Note: Descriptions are shown in the official language in which they were submitted.
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RECOMBINANT HEREGULINS
AND THEIR BIOLOGICAL
FUNCTIONS UPON RECEPTOR ACTIVATION
C .
The present invention relates to the generation of
versatile recombinant heregulins (HRGs) and some of the
biological functions and intracellular signaling pathways that these
proteins trigger following receptor activation.
Heregulins (HRGs) are mosaic glycoproteins that bind
10 to, and induce tyrosine phosphorylation of the HER4/P180erbB4
receptor. Heregulins (HRGs), Holmes, W. E. et al., (1992),
Science 256, 1205-1210, neu differentiation factor (NDF), Peles,
E. et al., (1992), Cell 69, 205-216; Wen, D. çt al., (1992), Cell 69,
559-572 and Wen, D. et al., (1994), Mol. Cell Biol. 14, 1909-1919,
15 glial growth factors (GGFs), Marchionni, M. A. et al., (1993) Nature
362, 312-318, and acetylcholine receptor-inducing activity (ARIA),
Falls, D. L. et al., (1993) ~!! 72, 801-815, are homologous
multifunctional proteins. The HRG isoforms originate from a
single gene by alternative RNA splicing. HRG cDNAs encode
20 large transmembrane precursors with multiple domains including
an immunoglobulin-like domain, a spacer domain with several
glycosylation sites, an EGF-like domain, a juxtamembrane domain
of variable length, a transmembrane region, and a cytoplasmic
domain. The solu~le mature HRGs are released from the cell
25 surface by proteolytic cleavage. GGFs, Marchionni, M. A. et al.,
(1993) Nature 362, 312-318, and ARIA, Falls, D. L. et al., (1993)
Ceil 72, 801-815, which were isolated from brain tissues, also
contain a kringle-like domain that is absent in HRGs and NDFs. a
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and B HRG isoforms display sequence differences in the third loop
of the EGF-like domain, and the juxtamembrane domain. The
EGF-like domain of HRGs contains six cysteine residues that are
characteristic of the EGF family of growth factors including EGF, 0
Carpenter, G. et al., (1979) Ann. Rev. Biochem.. 48,193-216,
transforming growth factor-o~, Derynck, R. et al., (1984) ~11 38,
287-297, vaccinia virus growth factor, Blomquist, C. l. et al., (1984)
Proc. Natl. Acad. Sci. USA 81, 7363-7367, amphiregulin, Shoyab,
M. et al., (1989) Science 243, 1074-1076, heparin-binding EGF-
like growth factor, Higashiyama, S. et al., (1991) Science 251,
936-939, and betacellulin, Shing, Y. et al., (1993) Science 259,
1604-1607.
Although HRGs contain an EGF-like motif, they do not bind
to EGFR/P170erbB1, Holmes, W. E. et al., (1992), Science 256,
1205-1210. The HRGs bind to HER4/pl80erbB4, a recently
isolated member of the epidermal growth factor receptor family,
Plowman, G. D. et al., (1993) Proc. Natl. Acad. Sci. U.S.A. 90,
1746-1750; Culouscou, J. M. et al., (1993) J. Biol. Chem. 268,
18407-18410 and Plowman, G. D. et al., (1993) Nature (London)
366, 473-475. The HER3/P180erbB3 receptor, another member of
this family has also been reported to be a receptor for HRGs,
Carraway, K. L. et al., (1994) J. Biol. Chem. 269,14303-14306.
HRGs do not directly interact with HER2/p185erbB2 receptor, as
originally proposed, Holmes, W. E. et al., (1992), Science 256,
1205-1210 and Peles, E. et al., (1992), Cell 69, 205-216. The
HER2/p185erbB2 indirectly participates in HRG-mediated
signaling through transphosphorylation or receptor
heterodimerization with HER4 and/or HER3, Plowman, G. D. et al.,
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(1993) Nature (London) 366, 473-475 and Carraway, K. L. et al.,
(1994) 1. Biol. Chem. 269, 14303-14306.
As a consequence of HRG/NDF binding to its receptors,
human mammary tumor cells have been shown to differentiate,
Peles, E. et al., (1992), Cell 69, 205-216 and Bacus, S. S. et al.,
(1993), Cancer Res. 53, 5251-5261, and up-regulate their
expression of the intercellular adhesion molecule-1 (ICAM-1),
Bacus, S. S. et al., (1993), Cancer Res. 53, 5251-5261. The
biological effects of HRGs are mediated through receptors that
possess an intrinsic tyrosine kinase activity and are
autophosphorylated upon HRG binding, Plowman, G. D. et al.,
(1993) Nature (London) 366, 473-475. The studies carried out on
receptor tyrosine kinases such as the epidermal growth factor
receptor, the platelet-derived growth factor receptor and the insulin
receptor, have demonstrated a crucial role for receptor
autophosphorylation in intracellular signal transduction following
ligand binding, Ullrich, A. et al., (1990) Cell 61, 203-212 and
White, M. F. et al., (1994) J. Biol. Chem. 269,1-4. It has been
demonstrated that specific autophosphorylation sites on receptor
tyrosine kinases serve as recognition structures for target
molecules containing Src homology 2(SH2) domains. SH2
domains are conserved noncatalytic sequences of approximately
100 amino acid found in various signaling molecules and
oncogenic proteins, Koch, C. A. et al., (1991) Science 252, 668-
674 and Songyang, Z. et al., (1993) Cell 72, 767-778. SH2
domain-containing proteins bind with high affinity to
phosphotyrosine residues in the context of specific flanking amino
acids. For example, the p85 subunit of phosphatidylinositol (P
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1)3 -kinase (P 1 3-K), the p21 ras GTPase activating protein (GAP),
and phospholipase C~(PLC-~) have been shown to contain SH2
domains. More recently, SH2 domain-containing proteins that
lack an apparent catalytic domain and seem to function as
5 adaptors linking proteins involved in signal transduction have
bee~scrlb~d, Lo-~ns~ein, E. J. et~., ~19~ C~li 70, ~31-442;
Pelicci, G. et al., (1992) Ç~! 70, 93-104; Egan, S. E. et al., (1993)
Nature (London) 363, 45-51; Skolnik, E. Y. et al., (1993) Science
260,1953-1955; Rozakis-Adcock, M. et al., (1993) Nature
(London) 363, 83-85 and Gale, N. W. et al., (1993) Nature
(London) 363, 88-92. One of them, Shc was identified and cloned
based on its homology to SH2 sequences from the human c-fes
gene, Pelicci, G. et al., (1992) Cell 70, 93-104. The Shc cDNA is
predicted to encode two proteins of 46 and 52 kDa that contain a
single C-terminal SH2 domain and a collagen-homologous region
that is rich in glycine and proline. No catalytic domain was
identified in Shc. Anti-Shc antibodies have been shown to
recognize three proteins of 46, 52, and 66 kDa in a wide range of
mammalian cells. A variety of growth factors and cytokines have
been shown to induce phosphorylation of Shc proteins, Pronk, G.
