From the
The heregulin family of polypeptides arise as splice variants
from a single gene and share a conserved epidermal growth factor
(EGF)-like domain thought to be the major determinant of their
biological activities. We report here the cloning of a novel member of
this family, termed sensory and motor neuron-derived factor or SMDF,
which is highly expressed in sensory and motor neurons in human and
rodent species. It contains a C-terminal
The search for a soluble ligand of the oncogene p185
In this paper, we report the
isolation of a human complementary DNA clone which encodes a new
heregulin variant containing a
The partial sequences of rat SMDF cDNA (802 bp, corresponding
to nt 552-1352 of human SMDF) and rat GGF cDNA (453 bp, corresponding
to nt 999-1451 of human GGF) were generated by PCR amplification of
cDNA fragments prepared from rat brain poly(A)
A fragment of cDNA corresponding to the entire coding
sequence of the human SMDF clone BS1 with added 5`-SalI and
3`- HindIII sites was generated by PCR (95 °C, 7 min; five
cycles at 95 °C, 1 min, 70 °C, 1 min, 72 °C, 2 min;
followed by 15 cycles at 94 °C, 1 min, 56 °C, 1 min, 72 °C,
2.5 min; 72 °C, 5 min) and inserted into the Epstein-Barr
virus-based expression vector pEBon (8) via the XhoI
and HindIII sites. Orientation was determined by restriction
enzyme digestion and confirmed by sequencing. Plasmid DNA was purified
on QIAGEN columns. 293 cells (ATCC CRL 1573) were transfected with the
SMDF/pEBon expression vector and the pEBon vector alone as controls,
using a modified CaPO
Total RNA was extracted from tissues or cells by the method
of Chomczynski and Sacchi(10) . Poly(A)
A diagramatic comparison
of SMDF, GGF, HRG
Using the human SMDF probe, strong SMDF mRNA
expression was revealed in isolated regions of the nervous system of
human, rat, and mouse embryos (Fig. 4). Intense expression was
observed in developing spinal motor neurons (Fig. 4, B and E), in dorsal root ganglia (Fig. 4, B and E), in cranial ganglia (Fig. 4B), and
in discrete foci of the brainstem which were likely to represent
developing cranial nerve nuclei (Fig. 5, D and G). Lower levels of expression were observed in other
developing brain structures, including the ganglion eminence, the
posterior portion of the cortical plate, and the anlage of the
hippocampal formation (Fig. 5, A and D). In the
adult rat, high levels of expression were maintained in both motor
neurons and sensory neurons of dorsal root ganglia (Fig. 4, C and F). Among dorsal root ganglion neurons, expression
was most prominent in large cells, but detectable signals were present
in nearly every cell. In the adult rodent brain, SMDF mRNA was
detectable in brainstem nuclei.
Comparisons of the signal
intensity of GGF and SMDF mRNAs were also carried out in the brain,
spinal cord, and dorsal root ganglia of the rat embryos (Fig. 5).
Hybridization signals for SMDF mRNA in embryonic ventral spinal cord
motor neurons were strong in comparison with those for the N-terminal
probe of GGF (Fig. 5, H and I). In developing
dorsal root ganglia, GGF was strongly expressed in a subset of neurons,
while SMDF mRNA was present at high levels throughout the entire
population of neurons (Fig. 5, H and I). Within
ventral regions of the brainstem, intense hybridization was seen for
SMDF in several distinct nuclei (Fig. 5, D and G). These regions of hybridization were likely to represent
developing motor nuclei and did not exhibit appreciable hybridization
for GGF (Fig. 5E). In contrast to the strong expression
of SMDF in the motor neurons of the adult rat (Fig. 4), the
signals for GGF were barely detectable (not shown). The lower
hybridization intensity of GGF compared to SMDF in these neurons was
not likely to reflect technical difficulties with the GGF probe, as
hybridization signals were seen for GGF, and not SMDF, in ventricular
zone of frontal cortex and in non-neuronal tissues (Fig. 5, D, E, H, and I).
The SMDF cDNA was transiently expressed in 293 cells using
the pEBon vector system(8) . In view of the presence of an
EGF-like domain in SMDF, unconcentrated (1
We have isolated a cDNA clone which encodes a protein termed
SMDF. SMDF possesses a
Like most heregulins, SMDF does not
possess an N-terminal signal peptide characteristic of secreted
proteins, and like HRG
When hybridization probes
are designed such that SMDF is represented by its entire unique
N-terminal sequence while other heregulins are represented by the
Ig-like sequence of GGF (which shares 100% identity with the Ig domain
of HRG
The findings that certain ovarian cells expressing ErbB2 and ErbB2-transfected fibroblasts did not bind
nor cross-link to NDF, nor did they respond to NDF to undergo tyrosine
phosphorylation (5), suggested that heregulins might interact first
with another molecule in order to stimulate the tyrosine
phosphorylation of p185
The existence of multiple forms of heregulins
points to the possible diversity of their biological functions.
