8. This similarity in the transcriptional profiles indicates a plausible interaction between meltrin beta  and NRGs. Many NRGs are derived from membrane-anchored precursor proteins via proteolytic cleavage. We therefore investigated whether the metalloprotease activity of meltrin beta  is involved in the processing of NRGs.


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Fig. 2.   Expression of meltrin beta  and NRGs mRNAs in differentiated P19 cells. Undifferentiated P19 cells (day 0) were cultured on bacterial grade dishes with retinoic acid for 4 days and subsequently cultured on culture grade dishes without retinoic acid for 6 days. Differentiated P19 cells were harvested at the indicated times, and the total RNA fractions of the cells were subjected to RT-PCR analysis using primer sets for meltrin beta  (top panel) and NRGs (middle panel). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) levels served as a control for template levels (bottom panel).

Proteolytic Processing of NRGs by Meltrin beta -- Alternative splicing of a single gene gives rise to multiple isoforms of NRG. Many of these encode transmembrane, glycosylated precursors of soluble NRGs. In this study, we used three transmembrane isoforms of NRG (alpha 2, beta 1, and beta 4) and the domain structure of the NRGs used here is shown schematically in Fig. 3A. The extracellular portion of these NRGs contains an immunoglobulin motif (Ig), a glycosylated spacer domain (Glyco.), and an EGF-like domain (EGF) (25). The two major classes of NRGs diverge in the C terminus of the EGF-like domain giving rise to the alpha - and beta -isoforms. Additional variation is seen in the juxtamembrane region following the EGF domain by the insertion of one of three different sequences (numbered 1, 2, or 4). To detect the ectodomains of NRGs released into the culture medium, the N terminus of NRGs was tagged with HA epitope.


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Fig. 3.   Meltrin beta  participates in the processing of NRG-beta 1. A, diagram of the domain organization of NRG isoforms used in this study. Boxes represent the major structural motifs of NRGs: an immunoglobulin-like domain (Ig), a glycosylated spacer domain (Glyco.), an EGF-like domain (EGF), and a transmembrane domain (TM). Variation among these isoforms occurs in the C terminus of the EGF-like domain and in the juxtamembrane domain. Two variant C terminus of the EGF-like domain characterize the alpha - and beta -isoforms. Further differences arise in the juxtamembrane domain to generate isoforms alpha 2, beta 1, and beta 4. HA epitope was attached to the N terminus of all NRGs. B and C, Western blotting of CM and cell extracts from L929 cells transfected with plasmids expressing HA-NRG-beta 1 and meltrin beta . Cells were transfected with the indicated plasmids and cultured for 48 h and then CM and cell extracts were harvested as described under "Experimental Procedures." CM was subjected to Western blotting using anti-HA antibody to detect soluble NRG-beta 1 (B). Cell extracts were subjected to Western blotting using anti-NRG C terminus antibody (C, upper panel) and anti-meltrin beta  antibody (C, lower panel). In lane 6 of the upper panel, 5-fold amount of the extract of NRG-beta 1 and Delta MP meltrin beta  expressing cells was used. The migration of prestained molecular mass standards is shown on the right. D, cells transfected with plasmid expressing HA-NRG-beta 1 together with pBIE (vector), pBIE-meltrin beta  (WT meltrin beta ), or pBIE-Delta MP meltrin beta  (Delta MP meltrin beta ) were stained with anti-HA antibody in the nonpermeabilized condition. Cell surface NRG-beta 1 was detected by anti-HA staining (red signals in upper panels). Transfected cells were identified by the existence of green fluorescent protein (GFP, green signals in lower panels). Note that most of GFP-positive cells expose the N-terminal HA-epitope on the cell surface in pBIE or pBIE-Delta MP meltrin beta -transfected cells but not in pBIE-meltrin beta -transfected cells.