J- ~L, (1993) J. Biol. Chem. 268,5748-5753; Yokote, K. et al.,
(1994) J. Biol. Chem. 269,15337-15343; Schorb, W. et al., (1994),
J. Biol. Chem. 269,19626-19632; Cutler, R. L. et al., (1993) J.13iol.
Chem. 268, 21463-21465; Burns, L. A. et ~I., (1993) J. Biol. Chem.
268, 17659-17661 and Damen, J. E. et al., (1993) Blood 82,2296-
2303. The overexpression of the Shc proteins is associated with a
transformed phenotype in fibroblasts, Pelicci, G. et al., (1992) Cell
70, 93-104, and neuronal differentiation of PC12 cells, Rozakis-
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Adcock, M. et al., (1992) Nature (London) 360, 689-692, strongly
suggesting that Shc is involved in cell growth regulation.
The present invention is directed to the generation of
~, versatile recombinant heregulins (HRGs) and the biological
5 functions and intracellular signaling pathways that these proteins
trigger following receptor activation.
Because the EGF-like domain of HRG is sufficient for
receptor binding, the present invention relates to the cloning of the
cDNA fragments encoding the EGF-like domains of HRG-oc,-B2, or
10 -B3 into an eukaryotic expression vector containing sequences
encoding a thrombin cleavage site, followed by the Fc portion of a
hurnan IgGI. The present invention also relates to the production
of the recombinant fusion proteins rHRGs-T-Fc which can be
used as chimeric proteins or as EGF-like domains (reHRGs) after
15 thrombin cleavage and removal of the Fc portion of the molecule.
The present invention demonstratesthat the
recombinant HRGs, in either form bind to and activate the HER4
rec0ptor and that the Shc proteins are tyrosine phosphorylated
following HRG stimulation. The present invention also
20 demonstrates that rHRG-o~-T-Fc bound to human breast cancer
cells that express HER4 receptors and induced the expression of
the intercellular adhesion molecule-1. Moreover, reHRG-B2
markedly induced phosphorylation of Shc proteins on tyrosine,
suggesting a role for these adaptor molecules in HRG-mediated
25 signaling.
Figure 1 illustrates tyrosine autophosphorylation of the
HER4 receptor following rHRGs-T-Fc stimulation.
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Figure 2 illustrates the binding of rHRG-a-T-Fc to MDA-
MB-453 cells.
Figure 3 illustrates the induction of ICAM-1 expression in
response to rHRG-a-T-Fc.
Figure 4 illustrates thrombin cleavage of rHRG-,~3-T-Fc.
Figure 5 illustrates the stimulation of protein
phosphorylation in response to reHRGs.
Figure 6 illustrates tyrosine phosphorylation of Shc
proteins upon HER4 activation.
Figure 7 shows the nucleotide and amino acid
sequences (SEQ. ID. NOS. 8 and 9) of a heregulin alpha fusion
protein. In this particular protein, the nucleotide bases correspond
to the following:
Bases Re~ion
1 to 178 CD5 signal sequence
179 to 373 Heregulin alpha EGF-like
binding domain
374-424 Thrombin cleavage site
425 to 1129 Human lg-constant region
The present invention is directed to the generation of
recombinant EGF-like domains of HRGs and the biological effects
induced by them as well as identifying intracellular molecules
involved in HER4 signaling. In preferred embodiments, the
present invention relates to the cloning of the EGF-like domains of
HRG-a,-~2 and -~3 into an eukaryotic expression vector in frame
with sequences encoding a thrombin cleavage site followed by the
Fc portion of a human IgGI. More preferably, the vector further
comprises the signal sequence of the CD5 protein which allows
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efficient processing and secretion of the proteins. The vector is a
mammalian expression vector.
The present invention relates to the creation of chimeric
genes which direct the expression of recombinant fusion proteins,
5 rHRGs-T-Fc. The recombinant fusion proteins are expressed in
large amounts by transfecting the vector onto a suitable host. The
preferred host is mammalian COS cells. These proteins are
shown to stimulate the phosphorylation of HER4/P180erbB4. The
bivalent fusion proteins generated are useful as growth factors
10 since they activate growth factor receptors. These fusion proteins
are also useful in being detected like antibodies, via their Fc
domain.
The recombinant fusion proteins can also be used in
high throughput screening assay for identifying low molecular
15 weight agonist or antagonist of HER3 and HER4 receptors. The
high throughput screening assay involves screening several
hundreds of compounds in a short period of time in microliter well
plates using reagents. The assay is carried out with the
assistance of robotics and automation. The assay could be a
20 binding assay or an enzyme assay, etc., depending on the
compound being screened.
The present invention is also useful for the production of
larg0 amounts of of other recombinant growth factors, such as
epidermal growth factor, transforming growth factor-alpha,
25 amphiregulin, betacellulin, heparin-binding epidermal growth
factor, vaccinia growth factor, cripto, insulin growth factor, insulin-
like growth factor, transforming growth factor-beta, platelet-derived
growth factor, fibroblast growth factor, and nerve growth factor.
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The present invention also relates to purified EGF-like
domains (reHRGs) after thrombin protease cleavage of the
rHRGs-T-Fc fusion proteins. These reHRGs are shown to
stimulate protein phosphorylation in HER4 expressing cells.
The present invention is also directed to a method of
purifying the fusion proteins rHRGs-T-Fc in a single step by protein
A-Sepharose chromatography.
The present invention demonstrates that reHRG-B2
markedly induces phosphorylation of Shc proteins on tyrosine.
This suggests a role for these adaptor molecules in HRG-mediated
signaling.
Technical terms used throughout this application are
well known to those skilled in the art of molecular genetics.
Definition of these terms are found in many textbooks dedicated to
the molecular biology field, such as "Genes," Second Edition, by
Dr. Benjamin Lewis, 1985, John Wiley & Sons, Inc., New York.