However, although the isolation of some heregulin variants is based on
a specific biological activity in vitro, it is highly probable
that many of them share the same activities. ARIA stimulates the
synthesis of muscle acetylcholine receptors at the neuromuscular
junction and increases the number of voltage-gated sodium channels in
cultured chick muscle(37) , and is suggested to enhance the
development of oligodendrocytes from bipotential (O2A) glial progenitor
cells(38) . GGF promotes the proliferation and tyrosine
phosphorylation of a 185-kDa protein of Schwann cells(4) . NDF
is reported to be expressed in neurons and glial cells in embryonic and
adult rat brain and primary cultures of rat brain cells, and is
suggested to act as a survival and maturation factor for
astrocytes(39) . It is very likely that all heregulin variants,
including SMDF, possess some or all of these activities.
The unique
nervous tissue-specific expression of SMDF mRNA distinguishes itself
from other heregulins in its possible neural-specific functions. Its
high expression in the spinal motor neurons and dorsal root ganglia in
the developing human and rodents suggests an action at the developing
neuromuscular junction and possible roles in motor and sensory neuron
development. It is of particular interest that within adult dorsal root
ganglion SMDF is more highly expressed in large neurons, consistent
with an action of SMDF on Schwann cell proliferation. At the
neuromuscular junction, specializations of three cell types constitute
the synapse: the motor neuron (nerve terminal), muscle fiber, and
Schwann cell (see review, 40). Like other neuronal peptides, SMDF may
be produced in the motor neuron cell body but is transported through
the motor axons to the nerve terminal, where it exerts its effects on
muscle acetylcholine receptor synthesis and/or the proliferation of
synapse-associated Schwann cells which cap the nerve terminals. Using
antibodies against a peptide within the conserved EGF domain versus an unique N-terminal peptide of heregulin, Jo et al.(41) demonstrate that some, but not all heregulins (e.g. GGF), are concentrated at the neuromuscular synapses in
innervated and denervated muscle and activate acetylcholine receptor
gene expression. The expression of SMDF in the adult rat spinal cord
motor neurons suggests that it may also act at mature neuromuscular
junctions via reinnervation of muscle fibers by motor neurons following
nerve damage.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We express our sincere appreciation to V. Chisholm, P.
Godowski, and C. Crowley for their advice on mammalian expression of
SMDF. We are grateful to B. Popko for providing the oligodendrocyte
cell line and to D. Finkle for harvesting the embryonic rat spinal
cords used for this study. We also thank M. Sadick, M. Sliwkowski, J.
Lofgren, and W. L. T. Wong for making the KIRA assay available for our
mammalian expression screening process. We appreciate very much the
encouragement and support of R. Vandlen and F. Hefti.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-type EGF-like domain and
an unique N-terminal sequence which lacks an Ig-like domain and is
distinct from all known heregulin variants. Mammalian cell-expressed
SMDF activates tyrosine phosphorylation of a 185-kDa protein in cell
lines expressing p185
, indicating
that it is biologically active. Analyses of expression patterns suggest
that, unlike other heregulin variants, SMDF is expressed mainly in the
nervous system. In situ hybridization signals with the unique
SMDF sequence probe and with a probe to the conserved EGF-like domain
are comparable, suggesting that SMDF is the predominant isoform
expressed in sensory and motor neurons. Expression of SMDF is
maintained in both adult motor neurons and dorsal root ganglion
neurons. These findings suggest that SMDF may mediate biological
responses such as Schwann cell proliferation and acetylcholine receptor
induction in the peripheral nervous system.
(also called neu/HER2) receptor tyrosine kinase led to
the purification and cloning of multiple members of a family of
polypeptide factors including heregulin (HRG)
(
)(1) and Neu differentiation factor (NDF, 2).
Two additional members were subsequently purified and cloned based on
acetylcholine receptor inducing activity (ARIA, 3), and Schwann cell
proliferative activity (glial growth factor (GGF), 4). It is apparent
that all these factors arise from alternatively spliced mRNAs of a
single gene. These proteins, referred to collectively as heregulins,
possess various combinations of six structural domains including the
signal peptide, kringle-like, immunoglobulin-like (Ig),
glycosylation-rich, epidermal growth factor-like (EGF-like),
transmembrane, and cytoplasmic domains. Heregulins are classified into
two major types,
and
, based on two variant EGF-like domains
which differ in sequences, yet are identical in the spacing of the 6
cysteines contained in the domain. The EGF domain appears to be one of
the regions which interact with specific receptors activated by the
heregulins, since this EGF domain alone, when expressed in Escherichia coli, is enough to stimulate
p185
receptor tyrosine
phosphorylation(1, 5) and to undergo covalent
cross-linking to a protein immunoprecipitable with an
anti-p185
antibody. Although some
other factors have been reported to activate
p185
(see review, 6), their
structures have not been determined.
