©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biosynthetic Processing of neu Differentiation Factor
GLYCOSYLATION, TRAFFICKING, AND REGULATED CLEAVAGE FROM THE CELL SURFACE (*)

(Received for publication, May 15, 1995; and in revised form, June 14, 1995)

Teresa L. Burgess (§) Sandra L. Ross Yi-xin Qian David Brankow Sylvia Hu

From theDepartment of Mammalian Cell Molecular Biology, Amgen Inc., Thousand Oaks, California 91320-1789

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

neu differentiation factor (NDF), also known as heregulin, is structurally related to the epidermal growth factor family of growth factors; it stimulates tyrosine phosphorylation of the neu/HER-2 oncogene and causes differentiation of certain human breast cancer cell lines. Alternative splicing of a single gene gives rise to multiple isoforms of NDF/heregulin, as well as the neuronal homologues, designated ARIA (acetylcholine receptor inducing activity) and GGF (glial growth factor); at least 15 structural variants are known. All but two of the NDF/heregulin cDNAs are predicted to encode transmembrane, glycosylated precursors of soluble NDF.

In this report we characterized the biosynthetic processing of different NDF isoforms in stably transfected Chinese hamster ovary cells expressing individual NDF isoforms, and in the native cell line Rat 1-EJ, which expresses at least six different NDF isoforms. We found that the precursors for NDF undergo typical glycosylation and trafficking. A portion of the molecules are proteolytically cleaved intracellularly leading to the constitutive secretion of soluble, mature NDF into the culture media. However, a significant portion of the newly synthesized NDF precursor molecules escape intracellular cleavage and are transported to the cell surface of both transfected and native cells, where they reside as full-length, transmembrane proteins. Finally we show that these full-length, transmembrane NDF molecules can undergo phorbol ester regulated cleavage from the membrane, releasing the soluble growth factor into the medium.


INTRODUCTION

neu differentiation factor (NDF) (^1)was identified and purified from the conditioned medium of Rat 1-EJ cells, based on its ability to stimulate tyrosine phosphorylation of the neu oncogene (also known as HER-2 and c-erbB-2), and was shown to cause differentiation of certain human breast cancer cell lines(1, 2) . A similar strategy was employed to identify the human homologue of NDF, heregulin(3) . In addition to growth and differentiation activities, one recent study found that NDF/heregulin may function as a paracrine mediator in wound healing and tissue repair in vivo(4) . Subsequent cloning and sequencing of NDF and heregulin predicted that the secreted factor was derived from a membrane-bound precursor protein belonging to the EGF family of growth factors(2, 3) .

Until recently, NDF/heregulin was thought to be the direct ligand for HER-2/neu(1, 2, 3) ; however, several lines of investigation suggested that NDF did not directly stimulate the phosphorylation of HER-2/neu(5, 6) . It now appears that NDF binds directly to two other members of the EGF receptor family, HER-3 and HER-4, and stimulates phosphorylation of HER-2, HER-3, and HER-4 through the formation of homo- and heterodimeric receptor molecules at the cell surface ((6, 7, 8, 9, 10) ; for review, see (11) ).

The HER-2/neu oncogene has been of keen interest to tumor biologists because specific mutations in this receptor tyrosine kinase lead to the development of neuroblastomas and glioblastomas in rats. In humans, it has been shown that amplification and/or overexpression of neu/HER-2 occurs in approximately 25% of breast, ovarian, stomach, pancreatic, and bladder carcinomas. Furthermore, HER-2 overexpression in breast and ovarian cancer is associated with poor prognosis for survival ((12, 13, 14) ; see (15) for review). Less is known about the expression of the newer members of this family in human cancers (HER-3 and HER-4), but studies suggest that these too may be amplified in mammary tumors(16) . The potential role played by the ligand(s) for these receptors, including NDF/heregulin, in normal and malignant cell growth is of great interest and is an area of intense research (for reviews, see (11) and (15) ).

Two other homologues of NDF have been identified from neuronal sources, one from chicken brain called ARIA (for acetylcholine receptor inducing activity; (17) ), and one from bovine brain named GGF (for glial growth factor; (18) ). ARIA was discovered by its ability to induce the synthesis of acetylcholine receptors in muscle. It has subsequently been shown that ARIA also increases the number of sodium channels in muscle (19) and causes a change in acetylcholine receptor subunit expression from embryonic to adult isoforms(20) . Thus, ARIA may be one of the important factors regulating the architecture of the developing and adult synapse. GGF was identified based on its ability to stimulate the growth of Schwann cells (21, 22) and was recently cloned(18) . The genomic cloning of GGF suggested that extensive alternative splicing of different functional domains within this single gene gives rise to all of the NDF/heregulin isoforms of this growing family of EGF-related growth/differentiation factors(18) .

At least 15 variants of the NDF/heregulin family have been cloned from mesenchymal and neuronal sources, and many have been shown to display a tissue-specific pattern of expression (for review see (23) ). Six different isoforms of rat NDF have been identified by exhaustive cDNA cloning from the ras-transformed Rat 1-EJ cells (24) . Based on their DNA sequences, all but one of the NDF cDNAs isolated from Rat 1-EJ were predicted to encode membrane-bound, glycosylated precursors of soluble NDF, henceforth referred to as proNDF.

As a prerequisite to understanding the potential functional variation of this structurally diverse family, we have begun to characterize the biosynthetic processing of different NDF isoforms in stably transfected CHO cells expressing individual NDF isoforms, and in the native cell line, Rat 1-EJ. Because of the structural similarity of the proNDFs to proTGFalpha and other membrane-anchored growth factors(25, 26) , we have also explored the possibility that unprocessed proNDF might be transported intact to the cell surface, where it could undergo regulated proteolysis to control release, and/or be involved in juxtacrine signaling via cell-cell interactions with receptors on neighboring cells(27) .

Using a combination of pulse-chase, deglycosylation and cell surface labeling techniques we provide evidence that proNDFs undergo typical glycosylation and trafficking. However, only a portion of the molecules undergo intracellular proteolysis, such that at steady state a significant level of full-length, membrane-bound proNDF is exposed at the cell surface in both native and transfected cells. Finally, we show that proNDF can undergo phorbol ester regulated cleavage from the membrane releasing the soluble growth factor into the medium. The biological implications of these results are considered in the discussion.


EXPERIMENTAL PROCEDURES

Cell Lines, Transfection, and Cell Culture

Chinese hamster ovary cells deficient in dihydrofolate reductase activity (CHO d, (28) ) were transfected with calcium phosphate using pDSRalpha2-derived expression vectors (29) containing the rat NDF or NDF cDNAs ((24) ; see Fig.1). Transfected colonies were cloned and analyzed by Western blot using an antibody directed against Escherichia coli-derived rat NDF (see below). High expressing clones for NDF and NDF were chosen for further analysis. The expression of NDF was gradually amplified with methotrexate(30) . CHO d cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, nonessential amino acids, glutamine, penicillin and streptomycin and hypoxanthine/thymidine. Transfected clones were grown in similar media using 5% dialyzed fetal bovine serum and lacking hypoxanthine/thymidine; in addition, methotrexate was added to 70 nM for the NDF-expressing clone. Rat 1-EJ, a ras-transformed Rat 1 cell line(2) , was grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, glutamine, penicillin, and streptomycin.


