(Received for publication, May 15, 1995; and in revised form, June 14, 1995)
From the
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.
neu differentiation factor (NDF) ()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 proTGF 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.
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
and
forms as shown. Further differences arise in the
juxtamembrane domain to generate isoforms
2,
2, and
4;
the
3 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
forms yielding
2a,
2b, or
2c. Figure is redrawn
with permission from Ben-Baruch and
Yarden(23) .
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
and
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.
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) .
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
and/or
isoforms are represented at
the cell surface of Rat 1-EJ cells, since the antibody used here
recognizes both
and
forms equally well.
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
SO (lanes 4-6 and 10-12) or 0.1% Me
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
proTGF
.
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, TGF, etc.), but also
colony stimulating factor-1, SCF, tumor necrosis factor-
, 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. TGF
(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) mutation in
mouse. The Sl
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
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. ()
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 proTGF has been extensively studied, and
a recent report shows that valine at the C terminus of the cytoplasmic
tail of proTGF
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 proTGF
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 proTGF
.
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 proTGF
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 proTGF
is unlikely. First, PMA-stimulated
phosphorylation of proTGF
could not be detected(46) , and
second, mutation or deletion of any of the four Ser and Thr residues in
the cytoplasmic tail of TGF
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. ()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 proTGF
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
proTGF
, 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.