Since DRG neurons express meltrin beta  and NRG simultaneously (Fig. 1), we first examined the processing of a neuronal type of NRG, NRG-beta 1. In this study, we used mouse L929 fibroblast which expresses a low level of endogenous meltrin beta  (data not shown). L929 cells were transfected with an expression plasmid encoding NRG-beta 1 and then the conditioned medium (CM) was subjected to Western blotting using anti-HA antibody. Released soluble NRG-beta 1 (~46 kDa) was detected in the CM of NRG-beta 1 expressing cells (Fig. 3B, lane 2). This released polypeptide could induce the tyrosine phosphorylation of ErbB2 and -3 when added to differentiated muscle cells, C2C12 (data not shown), which shows that this 46-kDa polypeptide is a functionally mature NRG-beta 1. The broad appearance of processed NRG-beta 1 band might represent the variety of multiple N-linked and O-linked glycosylation in its spacer region (26). We further investigated whether coexpression of meltrin beta  affects the release of mature NRG-beta 1. Overexpression of wild-type meltrin beta  considerably increased the release of mature NRG-beta 1 (Fig. 3B, lane 3). Western blotting of cell extracts using the anti-C terminus of NRGs antibody showed that overexpression of wild-type meltrin beta  increased the ratio of processed cytoplasmic tail of NRG-beta 1 (74 kDa, open triangle) and decreased the ratio of full-length NRG-beta 1 (120 kDa, filled triangle) (Fig. 3C, upper panel, lane 3). These results strongly suggest that meltrin beta  could potentiate the basal processing activity of NRG-beta 1.

To investigate whether the meltrin beta  protease activity is necessary for the processing of NRG-beta 1, several mutants of meltrin beta  were constructed. In E347Q and H346A,H350A meltrin beta , glutamine, and alanine residues were substituted for the glutamic acid and histidine residues, respectively, which are essential for the metalloprotease activity. In Delta MP meltrin beta , metalloprotease domain is completely deleted. Western blotting using anti-meltrin beta  antibody revealed two immunoreactive species with apparent molecular masses of 125 and 100 kDa in the cell expressing E347Q meltrin beta  as shown in the cell expressing wild-type meltrin beta  (Fig. 3C, lower panel, lanes 3 and 4). The 100-kDa form is considered to be generated by removal of the prodomain from the 125-kDa form, probably by a furin-like pro-protein convertase, which cleaves ADAMs at the sequence motif RXKR in a late Golgi compartment (27, 28). Western blotting of the cells expressing H346A,H350A meltrin beta  revealed mainly the 125-kDa unprocessed form (Fig. 3C, lower panel, lane 5).

Expression of E347Q meltrin beta  made no change in the basal processing of NRG-beta 1 (Fig. 3, B, lane 4, and C, upper panel, lane 4). This observation clearly demonstrates that protease activity of meltrin beta  is essential for the increase of NRG-beta 1 processing. On the other hand, expression of H346A,H350A meltrin beta  remarkably suppressed the release of mature NRG (Fig. 3B, lane 5). At the same time, expression of H346A,H350A meltrin beta  increased the ratio of the unprocessed form of NRG-beta 1 and decreased the ratio of its processed cytoplasmic tail in the cells (Fig. 3C, upper panel, lane 5). Expression of Delta MP meltrin beta  decreased the production of NRG-beta 1 by unknown reasons (data not shown). However, Western blotting of an increased amount of the extract revealed that expression of Delta MP meltrin beta  also increased the ratio of the unprocessed form of NRG-beta 1 and decreased the ratio of the processed form of NRG-beta 1 (Fig. 3C, upper panel, lane 6). Thus, expression of these mutants of meltrin beta  exert dominant negative effects on the basal processing of NRG-beta 1. Taken together, these results indicate that meltrin beta  participates in the processing of NRG-beta 1 through its metalloprotease activity.

Small proportion of unprocessed membrane-anchored NRGs expose their extracellular domains on the cell surface (23). To investigate the effect of meltrin beta  on appearance of the extracellular domains on the cell surface, the cells expressing HA-NRG-beta 1 together with or without meltrin beta  were stained with anti-HA antibody under the nonpermeabilized condition. In this experiment, another type of expression plasmids were constructed in which wild-type or Delta MP meltrin beta  was expressed together with green fluorescent protein (GFP) by inserting internal ribosomal entry site sequence between cDNAs encoding these proteins. The result shown in Fig. 3D showed that efficient exposure of the N-terminal HA-tag of HA-NRG-beta 1 significantly decreased in the cells expressing wild-type meltrin beta . Such an effect could not be seen in Delta MP meltrin beta  expressing cells. Thus, enhanced processing of memrane-anchored NRG-beta 1 by meltrin beta  resulted in the decreased exposure of extracellular domains on the cell surface.