Figure 1: Schematic representations of proNDF isoforms expressed in Rat 1-EJ cells. Boxes represent the major structural motifs of proNDF and are roughly to scale. The extracellular N terminus (N-term.), immunoglobulin (Ig), and glycosylation (Glyco.) domains are conserved for all isoforms. Variation among isoforms occurs in the C-terminal region, beginning in the EGF domain giving rise to alpha and beta forms as shown. Further differences arise in the juxtamembrane domain to generate isoforms alpha2, beta2, and beta4; the beta3 isoform terminates before the transmembrane domain (TM). All membrane-bound forms share a common cytoplasmic portion (Cyto.) with final variation at the C terminus of the alpha forms yielding alpha2a, alpha2b, or alpha2c. Figure is redrawn with permission from Ben-Baruch and Yarden(23) .



Antibody Preparation

Recombinant E. coli-derived rat NDF used in competition experiments, the generation of rabbit polyclonal antibody 1872 and the generation and purification of CHO d-derived NDF have been described(24, 31) . The rabbit polyclonal antiserum 1219 was generated using CHO d-derived NDF as the immunogen. The antibody was purified by affinity chromatography with Actigel ALD as recommended by the manufacturer (Sterogene Bioseparation, Inc., Arcadia, CA). 1.3 mg of recombinant CHO d-derived rat NDF was coupled to 2 ml of Actigel ALD superflow resin. 20 ml of crude antiserum was bound, the column washed with PBS, and eluted with Immunopure gentle antigen/antibody elution buffer (Pierce). The eluted antibodies were pooled and dialyzed overnight against PBS and concentrated with an Amicon protein concentrator to approximately 1 mg/ml. The rabbit polyclonal antiserum 1310 is an anti-peptide antibody, generated using a peptide from the cytoplasmic domain of (pro)NDF (sequence: CNSFLRHARETPDSYRDS) covalently coupled to keyhole limpet hemacyanin as the immunogen. The affinity purification and characterization procedures were similar to antibody 1219 except that the peptide was coupled to the column matrix. We have determined that antibodies 1872, 1219, and 1310 react specifically with the alpha and beta forms of (pro)NDF in Western blotting and quantitative immunoprecipitation.

Metabolic Labeling of Cells and Quantitative Immunoprecipitation

Cells were grown to 70-80% confluence and then starved in medium lacking methionine and cysteine (ICN, Costa Mesa, CA) for 1 h. Cells were labeled with [S]methionine and -cysteine (TranS-label, ICN) at 0.5 mCi/ml. After a 30-min pulse, cells were either extracted in nonionic detergent (N-det) buffer (32) or chased with media containing excess unlabeled methionine and cysteine (Life Technologies, Inc.). Media and cell lysates were collected and protease inhibitors added immediately (aprotinin, leupeptin, pepstatin, and o-phenanthroline; Boehringer Mannheim). Samples were brought to 0.3% SDS and 1 N-det and incubated at room temperature with preimmune serum and protein A-agarose (Boehringer Mannheim) for 5 h. Antibody 1219 (5 µg) or 1872 (25 µg) was prebound to protein A-agarose in immunoprecipitation buffer (N-det with 0.3% SDS and protease inhibitors) for 5 h at room temperature (immune beads). Preimmune treated samples were then added to washed immune beads and incubated overnight at 4 °C. Immune complexes were washed, eluted in SDS-PAGE sample buffer, and analyzed by 10% reducing SDS-PAGE (Novex, San Diego, CA; (33) ). Quantitative depletion of NDF from the samples was shown by re-immunoprecipitation of the supernatants. To reduce the background, Rat 1-EJ samples were eluted and re-immunoprecipitated before SDS-PAGE analysis. Enhancement and fluorography was carried out as described(32) . For competition, 10 µg of recombinant rat NDF was added to the precleared samples prior to adding to the immune beads. Prestained protein molecular weight markers were from Bio-Rad and Amersham Corp.

Enzymatic Deglycosylation

Radiolabeled pulse-chase samples from CHO d clones were immunoprecipitated and NDF proteins eluted with 60-75 µl of elution buffer (10 mM Tris, 1 mM EDTA, 0.5% SDS, 1% beta-mercaptoethanol). 15-µl aliquots were diluted in water, and CHAPS (Sigma) was added to 19 µM. Glycosidase enzymes were added (endoglycosidase H, 2 milliunits; N-glycanase, 500 milliunits; neuraminidase, 20 milliunits; O-glyconase, 2 milliunits) (Genzyme, Boston, MA) in a final volume of 30 µl. After overnight digestion at 37 °C, samples were analyzed by 10% SDS-PAGE. For partial digestion of secreted NDF and NDF with N-glycanase, the enzyme was used at 62.5, 250, and 1000 milliunits/reaction, then incubated at 37 °C for 4 h.

Western Blot Analysis

Western blotting was carried out essentially as described(1) , except that blots were blocked, then incubated with affinity-purified 1219 at 2 µg/ml in 5% nonfat dry milk for 2 h, washed three times for 5 min each in 1% nonfat dry milk, and reacted with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Corp.) in 1% nonfat dry milk for 30 min.

Cell Surface Immunofluorescence

Transfected and untransfected CHO d cells were grown on Permanox chamber slides (Nunc, Naperville, IL). PBS-rinsed, live cells were incubated at 0 °C with antibody 1219 at 1 µg/ml for 1 h. Cells were washed three times with PBS and then incubated with fluorescein-labeled goat anti-rabbit secondary antibody for 30 min at 0 °C. Cells were washed and fixed with 4% formaldehyde for 20 min at room temperature. Slides were mounted with FITC-Guard (Testog, Chicago, IL) and then viewed and photographed on a Zeiss Axiovert 10 microscope.

Cell Surface Biotinylation and Immunoprecipitation

Cell surface proteins were biotinylated with sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin, Pierce) following procedures recommended by the manufacturer. Following cell lysis, immunoprecipitation was performed as described above. Cell surface NDF was identified by a subsequent streptavidin Western blot. For cell surface immunoprecipitation, transfected CHO d cells were grown to confluence. Cells were removed from the culture dishes with PBS containing 5 mM EDTA and 5 mM EGTA at room temperature for 10 min. The intact, suspended cells were washed with PBS three times and then incubated with 5-10 µg/ml antibody 1219 on ice for 1 h. After extensive washing with PBS, cells were lysed with N-det buffer. The nuclei were removed by centrifugation, and supernatants were subjected to immunoprecipitation with protein A-agarose beads as described above.