-type EGF-like domain and a novel
N-terminal sequence which is distinct from all the known heregulins
reported so far. This clone, when expressed in mammalian cells,
activates tyrosine phosphorylation of a 185-kDa protein in cell lines
expressing p185
. Northern blot and in situ hybridization analyses show that this clone differs
from other heregulins in that it is expressed mainly within the nervous
system, in human and rodent sensory and motor neurons. We therefore
term it sensory and motor neuron-derived factor or SMDF.
Isolation of Human SMDF cDNA Clones
Screening of cDNA Libraries and Isolation of cDNA
clones
Two degenerate oligonucleotides corresponding to (A) a portion of coding segment 1 (nt 739-825) and (B) a portion of coding segments 8 and 9 (nt 1452-1507)
of the heregulin gene (clone GGFHBS5, 4) were labeled by random
oligonucleotide priming and used simultaneously to screen 5.5
10
plaques from two human brain stem (LMG2, American Type
Culture Collection 37432, in
gt11; Strategene, in
ZAP) and a
human cerebellum (Clontech, 5`-Stretch, in
gt11) cDNA libraries.
Positive clones were isolated by repetitive screening. Initially 13
clones were isolated: three hybridized to both probes, four hybridized
to probe A only, and six hybridized to probe B only. The insert size
was estimated after EcoRI digestion of purified phage DNA. The
insert of each clone was subcloned into the plasmid pBluescript SK(-)
(Stratagene) at the EcoRI site and subjected to DNA sequence
analysis. After initial analysis, seven clones were selected for full
sequence analysis.
DNA Sequence Analysis
Nucleotide sequences were
determined by the dideoxy chain termination method (7) using a
70770 Sequenase version 2.0 DNA sequencing kit (United States
Biochemicals Inc.). Both strands of the inserts were sequenced.
Generation of Rat SMDF and GGF Clones
RNA.
Amplification reactions were performed using Taq DNA
polymerase in a Perkin-Elmer model 480 thermocycler for 10 cycles at 95
°C, 1 min, 80 °C, 2.5 min; followed by 25 cycles at 95 °C,
1 min, 72 °C, 2.5 min. Amplified DNA fragments were cloned at the SmaI sites in pBluescript SK(-). Recombinants were identified
and sequenced. The partial rat SMDF sequences share 87% (nucleotide)
and 85% (amino acid) identities with the corresponding human sequences;
while the partial rat GGF sequences share 88% (nucleotide) and 96%
(amino acid) identities with the corresponding human sequences (data
not shown).
Expression of SMDF in Mammalian Cells
-mediated transfection
protocol(9) . After 4 days, transfected serum-free culture
supernatants were assayed for SMDF expression in a kinase receptor
activation enzyme-linked immunosorbent assay (KIRA) (see below). After
21 days, positive G418-resistant clones were expanded, and confluent
culture supernatants were analyzed for stimulation of receptor tyrosine
phosphorylation by KIRA and Western blot.
Northern Blot Analysis
RNA was
isolated from total RNA on oligo(dT)-cellulose columns (QIAGEN)
according to manufacturer's suggested procedures.
Ethanol-precipitated poly(A)
RNA was dissolved in 1
MOPS buffer, 50% formamide, 17.5% formaldehyde, heat-denatured
at 95 °C for 5 min, and electrophoresed in 1.2% agarose gels
containing 1.1% formaldehyde (11). The fractionated poly(A)
RNA was then capillary transferred onto nylon membranes (Hybond,
Amersham Corp.). The RNA blot was UV fixed and baked at 80 °C for 2
h and prehybridized with 5
SSC, 5
Denhardt's,
0.1% SDS, 100 µg/ml salmon sperm DNA at 65 °C for 4 h. The
blots containing 2 µg each of poly(A)
RNA from
fetal human tissues (Fig. 3, A and B) were
purchased from Clontech. The hybridization probes were generated by PCR
amplification of the following cDNA fragments: human SMDF, nt 507-1211
(705 bp) of clone BS1 (see ``Results'' and Fig. 1);
human GGF, nt 518-1058 (541 bp). The DNA probes were labeled with both
[
-
P]dATP and -dCTP by random priming using
a mixed population of hexamers (Promega) to a specific activity of
7.5-11
10
cpm/µg. The RNA blot was
hybridized in the same hybridization solution with 2
10
cpm/ml of probe at 65 °C for 20 h. The blot was washed
several times with 0.1
SSC, 0.1% SDS at room temperature, and
finally washed with the same solution at 65 °C for 10 min. The
blots were exposed to Kodak XAR-2 films with intensifying screens at
-80 °C for 5-7 days.