Abbreviations utilized in this invention are defined below:
List of Abbreviations
HRG heregulin
EGF epidermal growth factor
EGFR EGF receptor
HER Human EGF receptor
NDF neu differentiation factor
ARIA acetylcholine receptor
inducing activity
GGF glial growth factor
p1 85erbB2 HER2 encoded protein
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p1 8oerbB4 HER4 encoded protein
ICAM-1 intercellular adhesion
molecule-1
~ SH2 Src homology 2
CHO Chinese hamster ovary
SDS-PAGE sodium dodecyl sulfate-
polyacrylamide gel
electrophoresis
Bes N, N-bis(2-hydroxyethyl)-
1 0 2-aminoethanesulfonic
acid
P B S phosphate-buffered
saline.
The construction of the rHRGs-T-Fc fusion proteins to
15 generate versatile recombinant HRGs for studying the various
aspects of the biology of the HER4/HRG receptor/ligand pair is as
follows: since the EGF-like domain of HRG-,B1 had previously
been shown to be sufficient for receptor binding, Holmes, W. E. et
al., (1992), Science 256, 1205-1210, three chimeric genes are
20 constructed that encode soluble proteins consisting of the EGF-
like domain of HRG-a, -~2, or -~B3 linked to a thrombin cleavage
site followed by the hinge, CH2 and CH3 regions of a human IgGI
antibody, with secretion of the proteins directed by the signal
sequence of CD5. The EGF-like domain of HRG-a corresponds to
25 residue 177 to 241 of the mature protein, while that of HRG-~2
and -,~3 corresponds to residue 177 to 238 and residue 177 to
241, respectively. The three fusion proteins rHRGs-T-Fc are
prepared by transient expression in COS cells, purified from
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culture supernatants on protein A-Sepharose, and gave yields in
the range of 350 to 1900 ~lg/l. The CD5 signal peptide allowed
efficient processing and secretion of the rHRGs-T-Fc. All three
fusion proteins are secreted as disulfide-linked homodimers
similar to immunoglobulins and therefore are each capable of
presenting two HRG-EGF-like domains. To establish that the
rHRGs-T-Fc are able to bind and activate the HER4 receptor, a
study is preferably carried out of their potential to induce
phosphorylation of HER4 as well as morphological changes and
1 0 up-regulation of ICAM-1 .
For the production of large amounts of other
recombinant growth factors, the chimeric genes of other growth
factors such as, epidermal growth factor, transforming growth
factor-alpha, amphiregulin, betacellulin, heparin-binding
epidermal gro~th factor, vaccinia growth factor, cripto, insulin
growth factor, insulin-like growth factor, transforming growth factor-
beta, platelet-derived growth factor, fibroblast growth factor, and
nerve growth factor, can be constructed and expressed in a similar
manner as for rHRGs-T-Fc.
The activation of the HER4 receptor by rHRGs-T-Fc is
examined with CHO/HER4 cells that express high levels of
recombinant human p180erbB4/HER4 and have previously been
shown to respond to HRG, Plowman, G. D. et al., (1993) Proc.
Natl. Acad. Sci. U.S.A. 90, 1746-1750 and Culouscou, J. M. et al.,
(1993) 1. Biol. Chem. 268, 18407-18410. rHRG-o~-T-Fc, -,~2-T-Fc,
and -~3-T-Fc, are added to CHO/HER4 cells at 50 and 200 ng/ml
for 10 minutes at 37~C. Cells are then Iysed and then the pattern
of tyrosine phosphorylated proteins are analyzed by anti-
- 10-
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phosphotyrosine Western blotting as compared to untreated cells.
As shown in Fig. 1A, all three rHRGs-T-Fc induced the hyper-
phosphorylation of the HER4 receptor. Ligand activation not only
resulted in receptor autophosphorylation, but also in the tyrosine
- - 5 phosphorylation of several substrates, including a Mr 100,000
band, not identified (Fig. 1A). When tested on CHO/EGFR cells
that express high levels of recombinant human EGFR, rHRGs-T-Fc
(200 ng/ml) failed to activate the EGFR (Fig. 1 B). The EGF (200
ng/ml) markedly induced phosphorylation of the EGFR in
CHO/EGFR cells (Fig. 1 B, lane 2). These demonstrate that the
rHRGs-T-Fc are active molecules and are able to specifically
indl~ce HER4 tyrosine phosphorylation.
The binding of rHRG-a-T-Fc to HER4 expressing cells
can be shown in the MDA-MB-453 cells that are known to express
the HER4 receptor, Plowman, G. D. et al., (1993) Proc. Natl. Acad.
Sci. U.S.A. 90, 1746-1750, as well as in the related receptors
HER2 and HER3, Kraus, M. H.et al., (1987) EMBO J. 6, 605-610
and Kraus, M. H. et al., 1989) Proc. Natl. Aca. Sci. U.S.A. 86, 9193-
9197. These cells are incubated on ice with 1 or 10 ~Lg/ml rHRG-a-
T-Fc. Bound fusion proteins are detected by adding fluorescein-
conjugated anti-human IgG antibodies which recognize the
human Fc portion of rHRG-a-T-Fc. As shown in Fig. 2, rHRG-a-T-
Fc bound to MDA-MB-453 cells~(Fig. 2B, 10 ~lg/mL; and 2D, 1
,ug/mL). The fluorescence, as analyzed by confocal microscopy, is
localized at the periphery of the cells which is consistent with the
fact that the staining is performed on live cells kept on ice. When
no fusion protein is added, the fluorescein-conjugated anti-human
IgG showed no detectable binding to the MDA-MB-453 cells (Fin=
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2A). Minimal background staining is observed when an irrelevant
Tek-Fc fusion protein is used at 10,ug/ml (Fig. 2C). This result
indicates that rHRG-a-T-Fc binds to HER4 expressing cells, and
can be used to detect cells expressing HRG binding proteins in a
5 manner similar to monoclonal antibodies. rHRGs-T-Fc represent
an alternative to antibodies for cell staining.
NDF, the rat homologue of HRG, has been shown to
induce morphological changes in AU565 mammary tumor cells,
Peles, E. et al., (1992), Cell 69, 205-216, as well as the expression
of ICAM-1, Bacus, S. S. et al., (1993), Cancer Res. 53,5251 -5261.
For carrying out the ability of rHRG-oc-T-Fc to induce the
expression of ICAM-1 at the surface of MDA-MB-453 cells, these
cells are treated for 3 days with 50 ng/ml of the fusion protein and
stained with an anti-lCAM-1 antibody. Bound anti-lCAM-1
15 antibodies are detected using a fluorescein-conjugated anti-
mouse IgG antibody. As shown in Fig. 3B, rHRG-o~-T-Fc induced a
clear up-regulation of ICAM-1 expression in MDA-MB-453 cells as
compared to untreated cells (Fig. 3A) and cells treated with an
irrelevant Tek-Fc fusion protein (Fig. 3C). p45, a HRG isoform
20 purified from conditioned medium from HepG2 cells, Culouscou, J.