Regulated Processing

CHO d clones expressing NDF and NDF were grown to 70-80% confluence. Growth medium was replaced by serum-free medium for 1 h, followed by addition of fresh serum-free medium containing either 1 µM phorbol 12-myristate 13-acetate (PMA, Sigma) and 0.1% Me(2)SO or 0.1% Me(2)SO alone as a control. Medium and lysate samples were collected at 10 and 30 min. Medium samples were concentrated 10-fold by trichloroacetic acid precipitation. Samples were separated by SDS-PAGE and analyzed by Western blot.


RESULTS

Structural Variation of ProNDF

The domain structure of the NDF isoforms expressed in Rat 1-EJ cells is shown schematically in Fig.1(see also (24) ). The extracellular portion of all NDF isoforms consists of a common N terminus, which contains an immunoglobulin domain, a spacer region containing multiple N- and O-glycosylation sites, and an EGF-like repeat. The two major classes of NDF molecules diverge in the C terminus of the EGF 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 2, 3, or 4). Isoform beta3 terminates prior to the transmembrane domain and is predicted to encode a cytoplasmic version of NDF. The other isoforms all contain a common transmembrane domain. Finally, the alpha isoforms show further variation in the length of their cytoplasmic domains (Fig.1; i.e. alpha2a, alpha2b, or alpha2c), whereas the beta isoforms share the same cytoplasmic tail (Fig.1; i.e. beta2a and beta4a). Additional NDF isoforms are expressed in other tissues (for review see (23) ); for simplicity, only those identified in Rat 1-EJ cells are illustrated here (Fig.1).

Characterization of ProNDF Biosynthesis

Quantitative immunoprecipitates of pulse-chase samples from transfected CHO cells expressing proNDF were analyzed by reducing SDS-PAGE and are shown in Fig.2a (see ``Experimental Procedures''). proNDF is initially synthesized as a precursor of 63 kDa (Fig.2a, lane1, openarrowhead). Within 30 min the majority of the precursor shows a reduced mobility of 66 kDa consistent with maturation and addition of carbohydrate moieties (Fig.2a, lane2, solidarrowhead; see also Fig. 3). After 30 min of chase, and continuing for several hours, this glycosylated species is proteolytically cleaved to yield the mature 44-kDa NDF molecule, a clear precursor-product relationship is seen (Fig.2a, lanes 2-6). The prominence of the 44-kDa species in the cell extracts at 30 min, 2 h, and 4 h of chase (Fig.2a, lanes2, 3, and 5) and the later appearance of mature NDF in the media samples (Fig.2a, lanes4 and 6) are consistent with intracellular cleavage and subsequent secretion of the mature 44-kDa NDF (secreted NDF is not detected at 30 min, but is seen in the media after 1 h of chase; data not shown). Note that by 4 h, most, but not all, of the pulse-labeled 66-kDa proNDF has been converted to the mature protein. The prominent, long-lived species at 60 kDa is immunologically related to proNDF (as demonstrated by competition with excess cold NDF, data not shown) and may be an unglycoslyated and/or improperly folded proNDF remaining in the endoplasmic reticulum.


Figure 2: NDF biosynthesis: pulse-chase analysis of transfected and native NDF isoforms. NDF was quantitatively immunoprecipitated from cell extracts and conditioned medium of S-labeled pulse-chase samples derived from NDF-transfected CHO cells (a), NDF-transfected CHO cells (b), and Rat 1-EJ cells (c), followed by reducing SDS-PAGE analysis. Panels a and b, lane1, 30-min pulse extract; lane2, 30-min chase extract; lane3, 2-h chase extract; lane4, 2-h chase medium; lane5, 4-h chase extract; lane6, 4-h chase medium; all immunoprecipitated with antibody 1219. Panel c, lane1, 30-min pulse extract; lane2, 30-min pulse extract plus 10 µg of unlabeled recombinant rat NDF; lane3, 2-h chase extract; lane4, 2-h chase extract plus 10 µg of unlabeled recombinant rat NDF; immunoprecipitated with antibody 1872; lane5, 1219 Western blot of conditioned medium collected over 1 day from Rat 1-EJ cells. The migration of molecular weight standards are shown at the left.




Figure 3: Deglycosylation analysis of intracellular and secreted NDF. Anti-NDF 1872 immunoprecipitated S-labeled NDF samples were digested with endoglycosidase H (Endo H), N-glycanase (N-glyc.), and O-glycanase (O-glyc.), as indicated, and analyzed by reducing SDS-PAGE. a, pro-NDF. Lanes 1-4, 30-min pulse extract; lanes 5-9, 30-min chase extract. b, mature intracellular and secreted NDF. Lanes 1-4, 1-h chase extract; lanes5-9, 4-h chase medium. c, partial digestion of mature, secreted NDF (lanes 1-4) and NDF (lanes 5-9) with N-glycanase, showing that one or two of the three potential N-glycosylation sites are utilized. Lanes1 and 5, mock-digested; lanes2 and 6, 62.5 milliunits of N-glycanase; lanes3 and 7, 250 milliunits of N-glycanase; lanes4 and 8, 1000 milliunits of N-glycanase was added to the digest. A schematic representation of all forms of NDF is shown at the right, and the region of each gel shown is indicated.



An identical experiment was performed with CHO cells expressing proNDF (Fig.2b). Similar to proNDF, proNDF is synthesized as a large, glycosylated precursor, which is proteolytically processed to release the 44-kDa mature NDF. Due to the extended cytoplasmic domain, proNDF has a significantly larger apparent molecular weight than the proNDF (Fig.1; (24) ). Immediately following the 30-min pulse, most of the precursor migrates at about 105 kDa (Fig.2b, lane1, openarrowhead). Following the chase, proNDF migrates at about 110 kDa (Fig. 2b, lanes2, 3, and 5, solidarrowhead). Proteolytic processing of proNDF begins at a similar time as proNDF; after 30 min of chase, 44-kDa mature NDF is detected in the cell extract (compare Fig.2a, lane2 to Fig. 2b, lane2), and is later detected in the media (Fig.2b, lanes4 and 6). However, the half-life of proNDF is significantly longer than that of proNDF; even after 4 h of chase, there is still a large portion of proNDF that is uncleaved. This raised the possibility that proNDF may be transported to and expressed as a full-length, transmembrane cell surface protein (see below). Nonetheless, the pulse-chase data indicated that the precursors of proNDF and proNDF undergo at least partial intracellular cleavage with subsequent constitutive secretion of mature NDF (Fig.2, a and b, lanes 2-6).