Figure 3:
Northern blot analysis. Hybridization of
Poly(A) RNA from human fetal brain, lung, liver, and
kidney (2 µg each) with
P-labeled DNA probes
corresponding to the coding sequences of SMDF 5` to the EGF-like domain (panel A), and the kringle and part of the Ig domains of GGF (panel B) as described under ``Experimental
Procedures.'' Two SMDF transcripts of 2.5 and 8.5 kb are detected
in fetal brain while two GGF transcripts of 1.3 and 4.4 kb are detected
in all four tissues. The sizes of DNA markers in kilobases are as
indicated.
Figure 1:
A, the cDNA and amino acid
sequences of human SMDF. The EGF-like domain and the hydrophobic
segment are underlined. Cysteines in the EGF-like domain are boxed. The stop codon is denoted by the letter O. B, hydropathy analysis of SMDF.
In Situ Hybridization
Preparation of RNA Probes
Hybridization probes
were generated by PCR amplification of the following cDNA fragments 3`
to an added T7 promotor sequence (Promega): human SMDF, nt 507-1172
(666 bp) of clone BS1 (see ``Results'' and Fig. 1);
human GGF, nt 781-1305 (525 bp); rat GGF, nt 1006-1299 (294 bp)
of the rat GGF clone. Antisense and sense RNA probes were transcribed in vitro by T7 RNA polymerase as described (12) and
incorporated [-
P]UTP (5000 Ci/mmol,
Amersham). The DNA template was removed by incubation with 1 unit of
RNase-free DNase (Promega) at 37 °C for 15 min. RNA probes were
extracted twice with phenol-chloroform using yeast tRNA (Sigma) as
carrier and precipitated with 100% ethanol in the presence of 0.3 M sodium acetate, rinsed with 70% ethanol, and taken up with
10 mM Tris, 1 mM EDTA, pH 7.4, at a concentration of
4
10
cpm/µl.
Hybridization
Hybridization was performed by a
modification of previously described
procedures(13, 14) . Prior to cryosectioning, tissue was
either fresh frozen (human embryos, adult brain, and spinal cord) or
fixed in 4% formaldehyde (mouse and rat embryos, adult dorsal root
ganglia). Frozen, dessicated tissue sections of 10-20 µm
thickness in sealed slide boxes were stored at -70 °C prior
to use. On the day of hybridization, sections of unfixed tissue were
fixed in 4% formaldehyde, 1% glutaraldehyde at 4 °C for 15 min,
while those of fixed tissue were treated for 30 min in 4% formaldehyde.
After two washes in 0.5 SSC, the sections were covered with
hybridization buffer (20 mM Tris-HCl, pH 8, 5 mM EDTA, 0.1 M NaCl, 1
Denhardt's, 10% dextran
sulfate, 10 mM dithiothreitol, 50% formamide; 0.3 ml/slide)
and incubated at 42 °C for 3 h.
P-Labeled RNA probes
in hybridization buffer containing tRNA as carrier were then added
directly into the hybridization buffer on the slides to a final
concentration of 8
10
cpm/ml and incubated at 55
°C overnight in humidified tightly covered boxes. After two washes
in 2
SSC solution containing 1 mM EDTA, the sections
were treated with RNase A solution (20 µg/ml in 10 mM
Tris-HCl, pH 8, 0.5 M NaCl) at room temperature for 30 min.
After two more washes in 2
SSC-EDTA solution at room
temperature, the sections were washed with high stringency buffer (0.1
SSC, 1 mM EDTA) at 55 °C for 1 h. The sections
were then washed two times with 0.5
SSC at room temperature and
dehydrated briefly each in 60, 75, 85, and 90% ethanol containing 0.3 M ammonium acetate, 95% ethanol, air-dried, and exposed to
Hyperfilm
(Amersham) followed by dipping in NTB2
emulsion (Kodak). After exposure times of 4-8 weeks and
development, the emulsion-dipped slides were counterstained with cresyl
violet.
Tyrosine Phosphorylation Assay
Western Blot Analysis
Western blot analysis of
ligand-stimulated receptor tyrosine phosphorylation was performed
essentially as described(1) . MCF-7 breast tumor cells (ATCC HTB
26) grown in Dulbecco's minimum essential medium (50%), F-12
(50%), 10% fetal bovine serum (Hyclone) to confluence in 24-well plates
were changed to medium without serum (assay medium) and incubated at 37
°C for 2 h. The cells were stimulated for 15 min at 37 °C with
SMDF-transfected culture supernatants or purified recombinant
HRG (rHRG
1, purified EGF-like domain
of HRG
1 (amino acid residues 177-241) expressed in E.