We further investigated whether meltrin beta  participates in the processing of other isoforms of NRGs. The expression plasmids encoding wild-type or H346A,H350A meltrin beta  were co-transfected into L929 cells with an expression plasmid encoding NRG-alpha 2, beta 1, or beta 4. Then the amount of released mature NRGs was determined by Western blotting of CM (Fig. 4A). The amount of mature NRG-beta 1 and -beta 4 was increased by overexpression of wild-type meltrin beta  and decreased by expression of H346A,H350A meltrin beta . On the other hand, the amount of mature NRG-alpha 2 was not affected by overexpression of either wild-type or H346A,H350A meltrin beta . We then examined the effect of meltrin beta  on the stability of full-length NRGs by a pulse-chase experiment. Full-length NRG-beta 1 is proteolytically cleaved in the presence of wild-type meltrin beta  (Fig. 4B, lower panel, lane 5). This cleavage is dependent on the protease activity of meltrin beta  (Fig. 4B, lower panel, lane 6). On the other hand, full-length NRG-alpha 2 is not cleaved by meltrin beta  (Fig. 4B, upper panel, lane 5). Taken together, these results demonstrate that meltrin beta  participates in the processing of beta -type, but not alpha -type, NRGs.


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Fig. 4.   Substrate specificity of meltrin beta . A, plasmids encoding several isoforms of NRG were transfected to L929 cells together with plasmid encoding wild-type or H346A,H350A (mut) meltrin beta . CM was subjected to Western blotting using anti-HA antibody as described in Fig. 3B. B, L929 cells were transfected with plasmids encoding NRG-alpha 2 (upper panel) or NRG-beta 1 (lower panel) and meltrin beta . Cells were labeled for 1 h with [35S]methionine/cysteine (PerkinElmer Life Sciences) and immediately frozen (0) or chased with cold media for 5 h (5). Cells were extracted, and NRGs were immunoprecipitated from extracts with anti-HA antibody. All samples were subjected to SDS-PAGE. Arrowheads indicate the migration of full-length NRGs. C, cells were transfected with plasmids encoding NRG-beta 1 and meltrin beta  or meltrin gamma . CM was subjected to Western blotting using anti-HA antibody.

Recently, it has been reported that meltrin gamma  is involved in the processing of a membrane-anchored growth factor, heparin-binding EGF (12). We examined whether meltrin gamma  also participates in the processing of NRG-beta 1. Coexpression of meltrin gamma  resulted in the release of 30- and 35-kDa HA-containing region of NRG-beta 1 into CM (Fig. 4C, arrowheads). These polypeptides are much smaller than the mature soluble NRGs reported previously (20, 29). This result indicates that meltrin gamma  cleaves NRG-beta 1 in a manner different from meltrin beta .

Brefeldin A-sensitive and Monensin-insensitive Cleavage of NRG-beta 1 by Meltrin beta -- To examine whether meltrin beta  is localized in the cell surface, we carried out a cell surface biotinylation analysis using cells transfected with meltrin beta -expressing plasmid. However, we could not detect any surface-exposed meltrin beta  (data not shown). In the same experiment, the fusion meltrin beta , which has an exogenous signal sequence of human granulocyte colony-stimulating factor, was efficiently biotinylated (data not shown), thereby excluding the possibility that the result was due to experimental failure. These findings indicate that meltrin beta  is mainly localized inside of the cell. Since it has been reported that some portions of NRGs undergo intracellular proteolysis (23), we investigated the subcellular compartment in which meltrin beta  processes NRG-beta 1 using two inhibitors of the secretory pathway, brefeldin A and monensin. In the presence of brefeldin A, the NRG-beta 1 processing induced by meltrin beta  was completely blocked (Fig. 5, lane 11). On the other hand, monensin did not block the processing of NRG-beta 1 induced by meltrin beta  (Fig. 5, lane 8). Thus the processing of NRG-beta 1 induced by meltrin beta  is a brefeldin A-sensitive and monensin-insensitive event. Brefeldin A blocks traffic from the endoplasmic reticulum to the Golgi by interfering with anterograde transport from the endoplasmic reticulum to Golgi (30, 31). On the other hand, monensin is expected to interfere with the transfer across Golgi compartments and compromise secretion from the trans-Golgi (30, 32). Taken together, our results suggest that meltrin beta  participates in the intracellular cleavage of NRG-beta 1 within the Golgi apparatus.