A similar experiment was performed on Rat 1-EJ cells, which express endogenous mRNAs encoding multiple isoforms of NDF(2, 24) . Due to the lower expression level, detection of the precursor protein molecules in the pulse-labeled cell extract is more difficult. However, by using excess cold NDF to compete for specific NDF signals, we obtained evidence for the synthesis of multiple proNDF species (Fig.2c, lanes1 and 2). Consistent with predictions based on cDNA cloning of NDF from Rat 1-EJ cells, we see several different size precursors, three of which may correspond to the ``a,'' ``b,'' and ``c'' cytoplasmic domain isoforms (Fig.1), with molecular masses in the range 110, 75, and 66 kDa, respectively (Fig.2c, lane 1, arrowheads; see also Fig. 5c). Additional, higher molecular weight species are also seen; these were not predicted from the known cDNA sequences, and their identity remains uncharacterized. This analysis does not distinguish between the alpha and beta isoforms because the antibody used here reacts well with both forms. After a 2-h chase, mature, 44-kDa NDF is present in cell extracts along with detectable levels of unprocessed 110-kDa precursor (the a form; Fig.2c, lane3). Again, the specificity of the immunoprecipitate is demonstrated by using excess cold NDF to compete the signal for both the precursor and mature forms of NDF (Fig.2c, compare lanes3 and 4). Secreted, pulse-labeled NDF is barely detected even after 2 h of chase (not shown), but clearly accumulates in the media of cells labeled for longer times (data not shown) or in concentrated conditioned media subjected to Western blot analysis (Fig.2c, lane5). Thus, proNDF, naturally expressed in Rat 1-EJ cells, appears to be processed similarly to proNDF expressed in transfected CHO cells: following glycosylation, a portion of the precursor molecules are proteolytically cleaved intracellularly, with subsequent secretion of mature 44-kDa NDF. However, some of the proNDF, especially the larger a isoform(s), appear to escape intracellular proteolysis, and thus may be displayed as transmembrane, cell surface molecules (see below).


Figure 5: Analysis of cell surface proNDF, proNDF, and native proNDF. a, biotinylation of CHO cells expressing proNDF (lanes2, 5, and 8), proNDF (lanes3, 6, and 9), and control, untransfected CHO cells (lanes1, 4, and 7). Intact cells (lanes 1-3 and 7-9) were biotinylated, lysed, and immunoprecipitated with anti-NDF antibody 1219 to isolate the cell surface-expressed proNDF. Total cellular NDF was detected by first lysing cells, then biotinylating and immunoprecipitating (lanes 4-6). Biotinylated NDF was detected by a streptavidin Western blot. Specificity was shown by competition during the immunoprecipitation with recombinant rat NDF (lanes 7-9). b, cell surface immunoprecipitation of proNDF from transfected CHO cells (lanes3 and 4). Lane3, intact cells were first treated with anti-NDF antibody, then lysed and immunoprecipitated. Lane4, cells were lysed prior to immunoprecipitation, and an anti-NDF Western blot was used to detect cell surface and total NDF, respectively. Lanes 1 and 2, anti-NDF Western blot of conditioned medium showing mature, secreted NDF (lane1), and total cell lysate, showing proNDF and mature, intracellular NDF (lane2). c, cell surface biotinylation of Rat 1-EJ cells. Lane1, CHO control; lane2, proNDF-expressing CHO cells; lane3, proNDF-expressing CHO cells; lane 4, rat 1-EJ cells expressing multiple cell surface proNDF isoforms.



Glycosylation Analysis of Intracellular and Secreted Forms of NDF

We performed deglycosylation assays to determine the extent and type of carbohydrates present on intracellular and secreted NDF during its biosynthesis. CHO cells transfected with and expressing proNDF were pulse-labeled and immunoprecipitated as above. The resulting immune complexes were digested with the indicated glycosidases, and the products were analyzed by SDS-PAGE (see ``Experimental Procedures'' and Fig.3).

Immediately following the 30-min pulse-labeling, two NDF species are detected (Fig.3a, lane1). The upper band at 63 kDa is sensitive to endoglycosidase H (lane2) and N-glycanase (lane3) digestion, indicating that it has one or more N-linked carbohydrate chain, and as expected the protein has not yet been transported to the medial Golgi and acquired resistance to endoglycosidase H digestion (lanes 1-3). In addition, at this stage, it appears that proNDF is not sensitive to O-glycanase since no further increase in mobility is detected following digestion (compare lanes3 and 4). The lower band at 60 kDa does not appear to be sensitive to deglycosylation and may be an unglycoslyated and/or improperly folded proNDF that remains cell-associated, perhaps in the endoplasmic reticulum (Fig.3a; see also Fig. 2a).

After 30 min of chase, most of the NDF precursor migrates more slowly at 66 kDa (Fig.3a, lane5), is resistant to endoglycosidase H (lane6), and is sensitive to both N- and O-glycanase digestion (lanes 7-9). Thus, proNDF is glycosylated at both N- and O-linked sites and is transported through Golgi compartments where maturation of the N-linked, and addition of O-linked, carbohydrates occurs before proteolytic cleavage.

Deglycosylation analysis of the intracellular and secreted mature 44-kDa NDF is shown in Fig.3b. Both intracellular (from 1-h chase extracts; lanes1-4) and secreted mature NDF (from 4-h media; lanes 5-9) are modified by N- (lanes2 and 7) and O-linked (lanes3 and 8) carbohydrates. All mature 44-kDa NDF is resistant to endoglycosidase H (lane6). Taken together, the pulse-chase (Fig.2) and deglycosylation analyses (Fig. 3) indicate that the majority of mature, secreted NDF is derived from larger, glycosylated, membrane-bound precursor molecules by intracellular proteolysis and subsequent secretion.

An identical set of deglycosylation reactions from pulse-labeled and chased samples of CHO cells expressing proNDF was analyzed in parallel to those shown for proNDF. The biosynthesis and glycosylation of proNDF (Fig.3) and proNDF (data not shown) were nearly identical. As noted above, the only detectable biosynthetic difference found between these two isoforms was the final extent of intracellular proteolysis (see Fig.2above).

The sequences of the cDNAs encoding proNDF and proNDF each contain three potential sites for N-linked carbohydrate addition(2) . To determine how many of these sites are actually used, we performed partial N-glycanase digestion on metabolically labeled mature, secreted NDF to create a ladder of partially N-glycosylated species, each differing by one carbohydrate chain (Fig.3c). Undigested mature NDF typically migrates as two bands (lanes1 and 5). Digestion with the lowest concentration of N-glycanase led to a complete loss of the upper band and the appearance of one additional, faster migrating band (lanes2 and 6). Upon further digestion all of the NDF migrates with this faster migrating species, presumably lacking any N-linked carbohydrate. Thus, it appears that all of the secreted NDF (lanes 1-4) and NDF (lanes 5-8) contain at least one N-linked carbohydrate (lowerband, lanes1 and 5), and about half of the molecules contain two (upperband, lanes1 and 5). We could not detect any secreted NDF without N-linked carbohydrate or any NDF molecules containing three carbohydrate chains. These results are completely consistent with the recently published biochemical characterization of purified recombinant NDF(31) .