coli, 1; kindly supplied by Process Sciences, Genentech) diluted
in assay medium containing 0.1% bovine serum albumin, as indicated. The
supernatants were removed, and 100-µl aliquots of SDS sample buffer
containing
-mercaptoethanol were added. Aliquots of the samples
(15 µl) were heated and electrophoresed in a 4-20%
polyacrylamide gel (Novex) and electroblotted onto a nitrocellulose
membrane. The membranes were blocked with 5% bovine serum albumin in
Tris-buffered saline containing 0.05% Tween-20 and incubated with an
anti-phosphotyrosine monoclonal antibody (4G10, Upstate Biotechnology)
for 1 h at room temperature. Bound anti-phosphotyrosine antibody was
probed with an alkaline phosphatase-conjugated goat anti-mouse
Immunoglobulin G antibody (Promega) for 30 min at room temperature and
visualized with 5-bromo-4-chloro-3-indoyl-1-phosphate and nitro-blue
tetrazolium (Promega).
Kinase Receptor Activation (KIRA) Enzyme-linked
Immunosorbent Assay
The assay was performed as described by
Sadick et al.,(
)similar to the method
of King et al.(15) . MCF-7 cells grown overnight in
microtiter plates were stimulated with SMDF-transfected culture
supernatants for 30 min at 37 °C. Tyrosine-phosphorylated
p185
in the cell lysates was bound
to rabbit anti-HER2 extracellular domain antibody on an enzyme-linked
immunosorbent assay microtiter plate and probed with a biotinylated
anti-phosphotyrosine antibody (4G10, Upstate Biotechnology) and
horseradish peroxidase-conjugated streptavidin using the substrate
tetramethyl benzidine. Sample concentrations were calculated from a
standard curve generated by parallel stimulation with known
concentrations of rHRG
1.
Isolation and Sequences of SMDF
Isolation of Human SMDF cDNA
Degenerate
oligonucleotides corresponding to portions of coding segment 1 (probe
A, 87-mer) and segments 8 and 9 (probe B, 56-mer), respectively, of the
heregulin gene (clone GGFHBS5, 4) were used to screen human brain stem
and cerebellum cDNA libraries. One of the resulting clones which
hybridizes strongly to both probes (clone BS4) has identical coding and
5`- and 3`-noncoding sequences as GGF (rhGGFII, 4). Two other clones
which hybridize to probe B only (clones BS1 and BS2) are independent
clones showing identical coding sequences. The 5`-untranslated
sequences of BS1 (506 nt) and BS2 (521 nt) are nearly identical except
for 18 nt adjacent to the 5`-polycloning site.
The cDNA and Deduced Amino Acid Sequences of
SMDF
Fig. 1A shows the nucleotide sequence of the
SMDF cDNA (clone BS1) and its predicted amino acid sequence. Beginning
with the ATG at nt 507 which starts the most extensive open reading
frame followed by the stop codon TAG at nt 1395, the cDNA encodes a
polypeptide of 296 amino acids, with a predicted M 31,686. The second ATG at nt 528 may be a stronger translation
initiator by sequence context criteria, but the
``first-AUG-rule'' holds for 93-95% of the eukaryotic
mRNAs, and theory predicts that ribosomes should initiate
(inefficiently) at the first as well as the second AUG(16) . In
addition, the G at nt 510 (following the first ATG) provides more
optimal translational efficiency for mammalian expressed
genes(17) . For these reasons it is assumed that the first ATG
is the translation initiator. The stop codon is followed by a 478 nt
3`-untranslated sequence ending with an A-rich region preceded by two
consensus polyadenylation signals AATAAA.
1, and ARIA is shown in Fig. 2A (only major structural characteristics are shown); and a
comparison of the amino acid sequences of the above proteins is shown
in Fig. 2B. SMDF has an EGF-like sequence that shares
100% identity with those of GGF (4), HRG
1,
2, and
3(1) , and human NDF-
1a,
2, and
3 (18); 94%
with certain rat NDF (clones 22, 40, 41, 42a; 18); and 85% with chicken
ARIA(3) . However, a comparison with the GenBank nucleotide data
base and protein data base using the BLAST program shows that the SMDF
sequence N-terminal to the EGF-like domain is novel and distinct from
all other reported heregulin sequences. Like HRG, NDF, and ARIA, SMDF
is also devoid of a N-terminal signal peptide typical of membrane and
secreted proteins. Like some variants (e.g. HRG
3 and
GGF), the SMDF sequence ends after an 8-10 variable amino acid
stretch which usually connects the EGF-like domain with the
transmembrane domain; it is therefore devoid of the latter and the
cytoplasmic tail. The major structural difference between SMDF and
other heregulins is the lack in SMDF of an Ig-like domain
characteristic of all the other heregulins. Another distinct feature of
SMDF is the apparent lack of N-linked glycosylation sites
(although there are abundant potential O-linked sites). A
third notable feature of SMDF is the presence of two stretches of amino
acids near the N terminus, residues Thr
-Leu
and Ile
-Val
, that are predominantly
hydrophobic in nature (see hydropathy analysis, Fig. 1B). It is possible that these sequences, in
particular Ile
-Val
, may act like internal,
uncleaved signal sequences which may mediate the translocation across
the membrane (see ``Discussion''). A fourth notable feature
is the presence of 8 cysteine residues scattered along the hydrophobic
stretch. The possible structural or functional roles of these cysteine
residues are not known.