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Fig. 5.   Brefeldin A-sensitive and monensin-insensitive cleavage of NRG-beta 1 by meltrin beta . Plasmids encoding NRG-beta 1 were transfected to L929 cells together with plasmid encoding wild-type or H346A,H350A (mut) meltrin beta . Cells were labeled for 1 h and then chased with cold media for 5 h in the absence or presence of 10 µg/ml brefeldin A or 2 µM monensin. Cells were extracted, and NRGs were immunoprecipitated from extracts with anti-HA antibody. All samples were subjected to SDS-PAGE and autoradiography. Arrowheads indicate the migration of full-length NRGs.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NRGs mediate a variety of biological functions including glial cell development, synaptogenesis, and cardiac development through the activation of the ErbB family of tyrosine kinase receptors (33). Most NRG isoforms encode membrane-anchored proteins that generate soluble ligands for the ErbB family by proteolytic cleavages. It is not yet clear, however, whether the functions of NRGs depend on actions of processed and released soluble NRGs or whether the transmembrane form is biologically active. Genetic disruption of only the intracellular domain of membrane-anchored NRG isoforms results in a similar phenotype of embryonic maldevelopment to that observed with disruption of the entire gene (34). Furthermore, deletion of the cytoplasmic tail of membrane-anchored NRGs completely abrogated the release of mature NRGs (34). These results strongly suggest that the proteolytic processings of membrane-anchored NRGs are critical regulatory mechanisms of NRG functions.

In the present study, we provided evidence that meltrin beta  participates in the processing of beta -type NRGs. Initially, both meltrin beta  and NRG proteins were found to be expressed in dorsal root ganglia at the same stages during embryogenesis (Fig. 1). During neurogenic differentiation of P19 cells, the expression of meltrin beta  and NRGs mRNA was activated in a similar fashion (Fig. 2). Next, overexpression of wild-type meltrin beta  potentiated the release of mature soluble NRG-beta 1 (Fig. 3B, lane 3) with a concomitant decrease in the cell surface expression of extracellular domains of NRG-beta 1 (Fig. 3D). The protease activity of meltrin beta  is indispensable for the potentiation of NRG-beta 1 processing (Fig. 3, B and C, lane 4). Furthermore, expression of H346A,H350A or Delta MP meltrin beta  remarkably suppressed the release of soluble NRG-beta 1 (Fig. 3B, lanes 5 and 6) with a concomitant increase in the ratio of full-length NRG-beta 1 and a decrease in the ratio of processed forms of NRG-beta 1 in the cells (Fig. 3C, lanes 5 and 6). We further confirmed the enhanced processing of NRG-beta 1 with meltrin beta  protease by the pulse-chase experiment shown in Fig. 4B. These results clearly demonstrate that meltrin beta  has functional processing activity of NRG-beta 1 and that the protease activity of meltrin beta  is necessary for constitutive processing of NRG-beta 1. This is the first report on the function of meltrin beta  and, at the same time, the first report that indicates the involvement of ADAM metalloproteases in the proteolytic processing of membrane-anchored NRGs. It is considered that meltrin beta  plays a pivotal role in the development of several organs through the processing of NRGs.

As reported previously, NRG-alpha 2 is the predominant isoform in mesenchymal cells, whereas NRG-beta 1 is the major neuronal isoform (35). The main cleavage sites in these NRG molecules are in exon-alpha and exon-beta , respectively (26). While L929 cells possess endogenous proteolytic processing activities for both alpha - and beta -type NRGs, both overexpression of wild-type and H346A,H350A meltrin beta  only affected the cleavage of beta -type NRGs. It is plausible that alpha -type NRG is cleaved by a protease(s) other than meltrin beta  in L929 cells. Alternatively, L929 cells may lack some regulatory factors that cooperate with overexpressed meltrin beta  to cleave alpha -type NRG efficiently.

Meltrin beta  expressed in L929 cells was mainly localized in the Golgi apparatus (data not shown) although intracellular localization of meltrin beta  remains to be determined precisely. Examinations of the effects of brefeldin A and monensin on the processing revealed that meltrin beta  participates in the intracellular processing of NRGs, probably in the Golgi apparatus or in monensin-insensitive secretory pathways. Recently, several reports demonstrated that some ADAMs are processed and activated in the trans-Golgi network (27, 28), and localized mainly in the Golgi apparatus (13, 27). Furthermore, Skovronsky et al. (36) have found activity of TACE and/or Kuzbanian in the trans-Golgi network. These observations and our results indicate that multiple ADAMs function in the trans-Golgi network as intracellular processing enzymes.