Cell Surface Expression of Full-length, Membrane-bound NDF

Our initial pulse-chase experiments (Fig.2) led us to suspect that the precursors of some NDF isoforms might be transported to and displayed at the cell surface. We could detect completely glycosylated precursor molecules even after relatively long chase periods (Fig.2, panels a and b, lanes3 and 5; panelc, lane3). Using immunofluorescence we detected NDF at the cell surface of transfected CHO cells (Fig.4). To specifically detect cell surface NDF, we incubated live cells at 0 °C with antiserum 1219 followed by a fluorescein-labeled secondary antibody. CHO cells expressing either NDF (Fig.4a) or NDF (Fig. 4b) showed strong cell surface labeling, whereas untransfected CHO cells were mostly negative; an occasional (probably dead) cell showed nonspecific staining (Fig.4c). However, since neither of these experimental approaches provided conclusive evidence for cell surface expression of transmembrane proNDF, we performed two additional types of analysis. First we used cell surface biotinylation followed by cell lysis, immunoprecipitation of all forms of NDF, and finally detected the biotinylated (cell surface) NDF species using streptavidin Western blots (Fig.5, a and c; see ``Experimental Procedures''). Second, we used cell surface immunoprecipitation to isolate the NDF molecules accessible on the cell surface and detected them by an anti-NDF Western blot (Fig.5b; see ``Experimental Procedures'').


Figure 4: Cell surface NDF immunofluorescence. Living CHO cells expressing proNDF (a) or proNDF (b) and untransfected CHO cells (c) were stained with anti-NDF antibody 1219 and a fluorescein-labeled second antibody at 0 °C. Strong cell surface NDF was detected in both transfected cell lines, but not in the untransfected CHO cells. Scalebar is 50 µm.



Two sets of samples, intact cells (Fig.5a, lanes 1-3 and 7-9) and cell lysates (lanes 4-6), were subjected to biotinylation, followed by anti-NDF immunoprecipitation. Using intact cells, we detected cell surface-exposed, (biotinylated) proNDF at 66 kDa (lane2) and proNDF at 110 kDa (lane3). Untransfected CHO cells were used as a negative control (Fig.5a, lane1), and as expected, no NDF immunoreactive protein was detected at the cell surface of untransfected cells. Unlabeled NDF was used to compete the NDF signals in an identical set of reactions (compare lanes 7-9 with 1-3).

Cell lysates were subjected to biotinylation to detect the total cell content of NDF (lanes 4-6). Untransfected CHO cells were again used as a negative control, and although some background bands were detected (lane4), lysates from cells expressing proNDF (lane5) unambiguously contained full-length, 66-kDa proNDF, which comigrates with the biotinylated, cell surface form in lane2. Lysates from cells expressing proNDF contained two apparently full-length species (lane6). The upper band at 110 kDa comigrates with the cell surface-exposed form, the lower band is only present in the cell lysate and may correspond to the 105-kDa species identified as a biosynthetic intermediate in Fig.2b, lanes1 and 2). In addition, 44-kDa NDF could be detected only in the cell lysates, not at the cell surface (data not shown, see also Fig.5b). The amount of biotin detected on proNDF in the cell lysates is much greater than the amount detected at the cell surface. However, we caution against the quantitative interpretation of these data, since additional biotinylation sites are exposed on the protein by membrane permeabilization. Nonetheless, the membrane-impermeant biotinylating reagent clearly has access to some of the proNDF molecules, suggesting that they are accessible at the cell surface.

To confirm the interpretation of the cell surface biotinylation experiments, we performed cell surface immunoprecipitations; the data for proNDF are shown in Fig.5b. Fully glycosylated, 110-kDa proNDF is the only NDF species detected when intact cells are incubated with monospecific antibodies to NDF on ice, followed by cell lysis and anti-NDF Western blot analysis (Fig.5b, lane3). In contrast, when the cells are first lysed and then subjected to anti-NDF immunoprecipitation and Western analysis, two proNDF species, as well as mature NDF are detected (Fig.5b, lane4). The upper 110-kDa form comigrates with the proNDF species detected by cell surface immunoprecipitation (Fig.5b, compare lanes3 and 4). The lower species is clearly not detected at the cell surface and may represent the 105-kDa biosynthetic intermediate described above. In addition the rabbit Ig heavy chain from the immunoprecipitation is detected by the secondary antibody just above, and partially overlapping with, mature NDF causing a slightly retarded mobility (Fig.5b, compare lanes2 and 4). The migration of mature, secreted NDF and the cell-associated mature and full-length (pro)NDF species are shown in Fig.5b (lanes1 and 2); they were directly detected by an anti-NDF Western blot of media and cell lysate samples, respectively. The amount of proNDF detected by whole cell immunoprecipitation (lane3) is less than that detected in the cell lysate. Again we caution against the quantitative interpretation of these data since the antibody may not have unhindered access to the cell surface form of NDF in the whole cell immunoprecipitation. These data show that a significant portion of proNDF is displayed at the cell surface as evinced by antibody accessibility. Very similar results were obtained using this technique on CHO cells expressing proNDF; however, the Ig heavy chain obscured the 60-66-kDa NDF precursors, making an unambiguous interpretation of the photographed data difficult (data not shown).

To determine if transmembrane proNDF was displayed at the cell surface of a non-transfected cell such as Rat 1-EJ, we used the biotinylation paradigm described above. As can be seen in Fig.5c (lane4), proNDF molecules are detected at the cell surface of NDF-expressing Rat 1-EJ cells. We can distinguish three major bands, which probably correspond to the three major size variants of proNDF, namely the a, b, and c forms at 66, 75 and 110 kDa (lane4). All three were specifically competed by the addition of excess CHO-derived NDF (data not shown). Untransfected CHO cells served as a negative control (Fig. 5c, lane1), and for comparison, the cell surface biotinylated forms of proNDF and proNDF expressed in transfected CHO cells were run on the same gel (lanes2 and 3, respectively). The lack of exact comigration of the cell surface Rat 1-EJ proNDF proteins with the CHO-derived isoforms may be due to the expression of other isoforms by the Rat 1-EJ cells (see Fig.1) or possibly to heterogeneity in the carbohydrate chains. As in the pulse-chase experiment shown above (Fig.2c), this analysis does not determine whether alpha and/or beta isoforms are represented at the cell surface of Rat 1-EJ cells, since the antibody used here recognizes both alpha and beta forms equally well.

Regulated Processing of Cell Surface ProNDF

To investigate whether proNDF could be cleaved from the cell surface by activating protein kinase C, we treated transfected CHO cells with PMA for 10 or 30 min and analyzed the effects on (pro)NDF using Western blots (Fig.6). Panela shows the results obtained using cells expressing proNDF. Samples from control cells (Me(2)SO-treated) are shown in lanes 1-3 (10 min) and 7-9 (30 min). Prominent proNDF is seen in the cell extract (Fig.6a, lanes1 and 7, solidarrowhead), and a small amount of mature NDF is detected in the concentrated medium sample (Fig.6a, lanes3 and 9, openarrowhead). Treatment of the cells with 1 µM PMA for 10 min (Fig. 6a, lanes 4-6) or 30 min (Fig.6a, lanes 10-12) led to the rapid and nearly complete loss of the 66-kDa cell surface form of proNDF (lanes 4 and 10, solidarrowhead) and a concomitant appearance of mature NDF in the media samples (lanes6 and 12, openarrowhead) indicative of protein kinase C-regulated cell surface processing. Using an anti-peptide antibody that recognizes the cytoplasmic tail of proNDFs (1310, see ``Experimental Procedures''), we detected the concomitant appearance of the cleaved, 20-kDa cytoplasmic tail fragment in the cell extracts following 10 or 30 min of PMA treatment (data not shown). The bands between 66 and 44 kDa probably represent biosynthetic intermediates or breakdown products of NDF; the distribution of these forms is not altered by PMA treatment. Thus, only the 66-kDa proNDF form, identified to be at the cell surface in the previous analysis (Fig.5), undergoes PMA-induced proteolytic release.