Figure 2:
A, diagramatic comparison of SMDF with
GGF, HRG1, and ARIA. Only major structural characteristics are
shown. The EGF-like domain of SMDF is 100% identical to that of GGF and
HRG
s, and 85% to ARIA at the nucleotide levels. Like GGF and
HRG
3, the SMDF sequence ends after an 8-10 amino acid
stretch which connects the EGF-like domain with the transmembrane
domain (TM) and is devoid of the latter and the cytoplasmic
tail, which are present in HRG
1 and ARIA. The sequences of SMDF
N-terminal of the EGF-like domain bear no identity to any known
heregulins. It lacks the Ig-like domain which is characteristic of all
known heregulins. It also lacks an N-terminal signal sequence of GGF
(denoted by hydrophobic) but possesses a stretch of apolar and
uncharged amino acid residues. B, amino acid sequence
comparison of SMDF with GGF, HRG
1, and ARIA. Homologous Ig-like,
EGF-like, and transmembrane domains are boxed. The EGF-like
domain of SMDF is identical to those of GGF and HRG
1, but differs
from ARIA by 7 amino acids (denoted by *). GGF and HRG
1 have
identical Ig-like domains and differ from ARIA by 30 and 35% at the
nucleotide and amino acid (denoted by *) levels, respectively. SMDF has
no Ig domain. The transmembrane domains of HRG
1 and ARIA are
identical.
Northern Blot and in Situ Hybridization Analysis of
SMDF mRNA
Northern Analysis
To look specifically for
transcripts encoding SMDF and not other heregulin isoforms which share
a type EGF-like domain, a
P-labeled cDNA fragment
corresponding to the human SMDF cDNA 5` to the EGF-like sequence was
used in Northern blot analysis. On a Northern blot of
poly(A)
RNA from human fetal brain, two major
transcripts of 2.5 and 8.5 kb were detected (Fig. 3A, lane 1). No hybridization signals were detected on the same
blot of poly(A)
RNA from human fetal lung, liver, and
kidney (Fig. 3A, lanes 2-4). The large
sizes of the transcripts suggested that SMDF mRNA might have long 5`
and 3`-untranslated regions common to growth factor mRNAs(19) .
When a
P-labeled probe corresponding to the kringle-like
and part of the Ig domain of human GGF cDNA (which was also 5` to the
shared EGF-like sequence) was used on a similar blot, two transcripts
of 1.3 and 4.4 kb were detected in all four of the above-mentioned
tissues (Fig. 3B, lanes 1-4). A single
GGF transcript of 4.4 kb was detected in adult human heart, brain,
placenta, lung, liver, skeletal muscle, kidney, and pancreas; while
SMDF transcripts were detected in the adult human brain only (data not
shown). This agreed with the wide distribution of mRNA in various
tissues and cells reported for some other heregulin
variants(1, 2, 3, 20, 21) .
Thus, there was a distinct difference in tissue distribution between
SMDF and other heregulin variants in that SMDF appeared to be expressed
mainly in the nervous system.
In Situ Hybridization
In situ hybridization experiments were performed on human, rat, and mouse
embryos as well as on adult rodent brain, spinal cord, and dorsal root
ganglia. The P-labeled RNA probes used correspond to the
unique N-terminal coding sequences of human SMDF or the Ig domain of
human and rat GGF.
Figure 4:
SMDF expression in human, rat, and mouse
sensory and motor neurons. In situ hybridization was performed
with P-labeled RNA probes corresponding to the coding
sequences 5` of the EGF domains of SMDF and GGF as described under
``Experimental Procedures.'' Prominent expression of SMDF is
seen in developing and adult rat and human primary sensory and motor
neurons. A and B, brightfield and darkfield images of
parasagittal sections of an 8-week human embryo. C, adult rat
dorsal root ganglia. D and E, transverse section of
E13.5 mouse embryo. F and G, adult rat spinal cord.
Sections in A-F were hybridized with antisense probe to SMDF. G, is a control section hybridized with the sense strand
probe. drg, dorsal root ganglia; lmc, lateral motor
column; tg, trigeminal ganglion. Scale bars are 4 mm for (A and B) and 0.5 mm for (C-G).