Expression of H346A,H350A or Delta MP meltrin beta  markedly suppressed the basal processing activity of NRG-beta 1 (Fig. 3). Genetic and biochemical characterization of other ADAM proteases also indicated such dominant negative effects of protease-deficient mutants (11-14, 37). In preliminary experiments, we found that small proportion of meltrin beta  and NRGs expressed in L929 cells could be coimmunoprecipitated (data not shown). H346A,H350A and Delta MP meltrin beta  might show dominant-negative effects through the interaction with NRGs, thereby blocking the interaction of endogenous proteases with NRGs. On the other hand, expression of E347Q meltrin beta  did not affect the basal processing activity (Fig. 3). As shown in Fig. 3C, the prodomain of E347Q meltrin beta  is removed precisely while those of H346A,H350A and Delta MP meltrin beta  are not removed. These meltrin beta  mutants might have different conformation from wild-type or E347Q meltrin beta , and their conformational abnormality might affect endogenous meltrin beta  or similar proteases to act on NRG-beta 1. The identification of the domain of meltrin beta  required for the dominant negative effect on the processing will provide further insight into the mechanism by which meltrin beta  recognizes and processes NRG-beta 1.

Phorbol ester induces the processing of several membrane-anchored proteins through the activation of protein kinase C (PKC). As reported previously in other cell types, we found that phorbol ester induces the processing and release of mature soluble NRG-beta 1 in L929 cells (Ref. 23, and data not shown). This induced processing was not suppressed by expression of H346A,H350A mutant of meltrin beta  (data not shown). Our observation indicates that meltrin beta  accounts for the constitutive processing but not for the PKC-regulated processing of NRG-beta 1. Thus, distinct pathways for the processing of NRG-beta 1 are suggested: one pathway is dependent on meltrin beta  protease while, in the other PKC-regulated pathway(s), processing is carried out by other proteases. Several reports have demonstrated that TACE, Kuzbanian, and meltrin gamma  take part in PKC-regulated processing (7, 10, 12, 13). As shown in Fig. 4C, meltrin gamma  is not able to process NRG-beta 1 as a mature form. Further studies are warranted to determine whether or not other ADAMs such as TACE and Kuzbanian participate in the PKC-regulated processing of NRG-beta 1.

In summary, we showed that meltrin beta  and NRGs are simultaneously expressed in the nervous system during development and meltrin beta  participates in the proteolytic processing of beta -type NRG isoforms which are involved in neurogenesis and synaptogenesis. During differentiation of P19 cells the activation of the meltrin beta  and NRG genes preceded that of glial fibrillary acidic protein (Fig. 2, data not shown), suggesting regulatory roles of meltrin beta  in glial cell differentiation through the release of mature NRGs. Further analysis including genetic disruption of meltrin beta  will be required to demonstrate the role of meltrin beta  in the development of the nervous system.

    FOOTNOTES

* This work was supported in part by a grant-in-aid for Scientific Research on Priority Areas (B) of The Ministry of Education, Science, Sports and Culture, research grants from the Japanese Health Science Foundation, the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare of Japan, and CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Research Fellow of the Japan Society for the Promotion of Science.

To whom correspondence should be addressed: Dept. of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Kawahara-cho 53, Shogo-in, Kyoto 606-8507, Japan. Tel.: 81-75-751-3826; Fax: 81-75-751-4646; E-mail: asehara@frontier.kyoto-u.ac.jp.

Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M007913200

    ABBREVIATIONS

The abbreviations used are: ADAM, a disintegrin and metalloprotease; NRG, neuregulin-1; EGF, epidermal growth factor; DRG, dorsal root ganglia; TACE, tumor necrosis factor-alpha converting enzyme; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; CM, conditioned medium; GFP, green fluorescent protein; PKC, protein kinase C.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Blobel, C. P. (1997) Cell 90, 589-592[CrossRef][Medline] [Order article via Infotrieve]
2. Black, R. A., and White, J. M. (1998) Curr. Opin. Cell Biol. 10, 654-659[CrossRef][Medline] [Order article via Infotrieve]
3. Blobel, C. P., Wolfsberg, T. G., Turck, C. W., Myles, D. G., Primakoff, P., and White, J. M. (1992) Nature 356, 248-252[CrossRef][Medline] [Order article via Infotrieve]
4. Cho, C., Bunch, D. O., Faure, J., Goulding, E. H., Eddy, E. M., Primakoff, P., and Myles, D. G. (1998) Science 281, 1857-1859[Abstract/Free Full Text]
5. Yagami-Hiromasa, T., Sato, T., Kurisaki, T., Kamijo, K., Nabeshima, Y., and Fujisawa-Sehara, A. (1995) Nature 377, 652-656[CrossRef][Medline] [Order article via Infotrieve]
6. Rooke, J., Pan, D., Xu, T., and Rubin, G. M. (1996) Science 273, 1227-1231[Abstract]
7. Peschon, J. J., Slack, J. L., Reddy, P., Stocking, K. L., Sunnarborg, S. W., Lee, D. C., Russell, W. E., Castner, B. J., Johnson, R. S., Fitzner, J. N., Boyce, R. W., Nelson, N., Kozlosky, C. J., Wolfson, M. F., Rauch, C. T., Cerretti, D. P., Paxton, R. J., March, C. J., and Black, R. A. (1998) Science 282, 1281-1284[Abstract/Free Full Text]
8. Black, R. A., Rauch, C. T., Kozlosky, C. J., Peschon, J. J., Slack, J. L., Wolfson, M. F., Castner, B. J., Stocking, K. L., Reddy, P., Srinivasan, S., Nelson, N., Boiani, N., Schooley, K. A., Gerhart, M., Davis, R., Fitzner, J. N., Johnson, R. S., Paxton, R. J., March, C. J., and Cerretti, D. P. (1997) Nature 385, 729-733[CrossRef][Medline] [Order article via Infotrieve]
9. Moss, M. L., Jin, S. L. C., Milla, M. E., Burkhart, W., Carter, H. L., Chen, W., Clay, W. C., Didsbury, J. R., Hassler, D., Hoffman, C. R., Kost, T. A., Lambert, M. H., Leesnitzer, M. A., McCauley, P., McGeehan, G., Mitchell, J., Moyer, M., Pahel, G., Rocque, W., Overton, L. K., Schoenen, F., Seaton, T., Su, J., Warner, J., Willard, D., and Becherer, J. D. (1997) Nature 385, 733-736[CrossRef][Medline] [Order article via Infotrieve]
10. Buxbaum, J. D., Liu, K. N., Luo, Y., Slack, J. L., Stocking, K. L., Peschon, J. J., Johnson, R. S., Castner, B. J., Cerretti, D. P., and Black, R. A. (1998) J. Biol. Chem. 273, 27765-27767[Abstract/Free Full Text]
11. Qi, H., Rand, M. D., Wu, X., Sestan, N., Wang, W., Rakic, P., Xu, T., and Artavanis-Tsakonas, S. (1999) Science 283, 91-94[Abstract/Free Full Text]
12. Izumi, Y., Hirata, M., Hasuwa, H., Iwamoto, R., Umata, T., Miyado, K., Tamai, Y., Kurisaki, T., Sehara-Fujisawa, A., Ohno, S., and Mekada, E. (1998) EMBO J. 17, 7260-7272[Abstract/Free Full Text]
13. Lammich, S., Kojro, E., Postina, R., Gilbert, S., Pfeiffer, R., Jasionowski, M., Haass, C., and Fahrenholz, F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3922-3927[Abstract/Free Full Text]
14. Koike, H., Tomioka, S., Sorimachi, H., Saido, T. C., Maruyama, K., Okuyama, A., Fujisawa-Sehara, A., Ohno, S., Suzuki, K., and Ishiura, S. (1999) Biochem. J. 343, 371-375[CrossRef][Medline] [Order article via Infotrieve]
15. Inoue, D., Reid, M., Lum, L., Kratzschmar, J., Weskamp, G., Myung, Y. M., Baron, R., and Blobel, C. P. (1998) J. Biol. Chem. 273, 4180-4187[Abstract/Free Full Text]
