©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sensory and Motor Neuron-derived Factor
A NOVEL HEREGULIN VARIANT HIGHLY EXPRESSED IN SENSORY AND MOTOR NEURONS (*)

Wei-Hsien Ho (1), Mark P. Armanini (2), Andrew Nuijens (3), Heidi S. Phillips (2), Phyllis L. Osheroff (1)(§)

From the (1)Departments of Protein Chemistry, (2)Neuroscience, and (3)Bioanalytical Technologies, Genentech, Inc., South San Francisco, California 94080

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -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.


INTRODUCTION

The search for a soluble ligand of the oncogene p185 (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.

In this paper, we report the isolation of a human complementary DNA clone which encodes a new heregulin variant containing a -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.


EXPERIMENTAL PROCEDURES

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

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) 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

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-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

Total RNA was extracted from tissues or cells by the method of Chomczynski and Sacchi(10) . Poly(A) 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 (rHRG1, purified EGF-like domain of HRG1 (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 rHRG1.


RESULTS

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.

A diagramatic comparison of SMDF, GGF, HRG1, 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), HRG1, 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. HRG3 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 HRGs, and 85% to ARIA at the nucleotide levels. Like GGF and HRG3, 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 HRG1 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, HRG1, 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 HRG1, but differs from ARIA by 7 amino acids (denoted by *). GGF and HRG1 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 HRG1 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.

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.


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).

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).

Expression of SMDF cDNA in Mammalian Cells and Stimulation of Tyrosine Phosphorylation of a 185-kDa Protein in Cell Lines Expressing p185

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 ) 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 rHRG1(1) . The responses to different concentrations of rHRG1 (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 rHRG1 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 rHRG1 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 rHRG1 (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.


DISCUSSION

We have isolated a cDNA clone which encodes a protein termed SMDF. SMDF possesses a -type EGF-like domain that is identical to those of the HRGs, GGF, and human NDFs, 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 rHRG1(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.

Like most heregulins, SMDF does not possess an N-terminal signal peptide characteristic of secreted proteins, and like HRG3 (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.

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, 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.

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. 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.

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.


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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L41827.

§
To whom correspondence and reprint requests should be addressed: Dept. of Protein Chemistry, Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080. Tel.: 415-225-2169; Fax: 415-225-5945; E mail: plo@gene.com.

The abbreviations used are: HRG, heregulin; rHRG1, 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.

M. D. Sadick, M. X. Sliwkowski, J. A. Lofgren, and W. L. T. Wong, unpublished results.


ACKNOWLEDGEMENTS

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.


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