Figure 5:
Comparison of SMDF expression with other
heregulins in the embryonic rodent nervous system. A and B are film autoradiographs from adjacent sections of E13.5 mouse
embryos hybridized with a probe to the unique N terminus (A)
or conserved EGF domain (B) of SMDF. C, is a
brightfield image of the section shown in B. D and E are darkfield images of adjacent sections of E15.5 rat
embryos hybridized with probes to SMDF (D) or GGF (E). F is a brightfield image. Arrowheads in D indicate nuclei likely to represent developing cranial motor
nuclei. The nuclei are shown at higher magnification in G. H and I are transverse sections of E15.5 embryos
demonstrating hybridization for SMDF (H) and GGF (I)
in lateral motor column and dorsal root ganglia. c,-cortex; ge, ganglionic eminence; sc, spinal cord; tg, trigeminal ganglion. Scale bars are 1 mm for (A-F), 0.5 mm for (G-I).
The striking expression observed
with the unique N-terminal SMDF sequence probe in developing sensory
and motor neurons suggested that a substantial portion of the
hybridization signals previously observed in sensory and motor neurons
using probes to the EGF domain of heregulins (4, 21) might represent
the expression of SMDF (and possibly other SMDF variants possessing the
same N-terminal sequence). To examine this possibility, rat embryos
were hybridized under identical conditions with probes to the conserved
-type EGF domain and the novel N-terminal sequence of SMDF. The
hybridization signals revealed with both probes were similar in
intensity in dorsal root ganglia, the lateral motor column, and in
brainstem nuclei, suggesting that SMDF (and other SMDF variants) was
more highly expressed in these sites than the Ig-containing heregulins (Fig. 5, A and B).
Expression of SMDF cDNA in Mammalian Cells and
Stimulation of Tyrosine Phosphorylation of a 185-kDa Protein in Cell
Lines Expressing p185
) and 10-fold
concentrated (10
) culture supernatants were assayed for their
ability to stimulate tyrosine phosphorylation in the MCF-7 human breast
tumor cell line expressing p185
. Fig. 6A shows that by Western blot analysis with an
anti-phosphotyrosine monoclonal antibody a 185-kDa protein was detected
in cells treated with SMDF-transfected culture supernatants and with
rHRG
1(1) . The responses to different concentrations of
rHRG
1 (lanes 2, 100 pM; lanes 3, 500
pM; lanes 4, 1 nM) and to unconcentrated (lane 6) and 10
concentrated SMDF-transfected
supernatants (lane 7) were concentration-dependent. Cells
treated with assay medium containing 0.1% bovine serum albumin (lane 1), serum-free transfection medium (lane 5), or
unconcentrated (lanes 8) and 10-fold concentrated
vector-transfected culture supernatants (lane 9) did not show
tyrosine phosphorylation of p185
.
Figure 6:
Stimulation of tyrosine phosphorylation by
SMDF-transfected 293 culture supernatants in MCF-7 human breast tumor
cell line and an erbB2-transformed mouse oligodendrocyte cell
line. The SMDF cDNA was transiently expressed in 293 cells using the
pEBon vector system. MCF-7 and transformed mouse oligodendrocyte cells
grown to confluence in 24-well plates were stimulated with various
agents as indicated below, and the stimulated cell lysates in SDS
sample buffer were electrophoresed in 4-20% SDS gels and
electroblotted onto nitrocellulose membranes. The blots were probed
with an anti-phosphotyrosine monoclonal antibody and detected with
alkaline phosphatase-conjugated goat-anti-mouse IgG. A, a
185-kDa protein is detected by the anti-phosphotyrosine antibody in
MCF-7 cell lysates treated with unconcentrated (lane 6), 10
concentrated (lane 7) SMDF-transfected 293 culture
supernatants, as well as with rHRG
1 at 100 pM (lane
2), 500 pM (lane 3), and 1 nM (lane
4). No tyrosine phosphorylation is seen in cells treated with
assay medium + 0.1% bovine serum albumin (lane 1),
serum-free transfection medium alone (lane 5), unconcentrated (lane 8) or 10
concentrated (lane 9)
vector-transfected 293 culture supernatants. B, tyrosine
phosphorylation of p185
in transformed mouse
oligodendrocytes treated with unconcentrated (lane 5) and 10
concentrated (lane 6) SMDF-transfected supernatants,
as well as rHRG
1 at 100 pM (lane 2), 500 pM (lane 3), and 1 nM (lane 4), all in
serum-free transfection medium. Medium control (lane 1), as
well as unconcentrated (lane 7) and 10
concentrated (lane 8) vector-transfected 293 supernatants, show no
effect.
SMDF-transfected supernatants also activated tyrosine
phosphorylation of a 185-kDa protein in a transformed erbB2-expressing mouse oligodendrocyte cell line derived from
a transgenic tumor (22) (Fig. 5B). The activation
with unconcentrated (lane 5) and 10 concentrated
SMDF-supernatants (lane 6), as well as different
concentrations of rHRG
1 (lane 2, 100 pM; lane 3, 500 pM; lane 4, 1 nM) were
concentration-dependent. Vector-transfected supernatants (lane
7, unconcentrated; lane 8, 10
concentrated) as
well as transfection medium controls (lane 1) had no effect.