Figure 6: Regulated cleavage of cell surface proNDFa, proNDF; b, proNDF. Transfected CHO cells were treated for 10 min (lanes 1-6) or 30 min (lanes 7-12) with 1 µM PMA in 0.1% Me(2)SO (lanes 4-6 and 10-12) or 0.1% Me(2)SO alone as a control (lanes 1-3 and 7-9). Cell lysates and media samples were collected and NDF detected by Western blot with antibody 1219. Lanes1 and 7, control lysate; lanes2 and 8, control medium; lanes 3 and 9, concentrated control medium; lanes4 and 10, PMA lysate; lanes 5 and 11, PMA medium; lanes6 and 12, concentrated PMA medium.



An identical experiment performed with proNDF-expressing cells is shown in Fig.6b. PMA treatment led to a loss of only the 110-kDa proNDF species between 10 and 30 min (compare lanes1 and 7 to lanes4 and 10, solidarrowhead), and a parallel appearance of mature NDF in the concentrated media samples (lanes6 and 12, openarrowhead). The kinetics of PMA induced processing of proNDF is slightly slower than that of proNDF (Fig.6a), but by 30 min the processing is essentially complete. In addition, using the cytoplasmic tail antibody(1310), we detected an increase in the amount of the 65-kDa cytoplasmic tail fragment in extracts from cells treated with PMA (data not shown). It is particularly striking to note that the 105-kDa band of proNDF is clearly not affected by PMA treatment, further supporting our conclusion that 110-kDa proNDF is specifically cleaved from the cell surface by a protein kinase C-regulated mechanism. Taken together, the data presented in Fig.4-6 provide the first direct evidence that multiple isoforms of proNDF are transported to the cell surface and can undergo regulated proteolysis by a process analogous to that of proTGFalpha.


DISCUSSION

NDF/heregulins are members of the EGF family of growth factors that were first identified as possible ligands for the HER-2/neu receptor tyrosine kinase(1, 3) . More recent evidence suggests, however, that NDF/heregulins activate HER-2 indirectly by binding to the related tyrosine kinase receptors HER-3 and HER-4(6, 7, 8, 9, 10) . At least 15 different isoforms are widely expressed in both mesenchymal and neuronal tissues (see (15) for review) but are apparently derived from a single gene(18) . Many of these isoforms were identified by cDNA cloning based on homology (e.g.(24) ), or by their functional actions in various assays (e.g. ARIA and GGF; (17) and (22) ). The wide tissue distribution and multiple in vitro activities suggest that the high degree of structural diversity may reflect functional variation as well.

In this study we have shown that proNDF isoforms undergo typical glycosylation and trafficking in Rat 1-EJ cells, which naturally express at least six different isoforms of (pro)NDF and in transfected CHO cells expressing individual isoforms (Fig.1-3). One or two N-linked and multiple O-linked carbohydrate chains are added to each proNDF molecule during biosynthesis (Fig.3; (31) ). Following glycosylation, most of the proNDF and some of the proNDF molecules undergo intracellular proteolytic processing and mature 44-kDa NDF is secreted into the culture medium (Fig.2). The proNDF species naturally expressed in Rat 1-EJ cells appear to follow a very similar biosynthetic path (Fig.2c). Interestingly, a significant portion of the full-length proNDF molecules in transfected CHO and Rat 1-EJ cells escape intracellular proteolytic cleavage and are transported to and displayed at the cell surface ( Fig.4and Fig. 5). Finally, we found that cells expressing surface-exposed proNDF can be stimulated to proteolytically release soluble mature NDF by treatment with PMA (Fig.6).

NDF/heregulin belongs to a growing family of membrane-anchored growth factors including not only a number of other EGF family members (e.g. EGF, TGFalpha, etc.), but also colony stimulating factor-1, SCF, tumor necrosis factor-alpha, bride of sevenless, and others (for review see (26) ). The intact, transmembrane form of some members of this group have been shown to bind to and signal through their receptors, e.g. TGFalpha(34, 35) , and tumor necrosis factor (36) by direct cell-cell contact in culture. This form of direct cell-cell signaling has been termed ``juxtacrine signaling'' (27) as it does not involve a diffusible growth factor.

Direct evidence for a similar, biologically relevant process in vivo is unavailable; however, several lines of evidence suggest that these membrane-bound forms are important and that juxtacrine signaling may occur in vivo. The most widely cited example of the importance of a membrane-anchored growth factor is that of the steel dickie (Sl^d) mutation in mouse. The Sl^d allele encodes a mutant form of SCF, which, although it completely lacks the cytoplasmic and transmembrane domains, shows a normal expression pattern in vivo and full biologic activity in vitro(37, 38, 39, 40) . However, as the result of the Sl^d mutation, affected mice suffer from macrocytic anemia, sterility, and white coat color, presumably because of the lack of functional, membrane-anchored SCF. Whether the transmembrane form of SCF is required for the full range of physiological signaling is still a matter of speculation; other functions of the transmembrane or cytoplasmic domains, such as regulation of intracellular trafficking or cell surface proteolysis, may be essential (see below).

Evidence for the importance of juxtacrine signaling in vivo comes from several examples in Drosophila (for review, see (41) ), especially for the development of the R7 cell of the retina. Correct differentiation of the R7 cell is dependent upon the expression of the sevenless receptor tyrosine kinase in R7 and the simultaneous expression of its transmembrane ligand, bride of sevenless, in the immediately adjacent R8 cell (for review, see refs. 42 and 43). These studies strongly suggest that R8 (bride of sevenless) controls the development of R7 (sevenless) by juxtacrine signaling via their respective membrane-anchored ligand and receptor molecules.