M. ~L. (1993) J. Biol. Chem. 268,18407-18410, is used at 50
ng/ml as a positive control and induced up-regulation of ICAM-1
(Fig. 3D). The result shows that the rHRGs-T-Fc elicited biological
responses similar to those elicited by the natural HRGs in breast
25 carcinoma cells expressing the HER4 receptor and can be used to
study the biological consequences of HRG binding to such cells.
The DNA fragments encoding the EGF-like domains of
HRGs are amplified by PCR, purified and inserted into a CDM7-
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derived expression vector containing sequences encoding a
thrombin cleavage site upstream and in frame with the Fc portion
of a human IgGI antibody. The addition of a thrombin cleavage
site in the expression vector is based on a system developed by
~ - 5 Hakes and Dixon, Hakes, D. J. et al., (1992) Anal. Biochem. 202,293-298, for recombinant protein expression in bacteria. The
presence of a thrombin cleavage site in the rHRGs-T-Fc allows for
separation of the two functional domains of the fusion proteins.
Following thrombin cleavage, the purified EGF-like domains are
recovered as monomeric proteins since the thrombin site is
located upstream of the hinge region of the Fc domain of the
fusion proteins. rHRG-,B2-T-Fc and -~3-T-Fc are incubated with
hunnan thrombin. The recombinant EGF-like domains of HRGs
(reHRGs) are then separated from the Fc portion of the molecules
by protein A-Sepharose chromatography. reHRGs are recovered
in tlle column flow-throughs. Fc portions are recovered from
protein A-Sepharose by acid elution. Fig. 4 shows a silver stained
polyacrylamide gel of the rHRG-~3-T-Fc before thrombin cleavage
(lane 1). The intact fusion protein displays an apparent molecular
mass of 40 kDa under reducing conditions, corresponding to its
monomeric form. After cleavage but before protein A-Sepharose
(lane 2), a 34 kDa band, corresponding to the Fc portion of the
fusion protein, and a 6 kDa band, corresponding to the EGF-like
dornain of HRG-,B3, are identified. The two fragments are
separated by protein A-Sepharose chromatography. The 6 kDa
EGF-like domain of HRG-~3 (reHRG-~3) is recovered in the
column flow-through (lane 3), and the 34 kDa Fc domain of the
fusion protein is acid eluted from the column (lane 4)
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The stimulation of protein phosphorylation in response
to reHRGs are carried out on the purified reHRG-~2, reHRG-,~3,
and Fc domains of rHRG-,~2-T-Fc and rHRG-,~3-T-Fc in MDA-MB-
453 cells. The intact rHRG-,~2-T-Fc and -,~3-T-Fc (Fig. 6B, lanes 3
and 6, respectively) are potent stimulators of tyrosine
phosphorylation of a 180 kDa protein, as compared to background
levels of phosphorylation observed in the absence of treatment
(lane 1) or following EGF treatment (lane 2). reHRG-~2 (lane 4)
and reHRG-~3 (lane 7) elicited an increase in the phosphorylation
level of the 180 kDa protein similar to that obtained with the rHRG-
~2-T-Fc and rHRG-,B3-T-Fc, whereas the Fc domains from rHRG-
,~2-T-Fc and rHRG-,~3-T-Fc failed to induce protein
phosphorylation (lanes 5 and 8, respectively).
Following cleavage of the fusion proteins, several amino
acid residues from the glycine rich region of the thrombin cleavage
site remain at the carboxy terminus of the reHRGs. Cleavage
occurs at the Proline-Arginine recognition sequence of the
thrombin cleavage site. See Hakes, D. J. et al., (1992), Anal.
Biochem. 202, 293-298. These additional residues did not affect
the properties of reHRGs. Among the multiple HRG/NDF isoforms,
the region proximal to the EGF domain (referred to as the
juxtamembrane region in the HRG/NDF precursor forms) can be
absent, e.g. HRG-,~2/NDF-,B2, or comprise up to 26 amino acids,
e.g. NDF-,~4, Holmes, W. E. et al., (1992), Science 256,1205-1210
and Wen, D. et al., (1994), Mol. Cell BjQI. 14,1909-1919. A
truncated form of NDF-a that lacks the juxtamembrane region
displays the same receptor binding affinity as the full-length NDF-
a isoform, implying that this region proximal to the EGF domain is
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not involved in receptor binding, Wen, D. et al., (1994), Mol. Cell
Biol.14,1909-1919. This shows that, after thrombin cleavage, the
reHRGs retain the activity displayed by the rHRGs-T-Fc fusion
proteins.
~ - 5 Because the MDA-MB-4~3 cells express HER3, HER4,
and also high levels of HER2, the exact identity of the 180 kDa
phosphorylated band is unknown. HER4 and HER3 have both
been identified as HRGs receptors, Plowman, G. D. et al., (1993)
Proc. Natl. Acad. Sci. U. S. A. 90,1746-1750; Culouscou, J. M. et
al., (1993) J. Biol. Chem. 268, 18407-18410; Plowman, G. D.
al., (1993) Nature (London) 366, 473-475 and Carraway, K. L. et
al., (1994) J . Biol. Chem. 269, 14303-14306. Since the samples
are electrophoresed under reducing conditions, the
phosphorylated species corresponds to a combination of
monomericforms of all three receptors, including HER2. In
response to HRG stimulation, HER2 is stimulated indirectly
through receptor transphosphory!ation, P!owman, G. D. vt al.,
(1993) Nature (London) 366, 473-475. In addition, it has been
indicated that a high affinity binding site for the EGF-like domain of
HRG-,~1 can be reconstituted by co-expression of HER2 and HER3
in COS-7 cells, and that binding of HRG results in tyrosine
phosphorylation of both HER2 and HER3, Sliwkowski, M. X. et al.,
(1994) J. Biol. Chem. 269, 1466~1-14665.
The results described clearly demonstrate that the EGF-
like domains of rHRGs-T-Fc mediate the obser~ed biological
effects and that those effects cannot be attributed to the Fc portion
of the fusion proteins.