-type EGF-like domain that is identical to
those of the HRG
s, GGF, and human NDF
s, but has a novel
N-terminal sequence that is distinct from all known members of the
heregulin family. The most notable structural difference is the absence
in SMDF of an Ig-like domain common to all known heregulins. SMDF also
lacks the region rich in N-linked glycosylation sites.
However, since the EGF-like domain alone, as in rHRG
1(1) ,
is capable of eliciting receptor tyrosine phosphorylation, SMDF would
be expected to show substantial overlap with other heregulins in its
range of biological activities.
3 (1) and GGFHFB1(4) , it also
lacks a C-terminal transmembrane domain with an immediate
amino-terminal proteolytic cleavage site. However, unlike GGFHFB1 which
is not released from the transfected COS-7 cells(4) , SMDF
appears to be released from transiently transfected 293 cells, as
suggested by the ability of SMDF-transfected culture supernatants to
stimulate tyrosine phosphorylation of a 185-kDa protein in cells
expressing p185
. Hydropathy
analysis of SMDF reveals a stretch of non-polar or uncharged amino
acids near the N terminus, Ile
to Val
, which
is sufficiently long and hydrophobic to act as an internal, uncleaved
signal sequence which may mediate the translocation across the
endoplasmic reticulum
membrane(23, 24, 25, 26) . The presence
of positively and negatively charged residues upstream of this
hydrophobic stretch in SMDF befits the typical features of internal
uncleaved signal peptides (26). Examples of such uncleaved, internal
signal peptides are the hydrophobic signal element near the N terminus
of ovalbumin (27) and synaptotagmin(28) . However, we
cannot rule out other release mechanisms.
,
s, and human NDF
2b,
3; 87% with rat NDF;
and 70% with ARIA; at the nucleotide level) in Northern blot and in
situ analysis, a major difference in tissue distribution is
revealed. While other heregulins are wide-spread in human tissues
including, in our study, the embryonic and adult brain, lung, liver,
kidney, adult heart, pancreas, placenta, and skeletal muscle, SMDF is
only found in the brain, spinal cord, and dorsal root ganglia. Thus
SMDF is likely neural tissue-specific. The high expression of SMDF mRNA
is maintained in the adult rat motor neurons and dorsal root ganglia,
where no clearly detectable signal is seen for GGF. Our in situ distribution of rat GGF mRNA in the embryonic rat brain, spinal
cord, and dorsal root ganglia is similar to that reported previously (20) using a mouse NDF probe which includes a partial Ig domain.
Using a similar Ig-domain probe, ARIA mRNA was also detected in
embryonic chick ventral horn spinal motor neurons but not in dorsal
root ganglia(3) . The particular expression pattern of ARIA in
the embryonic chick probably reflected species specificity of the
molecule. Note that in some of the reported northern or in situ analyses of heregulins, the hybridization probes used were
inclusive of the EGF-like sequence shared by
SMDF(1, 2, 4, 21) . Therefore, the SMDF
mRNA would have been co-localized with other heregulin isoform mRNAs in
these experiments, and the high intensity of the hybridization signals
observed in the spinal cord motor neurons and dorsal root ganglion
sensory neurons might not solely represent the particular heregulin
isoform in question. Indeed, Corfas et al.(29) observed a much more intense hybridization signal of
ARIA mRNA when an EGF-like domain probe of ARIA was used versus an Ig-like domain probe. These authors suggested that isoforms of
ARIA must exist without the Ig-like domain. SMDF may just be one of
such isoforms.
. Indeed, it
has subsequently been shown that two other related members of the
epidermal growth factor receptor family,
p180
(30) and
p180
(31) , are receptors for
the heregulins (32-36). The interaction of heregulins with either
receptor resulted in the tyrosine phosphorylation of
p185
and also the respective
ligand-binding receptor. In this study we show that SMDF-transfected
293 culture supernatant stimulates the tyrosine phosphorylation of a
185-kDa protein in a concentration-dependent manner in the human breast
tumor cell line MCF-7 (which expresses
p185
) and in a transformed
oligodendrocyte cell line derived from the tumor of a transgenic mouse
expressing erbB2(22) , suggesting that SMDF is
biologically active.
/EMBL Data Bank with accession number(s) L41827.
1, recombinant heregulin
1 amino acid residues
177-241; NDF, Neu differentiation factor; GGF, recombinant human
glial growth factor II; ARIA, acetylcholine receptor inducing activity;
EGF, epidermal growth factor; nt, nucleotide(s); bp, base pair(s); PCR,
polymerase chain reaction; KIRA, kinase receptor activation
enzyme-linked immunosorbent assay; cpm, counts/min; kb, kilobase(s);
MOPS, 4-morpholineethanesulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.