16. Kurisaki, T., Masuda, A., Osumi, N., Nabeshima, Y., and Fujisawa-Sehara, A. (1998) Mech. Dev. 73, 211-215[CrossRef][Medline] [Order article via Infotrieve]
17. Marchionni, M. A., Goodearl, A. D. J., Chen, M. S., Bermingham-McDonogh, O., Kirk, C., Hendricks, M., Danehy, F., Misumi, D., Sudhalter, J., Kobayashi, K., Wroblewski, D., Lynch, C., Baldassare, M., Hiles, I., Davis, J. B., Hsuan, J. J., Totty, N. F., Otsu, M., McBurney, R. N., Waterfield, M. D., Stroobant, P., and Gwynne, D. (1993) Nature 362, 312-318[CrossRef][Medline] [Order article via Infotrieve]
18. Meyer, D., and Birchmeier, C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1064-1068[Abstract]
19. Falls, D. L., Rosen, K. M., Corfas, G., Lane, W. S., and Fischbach, G. D. (1993) Cell 72, 801-815[Medline] [Order article via Infotrieve]
20. Holmes, W. E., Sliwkowski, M. X., Akita, R. W., Henzel, W. J., Lee, J., Park, J. W., Yansura, D., Abadi, N., Raab, H., Lewis, G. D., Shepard, H. M., Kuang, W. J., Wood, W. I., Goeddel, D. V., and Vandlen, R. L. (1992) Science 256, 1205-1210[Medline] [Order article via Infotrieve]
21. Wen, D., Peles, E., Cupples, R., Suggs, S. V., Bacus, S. S., Luo, Y., Trail, G., Hu, S., Silbiger, S. M., Levy, R. B., Koski, R. A., Lu, H. S., and Yarden, Y. (1992) Cell 69, 559-572[Medline] [Order article via Infotrieve]
22. Meyer, D., and Birchmeier, C. (1995) Nature 378, 386-390[CrossRef][Medline] [Order article via Infotrieve]
23. Burgess, T. L., Ross, S. L., Qian, Y., Brankow, D., and Hu, S. (1995) J. Biol. Chem. 270, 19188-19196[Abstract/Free Full Text]
24. Mizushima, S., and Nagata, S. (1990) Nucleic Acids Res. 18, 5322[Medline] [Order article via Infotrieve]
25. Peles, E., and Yarden, Y. (1993) BioEssays 15, 815-824[Medline] [Order article via Infotrieve]
26. Lu, H. S., Hara, S., Wong, L. W. I., Jones, M. D., Katta, V., Trail, G., Zou, A., Brankow, D., Cole, S., Hu, S., and Wen, D. (1995) J. Biol. Chem. 270, 4775-4783[Abstract/Free Full Text]
27. Lum, L., Reid, M. S., and Blobel, C. P. (1998) J. Biol. Chem. 273, 26236-26247[Abstract/Free Full Text]
28. Roghani, M., Becherer, J. D., Moss, M. L., Atherton, R. E., Erdjument-Bromage, H., Arribas, J., Blackburn, R. K., Weskamp, G., Tempst, P., and Blobel, C. P. (1999) J. Biol. Chem. 274, 3531-3540[Abstract/Free Full Text]
29. Peles, E., Bacus, S. S., Koski, R. A., Lu, H. S., Wen, D., Ogden, S. G., Levy, R. B., and Yarden, Y. (1992) Cell 69, 205-216[Medline] [Order article via Infotrieve]
30. Dinter, A., and Berger, E. G. (1998) Histochem. Cell Biol. 109, 571-590[CrossRef][Medline] [Order article via Infotrieve]
31. Chardin, P., and McCormick, F. (1999) Cell 97, 153-155[Medline] [Order article via Infotrieve]
32. Tartakoff, A. M. (1983) Cell 32, 1026-1028[Medline] [Order article via Infotrieve]
33. Burden, S., and Yarden, Y. (1997) Neuron 18, 847-855[Medline] [Order article via Infotrieve]
34. Liu, X., Hwang, H., Cao, L., Buckland, M., Cunningham, A., Chen, J., Chien, K. R., Graham, R. M., and Zhou, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13024-13029[Abstract/Free Full Text]
35. Wen, D., Suggs, S. V., Karunagaran, D., Liu, N., Cupples, R. L., Luo, Y., Janssen, A. M., Ben-Baruch, N., Trollinger, D. B., Jacobsen, V. L., Meng, S., Lu, H. S., Hu, S., Chang, D., Yang, W., Yanigahara, D., Koski, R. A., and Yarden, Y. (1994) Mol. Cell. Biol. 14, 1909-1919[Abstract]
36. Skovronsky, D. M., Moore, D. B., Milla, M. E., Doms, R. W., and Lee, V. M. Y. (2000) J. Biol. Chem. 275, 2568-2575[Abstract/Free Full Text]
37. Hattori, M., Osterfield, M., and Flanagan, J. C. (2000) Science 289, 1360-1365[Abstract/Free Full Text]


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