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The phosphorylation of Shc upon HER4 receptor
activation is carried out as follows: the activation of receptor
tyrosine kinases, such as the EGF receptor, the insulin receptor,
and the PDGF receptor results in phosphorylation of a number of
intracellular signaling molecules, Ullrich, A. et al., (1990) Ç~!! 61,
203-212 and White, M. F. et al., (1994) J. Biol. Chem. 269,1 -4. For
analyzing the molecules that are involved in HRG signaling, MDA-
MB-453 cells are stimulated with or without 200 ng/ml reHRG-,~2.
Cell Iysates are immunoprecipitated with the following antibodies:
anti-GAP, anti-PLC-gl, anti-P I 3-K, and anti-Shc. Precipitated
proteins are separated by SDS-PAGE, then immunoblotted with
antiphosphotyrosine antibodies. Shc and, to a lesser degree, P I
3-K immunoprecipitates displayed enhanced patterns of protein
phosphorylation following HRG stimulation. A further analysis of
Shc phosphorylation was carried out. Shc proteins are
ubiquitously expressed proteins containing a single SH2 domain.
Three structurally related Shc proteins, p46Shc, p52Shc, and
p66Shc have been described as adaptor molecules that are
implicated in Ras activation, Pelicci, G. et al., (1992) Ç~ 70, 93-
104 and Rozakis-Adcock et al., (1992) Nature (London) 360, 689-
692. CHO/HER4 cells and MDA-MB- 453 cells are exposed to
reHRG-~2, and Iysed. Equivalent amounts of cell Iysates are
immunoprecipitated with an anti-Shc antibody, and blotted with
either anti-Shc (Fig. 6A), or anti-phosphotyrosine antibodies (Fig.
6B). Fig. 6A shows that equal amounts of proteins from
stimulated and unstimulated cell Iysates are loaded per lane, and
that MDA-MB-453 cells (lanes 1 and 2) express only p46Shc and
p52Shc (p66Shc is not detected in the assay), whereas
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CHO/HER4 cells (lanes 3 and 4) express all three Shc isoforms.
p66Shc is translated from a different transcript than the other two
Shc isoforms and is not expressed in every cell type, for example it
is absent in human hematopoietic cell lines, Pelicci, G. et al.,
~ - 5 (1992) ~ell 70, 93 -104. As seen in Fig. 6B, reHRG-~2 induced
hyper-phosphorylation of Shc in both cell types. In MDA-MB-453
cells, reHRG-~2 stimulation resulted in tyrosine phosphorylation of
both p4~Shc and p52Shc (lanes 1 and 2). Following reHRG-132
stimulation, phosphorylation of p52Shc is markedly increased in
CHO/HER4 cells (lanes 3 and 4). p46Shc appeared to display a
relatively high endogenous level of phosphorylation in those cells,
and is only marginally affected following HRG treatment. Longer
exposure time of the blot shown in Fig. 6B, lanes 3 and 4, resulted
in a loss of resolution between the p46Shc and p52Shc bands
but revealed that p66Shc is phosphorylated in response to
reHRG-~2 (lane 6) as compared to unstimulated cells (lane 5).
The results presented in the instant invention indicate
recombinant EGF-like domains of HRG-a, -~2, and -,~3 fused to a
thrombin cleavage site followed by the Fc domain of a human IgGI.
These reagents are useful in in vitro assays as fusion proteins or
as a source of truncated recombinant HRGs. The results also
indicate that both forms in vitro can activate the HER4 receptor and
elicit known HRG biological responses. The present invention
shows for the first time, that following HRG stimulation, Shc
proteins which have been implicated in Ras activation pathway
are phosphorylated on tyrosine. The availability of the
recombinant HRGs will allow further experiments to dissect the
mechanism of HRG receptor signaling as well as compare the
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HER4 substrates to those of other members of the EGFR family of
tyrosine kinases.
In order to further clarify and enable the present
invention as illustrated in the Examples described herein below,
5 a general description of the materials and methods utilized in
producing the results disclosed is presented. These materials and
methods illustrate the technology utilized and are not intended to
be limiting of the present invention. Other variations and
modifications of these methods are well-known and are
10 contemplated by this invention.
Antibodies - RC20 recombinant antiphosphotyrosine
antibody (Transduction Laboratories) and PY20
antiphosphotyrosine antibody (ICN Biomedicals, Inc.) used in
Western blotting studies were purchased from Transduction
15 Laboratories and ICN Biomedicals, Inc. Polyclonal anti-Shc
antibodies were purchased from Upstate Biotechnology
Incorporated and the monoclonal anti-Shc antibody was
purchased from Transduction Laboratories. BBA 3, the anti-
human ICAM-1 monoclonal antibody, was purchased from R & D
20 Systems.
Cell Lines - MDA-MB-453 human breast cancer cells
were obtained from the American Type Culture Collection.
CHO/EGFR cells were generated by Dr. B. Thorne (Bristol-Myers
Squibb, Seattle, WA.) as follows: the complete recombinant
25 human EGF receptor coding sequence was inserted into a CDM8
expression vector containing the neomycin resistance gene. The
resulting construct was transfected into Chinese hamster ovary
cells (CHO-KI). G418 resistant clones were analyzed for EGFR
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expression. Levels of expression of functional EGFR in
CHO/EGFR stable cells were assessed by stimulating the cells
with EGF, immunoprecipitating the EGFR and determining its
phosphorylation level by phosphotyrosine Western blotting as
~ - 5 reported, by Plowman, G. D. et al., (1993) Proc. Natl. Acad. Sci.
U.S.A. 90, 1746-1750. CHO/HER4 cells expressing high levels of
recombinant human HER4 have previously been described, by
Plowman, G. D. et al., (1993) Proc. Natl. Acad. Sci. U.S.A. 90,
1746-1750; Culouscou, J. M. et al., (1993) J. Biol. Chem. 268,
18407-18410 and Plowman, G. D. et al., (1993) Nature (London)
366, 473-475.
Example 1
CONSTRUCTION OF HRG-T-FC EXPRESSION PLASMIDS
DNA fragments encoding part of the spacer domain of
the human HRGs, the EGF-like domains, the transmembrane
domain, and a few residues of the cytoplasmic domain wère
amplified by F~T-PGR from tota! RNA iso!ated from HApG2 cells.
The oligonucleotide primers were designed based on the
sequence of the human HRG-13, Holmes, W. E. et al., (1992),
Science 256, 1205-1210.