We have provided evidence that proNDF is expressed at the cell surface of transfected and native cells, suggesting the possibility that membrane-anchored proNDF may be functionally important in vivo as is the case for SCF and bride of sevenless. The structural conservation of the transmembrane domains of the NDF/heregulin family from chicken to man (23) also suggests an important function for membrane anchoring of this growth and differentiation factor; clearly, further studies are needed to provide direct evidence for a biologically important role for membrane-anchored proNDF. It is tantalizing to note that recent studies using an antibody to the cytoplasmic tail of NDF(1310) suggest that a transmembrane form(s) of proNDF is expressed at the cell surface in the rat nervous system, especially at presynaptic endings on motor neurons. (^2)

Another possible function for the transmembrane isoforms of growth and differentiation factors such as NDF/heregulin could be to temporally restrict the action of the soluble factor by regulating its release from the cell membrane. We have shown that proNDF can be cleaved and mature NDF released by a PMA-regulated proteolysis analogous to that observed for several other membrane-anchored growth factors (for review, see (26) ). PMA-induced processing of proTGFalpha has been extensively studied, and a recent report shows that valine at the C terminus of the cytoplasmic tail of proTGFalpha is essential for regulated processing(44) . Substitution of Ala, Met, Phe, Trp, Gly, Ser, or Glu for the terminal Val severely reduces the efficiency of proTGFalpha cleavage, it appears, however, that Leu and Ile function nearly as well. Interestingly, the a forms of proNDF contain a C-terminal valine, and the b forms contain a C-terminal leucine; the c form, however, encodes a C-terminal Arg (24) but undergoes PMA-induced processing (Fig.6). Given the non-conservative nature of the Val to Arg change, it is tempting to speculate that the mechanism (at least for the c form) of proNDF processing may differ from that of proTGFalpha.

The amino acid sequences surrounding the cleavage sites of a number of transmembrane growth factors are known, and they show some modest similarity to each other(26) . Recently Lu et al.(31) identified the cleavage site(s) for NDF and NDF, and both sequences share some limited similarity to the cleavage sites of the other membrane-bound growth factors. However, because the NDF protein(s) analyzed by Lu et al.(31) was not generated by PMA-induced cleavage but was purified after uninduced release of NDF into the cell medium (predominantly intracellular cleavage), we must speculate cautiously on this point since the actual cleavage site could be different.

Further studies have shown that one pathway activating proTGFalpha processing is regulated by a PMA-sensitive, Ca-independent protein kinase C, and involves an upstream heterotrimeric G-protein(45) . While the details of this signaling pathway are just now being elucidated, a role for direct phosphorylation of proTGFalpha is unlikely. First, PMA-stimulated phosphorylation of proTGFalpha could not be detected(46) , and second, mutation or deletion of any of the four Ser and Thr residues in the cytoplasmic tail of TGFalpha does not block cleavage(44) . Currently, the regulation of processing by direct phosphorylation of proNDF by protein kinase C cannot be ruled out. All of the different proNDF isoforms from Rat 1-EJ cells contain many Ser and Thr residues in their cytoplasmic tails, including two or three potential protein kinase C phosphorylation sites(24, 47) .

Although there is no direct evidence in vivo supporting temporal control of growth factor release by proteolysis, the in vitro data presented here and elsewhere (see above) suggest that stimulation of a protein kinase C-dependent signaling pathway could regulate such a process in vivo. Other mechanisms for locally restricting the action of growth factors exist; for example, the binding of basic fibroblast growth factor to extracellular matrix proteoglycans via its heparin binding domain may prevent widespread diffusion and action of the growth factor(48) . Members of the NDF/heregulin family are also strong heparin binding growth factors(1, 17) , and a recent study has shown that secreted chicken ARIA is in fact bound to the extracellular matrix via its heparin binding domain in vivo. (^3)Regulation of the release of NDF from the extracellular matrix would provide another level of control over the factor's action.

The existence of cell surface-anchored growth factors has led to the discussion of possible ``reverse signaling'' mechanisms, whereby the cytoplasmic tail of the growth factor could be involved in intracellular signaling. Stimulation of the extracellular domain might be via juxtacrine binding to its receptor, or perhaps through interaction with a soluble ``ligand.'' This notion is still controversial; however, a recent report (49) provides evidence for a specific interaction between the cytoplasmic tail of proTGFalpha with two other cellular proteins, one of which appears to have kinase activity. Further functional studies will be needed to determine if these associated proteins are involved in signaling or regulating proteolysis, or play some other role in the function of transmembrane proTGFalpha, and whether the cytoplasmic tails of other membrane-anchored growth factors are associated with similar proteins.

There are many questions yet to be answered regarding the role of anchoring certain transmembrane growth factors. The increasing size of this family, the sequence conservation of cytoplasmic tails, and a small amount of in vivo data strongly suggest that the tails themselves, or perhaps the cell surface display per se are functionally important. Our current challenge is to design definitive experiments to determine what role(s) membrane-anchored growth factors play in vivo.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Mammalian Cell Molecular Biology, Amgen Inc., 1840 DeHavilland Dr., Thousand Oaks, CA 91320-1789. Tel.: 805-447-2493; Fax: 805-499-7464.

^1
The abbreviations used are: NDF, neu differentiation factor; ARIA, acetylcholine receptor inducing activity; GGF, glial growth factor; SCF, stem cell factor; TGF, transforming growth factor; CHAPS, 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonic acid; CHO, Chinese hamster ovary; EGF, epidermal growth factor; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PMA, phorbol 12-myristate 13-acetate.

^2
A. Sandrock and G. Fischbach, personal communication.

^3
J. Loeb and G. Fischbach, personal communication.


ACKNOWLEDGEMENTS

We acknowledge our many terrific colleagues at Amgen for their support of this research, with special thanks to Barry Ratzkin and Rachel Yabkowitz for critically reading the manuscript, Joan Bennett for help in preparation of the manuscript, and Katherine Rubenstein for help with the figures. This work could not have been completed without generous support and reagents generated by Duanzhi Wen, Yi Luo, Rod Cupples, Hsieng Lu, Naili Liu, Larry Bennett, David Chang, Donna Yanagihara, Marynette Rihanek, and Aihua Zou (all from Amgen). We are grateful to Gerald Fischbach, Jeff Loeb, and Al Sandrock (Harvard Medical School) for lively discussions and for communicating their results prior to publication.