The PCR primers used were synthesized and have the
following sequences:
5'-GTGTCTTCAGAGTCTCCCATTAGA-3' (forward
primer, SEQ. I.D. NO.: 1 ) and
5'-CTTGGI I I I GCAGTAGGCCAC-3' (reverse primer,
SEQ. I.D. NO.: 2). Amplification was performed with Taq DNA
polymerase (Perkin-Elmer Roche) using 35 cycles, each cycle
being composed of a 1 minute at 95~C denaturing step, 1 minute
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at 65~C annealing step, and 30 sec at 72~C extension step. The
PCR products were blunt-ended using the Klenow fragment of E.
coli DNA polymerase 1, subcloned into a Sma-1 digested
pBluescript ll vector (Stratagene) and the nucleotide sequence of
5 individual clones was determined by the dideoxy-mediated chain
termination reaction. This procedure generated EGF-like domains
of HRG-a, -,~2, and -,~3
The EGF-like domains of HRG-a, -B2, and -B3 were
generated by PCR using HRG-a, or-,B2 template plasmids
10 generated as described above. The oligonucleotide primers
described below were designed to place a Spel site at the 5' end
and a BamH1 site at the 3' end of the amplified products for
cloning purposes. The epidermal growth factor-like domain of the
human HRG-a was amplified using the following sequences:
1 5 5'-GAGACTAGTAGCCATCTTGTAAAATGTGCG-3'
(forward, SEQ. I.D. N0.: 3), and
5 '-CCGTGGATCCTTCTGGTACAGCTCCTCCGC-3 '
(reverse, SEQ. I.D. NO.: 4. PCR conditions consisted of 40 cycles
of 30 sec at 94~C, 1 minute at 55~C, and 2 minutes at 72~C, using
20 Pfu polymerase and reagents recommended by the vendor
(Stratagene Corp.). The PCR product encoded complementary
sequences corresponding to residue 177 to 241 of HRG-a. The
epidermal growth factor-like dornains of human HRG-,B2 and -,~3
were amplified using a HRG-B~2 clone as a template. The
25 forward primer is shown above (SEQ. I.D. No.: 3). The HRG-~2
reverse primer had the sequence:
5 '-CCGTGGATCCTTCTGGTACAGCTCCTCCGCCTT-3 '
(SEQ. I.D. N0.: 5). Amplification was performed with Pfu
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polymerase using the same temperature conditions as that used
for HRGa. This PCR product encoded sequences corresponding
to residue 177 to 238 of HRG-,B2. The HRG-,~3 reverse primer
contained a silent point mutation introducing a Hindlll site for
5 diagnostic purposes and had the following sequence:
5 -CCGTGGATCCTCAGGCAAGCTTAGAAAGGGA
GTGGACGTACTGTAGMGCTGGCCATTAC-3
(SEQ. I.D. NO.:6). PCR conditions consisted of 40 cycles of 1
minute at 94~C, 2 minutes at 50~C, and 3 minutes at 72~C using
10 Pfu polymerase. The PCR product encoded sequences
corresponding to residues 177 to 241 of HRG-~3. All PCR
products were digested with BamHI and Spel and ligated to a
BamHI-~pel-cut CDM7-derived vector containing cDNA
sequences coding for the CD5 signal peptide 5 of the cloning site
15 for proper secretion of the expressed proteins, as well as cDNA
sequences encoding a thrombin cleavage site (amino acid
sequence DPGGGGGRLVPRGFGTG; Sequence l.D. No. 7) and
cDNA sequences encoding the hinge and constant regions of a
human IgGI, 3 of the cloning site. All constructs were sequenced
20 by the dideoxy-mediated chain termination reaction to confirm the
sequence of the EGF-like domains as well as to verify that their
sequences were in frame with the thrombin and Fc coding
sequences. This procedure re~ulted in the production of the
constnucts or the expression plasmids of HRGs-T-Fc. The
25 sequence of a rHRG-o~-T-Fc appears in Figures 7A and B.
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Example 2
TRANSFECTION OF CONSTRUCTS INTO COS CELLS
The above constructs were transfected into COS cells as
previously described, Seed, B. et al., (1987) Proc. Natl. Acad. Sci.
~!~ 87, 3365-3369, and the resulting fusion proteins recovered
from culture supernatants using protein A-Sepharose (Repligen).
Purified proteins were visuaiized on 8% SDS-PAGE under
reducing and non-reducing conditions. Protein concentrations
were determined using a protein assay kit (Bio-Rad Labs). This
experiment resulted in the fusion protein, rHRG-oc-T-Fc, rHRG-~2-
T-Fc or rHRG-,~3-T-Fc
Example 3
THROMBIN CLEAVAGE OF FUSION PROTEINS
Fusion proteins were incubated for 30 minutes at room
temperature with human thrombin (purchased from Sigma, St.
Louis, MO) at a 1 :50 (w/w) thrombin: fusion protein ratio. Cleaved
proteins were then loaded on a protein A-Sepharose column.
Column flow-throughs containing the recombinant EGF-like
domain of HRGs were stored at -20~C. This procedure gave
recombinant protein reHRG-a, reHRG-,B2 or reHRG-,~3.
Example 4
DETECTION OF TYROSINE-PHOSPHORYLATED PROTEINS BY
WESTERN BLOTTING
CHO/HER4 cells (5x104), CHO/EGFR cells (2x104), and
MDA-MB-453 cells (4x105) were seeded in 48-well plates. 24
hours later, cells were serum-starved for 8 hours and then
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stimulated with various samples for 10 minutes at 37~C.
Supernatants were discarded, and cells were Iysed by adding
boiling electrophoresis sample buffer. Lysateswere subjected to
SDS-PAGE on 8% polyacrylamide gels (purchased from Novex)
- 5 and then electroblotted onto nitrocellulose. PY20 monoclonal
anti-phosphotyrosine antibody (purchased from ICN) and
horseradish peroxidase-conjugated goat anti-mouse IgG F(ab )2
(purchased from Cappel) were used as primary and secondary
probing reagents, respectively. Immunoreactive bands were
visualized using enhanced chemiluminescence (purchased from
Amersham Corp.). The results showed the pattern of tyrosine
phosphorylated proteins in HER4 receptor which is illustrated in
Fig. 1.