REFERENCES

  1. Peles, E., Bacus, S. S., Koski, R. A., Lu, H. S., Wen, D., Ogden, S. G., Ben Levy, R., and Yarden, Y. (1992) Cell 69,205-216 [Medline] [Order article via Infotrieve]
  2. Wen, D., Peles, E., Cupples, R., Suggs, S. V., Bacus, S. S., Luo, Y., Trail, G., Hu, S., Silbiger, S. M., Ben Levy, R., Koski, R. A., Lu, H. S., and Yarden, Y. (1992) Cell 69,559-572 [Medline] [Order article via Infotrieve]
  3. 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]
  4. Danilenko, D. M., Ring, B. D., Lu, J. Z., Tarpley, J. E., Chang, D., Liu, N., Wen, D., and Pierce, G. F. (1995) J. Clin. Invest. 95,842-851 [Medline] [Order article via Infotrieve]
  5. Peles, E., Ben-Levy, R., Tzahar, E., Liu, N., Wen, D., and Yarden, Y. (1993) EMBO J. 12,961-971 [Abstract]
  6. Plowman, G. D., Green, J. M., Culouscou, J.-M., Carlton, G. W., Rothwell, V. M., and Buckley, S. (1993) Nature 366,473-475 [CrossRef][Medline] [Order article via Infotrieve]
  7. Carraway, K. L., III, Sliwkowski, M. X., Akita, R., Platko, J. V., Guy, P. M., Nuijens, A., Diamonti, A. J., Vandlen, R. L., Cantley, L. C., and Cerione, R. A. (1994) J. Biol. Chem. 269,14303-14306 [Abstract/Free Full Text]
  8. Sliwkowski, M. X., Schaefer, G., Akita, R. W., Lofgren, J. A., Fitzpatrick, V. D., Nuijens, A., Fendly, B. M., Cerione, R. A., Vandlen, R. L., and Carraway, K. L., III (1994) J. Biol. Chem. 269,14661-14665 [Abstract/Free Full Text]
  9. Tzahar, E., Levkowitz, G., Karunagaran, D., Yi, L., Peles, E., Lavi, S., Chang, D., Liu, N., Yayon, A., Wen, D., and Yarden, Y. (1994) J. Biol. Chem. 269,25226-25233 [Abstract/Free Full Text]
  10. Kita, Y. A., Barff, J., Luo, Y., Wen, D., Brankow, D., Hu, S., Liu, N., Prigent, S. A., Gullick, W. J., and Nicolson, M. (1994) FEBS Lett. 349,139-143 [CrossRef][Medline] [Order article via Infotrieve]
  11. Carraway, K. L., III, and Cantley, L. C. (1994) Cell 78,5-8 [Medline] [Order article via Infotrieve]
  12. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ullrich, A., and McGuire, W. L (1987) Science 235,177-182 [Medline] [Order article via Infotrieve]
  13. Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A., and Press, M. F. (1989) Science 244,707-712 [Medline] [Order article via Infotrieve]
  14. Berger, M. S., Locher, G. W., Saurer, S., Gullick, W. J., Waterfield, M. D., Groner, B., and Hynes, N. E. (1988) Cancer Res. 48,1238-1243 [Abstract]
  15. Peles, E., and Yarden, Y. (1993) Bioessays 15,815-824 [Medline] [Order article via Infotrieve]
  16. Kraus, M. H., Issing, W., Miki, T., Popescu, N. C., and Aaronson, S. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,9193-9197 [Abstract]
  17. 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]
  18. Marchionni, M. A., Goodearl, A. D. J., Chen, M. S., Bermingham-McDonough, 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]
  19. Corfas, G., and Fischbach, G. D. (1993) J. Neurosci. 13,2118-2125 [Abstract]
  20. Martinou, J.-C., Falls, D. L., Fischbach, G. D., and Merlie, J. P. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,7669-7673 [Abstract]
  21. Lemke, G. E., and Brockes, J. P. (1984) J. Neurosci. 4,75-83 [Abstract]
  22. Goodearl, A. D. J., Davis, J. B., Mistry, K., Minghetti, L., Otsu, M., Waterfield, M. D., and Stroobant, P. (1993) J. Biol. Chem. 268,18095-18102 [Abstract/Free Full Text]
  23. Ben-Baruch, N., and Yarden, Y. (1994) Soc. Exp. Biol. Med. 206,221-227 [Abstract]
  24. 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.-Y., 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]
  25. Bringman, T. S., Lindquist, P. B., and Derynck, R. (1987) Cell 48,429-440 [Medline] [Order article via Infotrieve]
  26. Massagué, J., and Pandiella, A. (1993) Annu. Rev. Biochem. 62,515-541 [CrossRef][Medline] [Order article via Infotrieve]
  27. Bosenberg, M. W., and Massagué, J. (1993) Curr. Opin. Cell Biol. 5,832-838 [Medline] [Order article via Infotrieve]
  28. Urlaub, G., and Chasin, L. A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,4216-4620 [Abstract]
  29. DeClerck, Y. A., Yean, T. D., Lu, H. S., Ting, J., and Langley, K. E. (1991) J. Biol. Chem. 266,3893-3899 [Abstract/Free Full Text]
  30. Kaufman, R. J., and Sharp, P. A. (1982) J. Mol. Biol. 159,601-21 [Medline] [Order article via Infotrieve]
  31. 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. , in press
  32. Burgess, T. L., Craik, C. S., and Kelly, R. B. (1985) J. Cell Biol. 101,639-645 [Abstract]
  33. Laemmli, U. K. (1970) Nature 227,680-685 [Medline] [Order article via Infotrieve]
  34. Wong, S. T., Winchell, L. F., McCune, B. K., Earp, H. S., Teixido, J., Massagué, J., Herman, B., and Lee, D. C. (1989) Cell 56,495-506 [Medline] [Order article via Infotrieve]
  35. Brachmann, R., Lindquist, P. B., Nagashima, M., Kohr, W., Lipari, T., Napier, M., and Derynck, R. (1989) Cell 56,691-700 [Medline] [Order article via Infotrieve]
  36. Perez, C., Albert, I., DeFay, K., Zachariades, N., Gooding, L., and Kriegler, M. (1990) Cell 63,251-258 [Medline] [Order article via Infotrieve]
  37. Flanagan, J. G., Chan, D. C., and Leder, P. (1991) Cell 64,1025-1035 [Medline] [Order article via Infotrieve]
  38. Brannan, C. I., Lyman, S. D., Williams, D. E., Eisenman, J., Anderson, D. M., Cosman, D., Bedell, M. A., Jenkins, N. A., and Copeland, N. G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,4671-4674 [Abstract]
  39. Huang, E. J., Nocka, K. H., Buck, J., and Besmer, P. (1992) Mol. Biol. Cell 3,349-362 [Abstract]
  40. Toksoz, D., Zsebo, K. M., Smith, K. A., Hu, S., Brankow, D., Suggs, S. V., Martin, F. H., and Williams, D. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,7350-7354 [Abstract]
  41. Woods, D. F., and Bryant, P. J. (1993) J. Cell Sci. Suppl. 17,171-181 [Abstract]
  42. Yamamoto, D. (1994) Bioessays 16,237-244 [Medline] [Order article via Infotrieve]
  43. Hafen, E., Dickson, B., Brunner, D., and Raabe, T. (1994) Prog. Neurobiol. 42,287-92 [CrossRef][Medline] [Order article via Infotrieve]
  44. Bosenberg, M. W., Pandiella, A., and Massagué, J. (1992) Cell 71,1157-1165 [Medline] [Order article via Infotrieve]
  45. Bosenberg, M. W., Pandiella, A., and Massagué, J. (1993) J. Cell Biol. 122,95-101 [Abstract]
  46. Pandiella, A., and Massagué, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,1726-1730 [Abstract]
  47. Woodgett, J. R., Gould, K. L., and Hunter, T. (1986) Eur. J. Bioch. 161,177-184 [Abstract]
  48. Yeoman, L. C. (1993) Oncol. Res. 5,489-499 [Medline] [Order article via Infotrieve]
  49. Shum, L., Reeves, S. A., Kuo, A. C., Fromer, E. S., and Derynck, R. (1994) J. Cell Biol. 125,903-916 [Abstract]

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