Example 5
IMMUNOPRECIPITATION
CHO/HER4 cells were seeded in 100-mm dish. 80-90%
confluent monolayers were washed and incubated with the
various recombinant HRGs for 10 minutes at 37~C. Monolayers
were washed with ice-cold PBS, and solubilized for 10 minutes on
ice in PBSTDS Iysis buffer (10 mM sodium phosphate, pH 7.3, 150
mM NaCI, 1% Triton-X100, 0.5% sodium deoxycholate, 0.1%
SDS) containing 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride,
1 mM Na3 V04, 20 ~lg/ml aprotinin, 20 ,~Lg/ml leupeptin, 20 ~g/ml
pepstatin. The protein concentrations of the clarified extracts were
determined using a BCA protein assay kit (purchased from
Pierce). Lysates (1 mg per immunoprecipitation) were incubated
overnight at 4~C with a rabbit anti-Shc antibody (purchased from
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UBI). Immune complexes were precipitated by adding protein G-
Plus/Protein A-Agarose (purchased from Oncogene Sciences) to
the suspensions. After one hour of incubation at 4~C, the
immunoprecipitates were washed three times with PBSTDS and
5 then resolved on 8% polyacrylamide gels (purchased from Novex)
under reducing conditions. Proteins were electroblotted onto
nitrocellulose and probed with RC20 recombinant anti-
phosphotyrosine antibody (purchased from Transduction
Laboratories) or anti-Shc monoclonal antibody (purchased from
1 0 Transduction Laboratories) . I mmunoreactive bands were
visualized using enhanced chemiluminescence (purchased from
Amersham Corp.). The results showed the precipitation of Shc
proteins and that these proteins are phosphorylated in response to
protein reHRG-~2.
Example 6
IMMUNOHISTOCHEMICAL STAINING
MDA-MB-453 cells were plated in 8-well borosilicate
chambered slides (purchased from Lab-Tek). For receptor binding
20 visualization, after a 48 hour-culture period, the cells were placed
on ice for 10 minutes, washed twice with ice-cold binding buffer
(DMEM supplemented with 44 mM sodium bicarbonate, 50 mM
Bes, pH 7.0,0.1% bovine serum albumin) and then incubated on
ice for 2 hours with rHRG-a-T-Fc, or as a negative control, an
25 irrelevant fusion protein consisting of the extracellular domain of
Tek receptor, Dumont, D. J. et al., (1993) Oncogene 8, 1293-1301,
fused to the thrombin cleavage site followed by the Fc region of an
IgGI as in rHRGs-T-Fc. The reagents, cloning vector, and
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mammalian cells used to construct and generate the Tek-Fc fusion
protein were identical to the ones used to make the rHRGs-T-Fc.
The cells were washed twice and incubated for 45 minutes on ice
with a fluorescein-conjugated goat anti-human IgG F(ab')2
5 (purchased from Tago). The cells were rinsed twice with PBS and
fixed for 20 minutes in PBS, 2% formaldehyde. The results
showed that the recombinant fusion protein can be used to stain
cells that express HER4 receptor.
Example 7
ICAM-1 EXPRESSION STUDIES
For ICAM-1 expression studies, after a 24 hour-culture
period, the MDA-MB-453 cells were incubated for three days with
50 ng/ml rHRG-oc-T-Fc, p45, Culouscou, J. M. et al., (1993) J. Biol.
Chem. 268, 18407-18410, Tek-Fc fusion protein as a negative
control, or culture medium alone. Staining was then performed on
live cells. The cells were washed and incubated for 1 hour on ice
with an anti-lCAM-1 antibody (purchased from R & D Systems)
diluted 1:500 in binding buffer. The cells were washed and
20 incubated for 45 minutes on ice with a fluorescein-conjugated goat
anti-mouse IgG F(ab')2 (purchased from Tago). The cells were
rinsed and fixed as described above. The levels of receptor
staining and ICAM-1 expression were analyzed using a Leica
confocal microscope. The results showed that fusion protein
25 induced the expression of ICAM-1 in breast carcinoma cells that
express HER4 receptor and is illustrated in Fig. 3.
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Example 8
TYROSINE AUTOPHOSPHORYLATION OF THE HER4
RECEPTOR
FOLLOWING rHRGS-T-FC STIMULATION - Figure 1
A. CHO/HER4 cells were incubated in the absence
(lane 1 ) or the presence of rHRG-o~-T-Fc (lanes 2 and 3), rHRG-,~2-
T-Fc (lanes 4 and 5), and rHRG-,~3-T-Fc (lanes 6 and 7) at 50
ng/ml (lanes 2, 4, and 6) or at 200 ng/ml (lanes 3, 5, and 7). Cells
were Iysed, proteins separated by SDS-PAGE, transferred to
nitrocellulose and immunoblotted with antiphosphotyrosine
antibodies. Horseradish peroxidase-conjugated goat anti-mouse
IgG antibodies and chemiluminescence reagents were used to
visualize the bound antibodies. The results illustrated in Fig. 1A
showed that all three rHRGs-T-Fc induced the hyper-
phosphorylation of the HER4 receptor. Also, ligand activation
resulted in receptor autophosphorylation as well in the tyrosine
phosphorylation of several substrates.
B. CHO/EGFR cells were incubated in the absence
(lane 1 ) or the presence of EGF (lane 2), rHRG-a-T-Fc (lane 3),
rHRG-,B2-T-Fc (lane 4), and rHRG-,~3-T-Fc (lane 5) used at 200
ng/ml. Cells Iysates were processed as described in A. The
positions of HER4 and EGFR are indicated. These results
illustrated in Fig. 1 B showed that rHRGs-T-Fc failed to activate the
EGFR. Fig. 1 B (lane 2) showed that EGF markedly induced
phosphorylation of the EGFR in CHO/EGFR cells.
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Example 9
BINDIN~ OF rHRG-a-T-FC TO MDA-MB-453 CELLS - Figure 2
Cells were plated in 8-well Lab-Tek (Nunc) chamber
slides at 2 x 105 cells/ well. After 2 days, the cells were placed on
5 ice and stained with rHRG-a-T-Fc at 10 ~g/ml (panel B) and 1
g/ml (panel D), or with an irrelevant fusion protein used at 10
,ug/ml (panel C). No fusion proteins were added in the experiment
shown in panel A. Fluorescein-labeled goat anti-human Fc
antibodies were used to visualize the bound fusion proteins.
10 Fluorescent staining was analyzed by confocal microscopy. The
results illustrated in Fig. 1A showed no binding to the cells when
fusion protein was not added to it. The results illustrated in Fig. 2B
showed that rHRG-a-T-Fc bound to MDA-MB-453 cells. The
results illustrated in Fig. 2C showed minimal background staining
15 when an irrelevant Tek-Fc fusion protein was used. The results of
this experiment indicated that rHRG-a-T-Fc bound to HER4
expressing cells and can be used to detect cells expressing HRG
binding proteins.
Example 10
THE INDUCTION OF ICAM-1 EXPRESSION IN RESPONSE TO
rHRG-oc-T-FC - Figure 3
MDA-MB-453 cells were cultured for 24 hours in 8-well
Lab-Tek (Nunc) chamber slides. Cells were treated with 50 ng/ml
rHRG-a-T-Fc (panel B), irrelevant fusion protein (panel C), p45,
Culouscou, J. M. et al., (1993) J. Biol. Chem. 268,18407-18410,
(panel D), or left untreated (panel A). Following three additional
days of incubation the cells were stained with an anti-lCAM-1
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monoclonal antibody. Fluorescein-labeled goat anti-mouse Fc
antibodies were used to visualize the bound anti-lCAM-1
antibodies. Staining was analyzed by confocal microscopy. Fig.
3B showed that rHRG-a-T-Fc induced a clear up-regulation of
5 ICAM-1 expression in MDA-MB-453 cells as compared to
untreated cells (Fig. 3A) and cells treated with an irrelevant Tek-Fc
fusion protein (Fig. 3C). Fig. 3D showed that p45 induced up-
regulation of ICAM-1. Thus, the results of this experiment showed
that rHRGs-T-Fc elicited biological responses similar to those
10 elicited by the natural HRGs in breast carcinoma cells expressing
the HER4 receptor.
Example 1 1
THROMBIN CLEAVAGE OF rHRG-~3-T-FC - Figure 4
The fusion protein was incubated with human thrombin
at room temperature for 30 minutes, and loaded on a protein A-
Sepharose column. The EGF-like domain of HRG-,~3 (reHRG-133)
was recovered in the column flow-through while the Fc portion of
the fusion protein was eluted from the column. The resulting
20 products were analyzed by SDS-PAGE, and silver stained. Lane
1, rHRG-,~3-T-Fc, untreated; lane 2, rHRG-,~3-T-Fc, after thrombin
cleavage; lane 3, protein A-Sepharose column flow-through
(reHRG-~3); lane 4, protein A-Sepharose column eluate (Fc
portion of the fusion protein). Fig. 4 (lane 1) showed a silver
25 stained polyacrylamide gel of the rHRG-~3-T-FC before thrombin
cleavage. Fig. 4 (lane 2) showed a 34 kDa band corresponding to
the Fc portion of the fusion protein and a 6 kDa band
corresponding to the EGF-like domain of HRG-~3. The 6 kDa
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EGF-like domain of HrG-,B3 (reHRG-,~3) was recovered in the
column-flow-through (lane 3) and the 34 kDa Fc domain of the
fusion protein was acid eluted from the column (lane 4).
Example 12
STIMULATION OF PROTEIN PHOSPHORYLATION
IN RESPONSE TO reHRGS - Figure 5
MDA-MB-453 cells were incubated in the absence (lane
1) orthe presence of EGF (lane 2), rHRG-,~2-T-Fc (lane 3),
reHRG-~2 (lane 4), Fc portion of the rHRG-~2-T-Fc fusion protein
(lane 5), rHRG-,~3-T-Fc (lane 6), reHRG-~3 (lane 7), Fc portion of
the rHRG-,~3-T-Fc fusion protein (lane 8), at 200 ng/ml. Cells were
Iysed, proteins were separated by SDS-PAGE, transferred to
nitrocellulose membrane and blotted with an anti-phosphotyrosine
antibody. Immunoreactive bands were visualized with enhanced
chemiluminescence reagents. The results showed that rHRG-~2-
T-Fc and -~3-T-Fc (lanes 3 and 6 respectively) were potent
stimulators of tyrosine phosphorylation of 180 kDa protein as
compared to background level of phosphorylation observed in the
absence of treatment (lane 1 ) or following EGF treatment (lane 2).
Fig. 5 (lanes 4 and 7) showed that reHRG-,~2 and reHRG-,~3
elicited an increase in the phosphorylation level of the 180 kDa
protein, whereas the Fc domains from rHRG-~2-T-Fc and rHRG-
,B3-T-FC failed to induce protein phosphorylation (Fig. 5, lanes 5
and 8).
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Example 13
TYROSINE PHOSPHORYLATION OF SHC PROTEINS UPON
HER4 ACTIVATION - Figure 6A and 6B
MDA-MB-453 cells (lanes 1 and 2) and CHO/HER4 cells
5 (lanes 3 to 6) were treated with (+) or without (--) 200 ng/ml of
reHRG-,~2 for 10 minutes at 37~C and solubilized. Cell Iysates
containing equal amounts of protein (1 mg) were precipitated with
a polyclonal rabbit anti-Shc antibody. Immune complexes were
washed, separated by SDS-PAGE, and transferred to
10 nitrocellulose.
A. Shc proteins were detected by immnunoblot using a
monoclonal anti-Shc antibody.
B. Tyrosine phosphorylation of Shc proteins was analyzed
by immunoblot using antiphosphotyrosine antibodies. The
15 positions of the three Shc isoforms are indicated.
The results illustrated in Fig. 6A showed that MDA-MB-
453 cells (lanes 1 and 2) express only p46Shc and p52Shc
whereas CHO/HER4 cells (lanes 3 and 4) express all three Shc
isoforms. Fig. 6B showed that reHRG-~2 induced hyper-
20 phosphorylation of Shc in both cell types. In MDA-MB-453 cells,
reHRG-,B2 stimulation resulted in tyrosine phosphorylation of both
p46Shc and p52Shc (lanes 1 and 2). Following reHRG-,B2
stimulation, phosphorylation of p66Shc was markedly increased in
CHO/HER4 cells (lanes 3 and 4). Longer exposure time of the
25 blot (Fig. 6B, lane 3 and 4) resulted in a loss of resolution between
the p46Shc and p52Shc bands, but revealed that p66Shc was
phosphorylated in response to reHRG-~2 (lane 6) as compared to
unstimulated cells (lane 5).
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Example 1 4
CONSTRUCTION OF EXPRESSION PLASMIDS
OF OTHER GROWTH FACTORS
- 5 The expression plasmids of other growth factors can be
constructed analogously from cDNA's of other growth factors, such
as epidermal growth factor, transforming growth factor-alpha,
amphiregulin, betacellulin, heparin-binding epidermal growth
factor, vaccinia growth factor, cripto, insulin growth factor, insulin-
10 like growth factor, transforming growth factor-beta, platelet-derived
growth factor, fibroblast growth factor, or nerve growth factor,
according to Example 1 and expressed according to Example 2.
The foregoing description and Examples are intended
as illustrative of the present invention, but not as limiting.
15 Numerous variations and modifications may be effected without
departing from the true spirit and scope of the present invention.
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