U.S. patent application number 10/323001 was filed with the patent office on 2003-07-10 for gdu, a novel signalling protein.
Invention is credited to Daly, Roger John, Sutherland, Robert Lyndsay.
Application Number | 20030129639 10/323001 |
Document ID | / |
Family ID | 25644943 |
Filed Date | 2003-07-10 |
United States Patent
Application |
20030129639 |
Kind Code |
A1 |
Daly, Roger John ; et
al. |
July 10, 2003 |
GDU, a novel signalling protein
Abstract
The present invention provides the nucleotide and amino acid
sequence of a previously unidentified erbB receptor target. The
nucleotide and amino acid sequence is set out in the figure.
Inventors: |
Daly, Roger John;
(Alexandria, AU) ; Sutherland, Robert Lyndsay;
(Lindfield, AU) |
Correspondence
Address: |
Shantanu Basu
Morrison & Foerster LLP
755 Page Mill Road
Palo Alto
CA
94304-1018
US
|
Family ID: |
25644943 |
Appl. No.: |
10/323001 |
Filed: |
December 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10323001 |
Dec 18, 2002 |
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10242332 |
Sep 11, 2002 |
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10242332 |
Sep 11, 2002 |
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08945771 |
Apr 22, 1998 |
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6465623 |
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08945771 |
Apr 22, 1998 |
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PCT/AU96/00258 |
May 2, 1996 |
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Current U.S.
Class: |
435/6.14 ;
435/7.23; 530/388.26; 536/23.2; 536/24.3 |
Current CPC
Class: |
C07K 14/4705 20130101;
B65D 65/38 20130101 |
Class at
Publication: |
435/6 ; 435/7.23;
536/23.2; 530/388.26; 536/24.3 |
International
Class: |
C12Q 001/68; G01N
033/574; C07H 021/04; C07K 016/40 |
Foreign Application Data
Date |
Code |
Application Number |
May 2, 1995 |
AU |
PN 2742 |
Claims
1. A polynucleotide encoding GDU, the polynucleotide having a
sequence which encodes a polypeptide having an amino acid sequence
as shown in FIG. 2 or a sequence which hybridises thereto.
2. A polynucleotide as claimed in claim 1 in which the
polynucleotide has a sequence as shown in FIG. 2.
3. A GDU polypeptide, the polypeptide having an amino acid sequence
as shown in FIG. 2.
4. An antibody which binds to the polypeptide as claimed in claim
3.
5. An antibody as claimed in claim 4 in which the antibody is a
monoclonal antibody.
6. An oligonucleotide probe of at least 12 nucleotides, the
oligonucleotide probe having a sequence such that the probe
selectively hybridises to the polynucleotide as claimed in claim 1
under stringent conditions.
7. An oligonucleotide probe as claimed in claim 6 in which the
oligonucleotide probe is labelled.
8. An-oligonucleotide probe as claimed in claim 6 or claim 7 in
which the oligonucleotide is of at least 18 nucleotides.
9. A method of detecting the presence of GDU in a sample, the
method comprising reacting the sample with an antibody as claimed
in claim 4 or claim 5 or an oligonucleotide probe as claimed in any
one of claims 6 to 8 and detecting the binding of the antibody or
the probe.
Description
[0001] The present invention relates to a previously unidentified
erbB receptor target designated GDU. The present invention relates
to a polynucleotide encoding GDU and to methods of detecting the
presence of GDU.
[0002] Many intracellular targets for receptor tyrosine kinases
(RTKs) contain one or more src homology (SH)2 domains. These are
conserved, non-catalytic domains of approximately 100 amino acids
which bind to short peptide sequences containing phosphotyrosine
(Cohen et al, Cell 80, 237-248, 1995). Since receptor
autophosphorylation on specific tyrosine residues follows RTK
activation, SH2 domains mediate receptor-substrate, as well as
other protein-protein interactions, during signal transduction. SH2
domains contain not only a pocket lined with basic residues which
binds the phosphotyrosine but also an additional binding pocket or
groove which interacts with amino acids C-terminal to this residue,
this determining the specificity of the interaction. The particular
autophosphorylation sites present on a given RTK therefore define
the SH2 domain-containing signalling proteins that it can recruit
and hence, to a large extent, the signalling specificity of the
receptor. SH2 domains are often accompanied in signalling proteins
by two other conserved protein modules; SH3 domains, which bind to
proline-rich peptide ligands, and pleckstrin-homology (PH) domains.
The function of the latter remains ill-defined, and both protein
and phospholipid ligands have been described.
[0003] SH2 domain-containing proteins can be divided into two
classes (Schlessinger and Ullrich Neuron, 9,383-301 1992); Class I,
which also possess a catalytic function e.g. phospholipase
C-.gamma.1 (PLC-.gamma.1) and the GTPase activating protein for Ras
(Ras-GAP), and Class II, which contain only non-catalytic protein
modules and are thought to function as adaptors, linking separate
catalytic subunits to receptors or other signalling proteins e.g.
Grb2. The tissue expression of particular SH2 domain-containing
proteins varies from ubiquitous, e.g. Grb2, which performs a
fundamental role in linking tyrosine kinases to Ras signalling, to
relatively restricted e.g. Grb7, which is mainly expressed in the
liver and kidney (Margolis et al Proc. Natl. Acad. Sci. USA, 89,
8894-8898, 1992). Presumably the latter protein performs relatively
specialised signalling functions. The CORT (cloning of receptor
targets) technique, in which cDNA expression libraries are screened
with the tyrosine phosphorylated C-terminus of the EGF receptor
represents a powerful methodology for the identification and
characterisation of novel, SHZ domain-containing, receptor
substrates (Skolnik et al Cell 65, 83-90, 1991).
[0004] Members of the erbB family of RTKs and their ligands are
implicated both in normal mammary gland development and the growth
and progression of human breast cancer. Furthermore, marked
alterations in the expression or activity of several SH2
domain-containing proteins have been observed in human breast
cancers or breast cancer-derived cell lines, suggesting that this
represents an additional level at which RTK signalling may be
modulated in this disease (Daly, Breast Cancer Res Treat, 34,
85-92, 1995). We therefore chose normal human mammary epithelial
cells as a basis for a CORT screening program and hence
identification of novel, and relatively tissue specific, erbB
receptor targets.
[0005] Screening of a HMEC 184 .lambda.EXlox cDNA library isolated
1 Ras-GAP, 2 Grb2 cDNAs and a cDNA encoding a novel SH2
domain-containing protein. This protein, designated GDU or Grb14
(the designations "GDU" and "Grb14" are used interchangeably
herein), is related both in molecular architecture and sequence
homology to Grb7 and Grb10, previously identified erbB receptor
targets. These three proteins also share significant sequence
homology, over an approximately 300 amino acid region encompassing
the PH domain, with the C. elegans gene F10E9.6. The latter gene
has recently been shown to encode a protein (mig 10) critical for
longitudinal neuronal migration in C. elegans; members of the Grb7
gene family, including GDU, may therefore be involved in the
regulation of cell migration in higher organisms.
[0006] Analysis of GDU gene expression in normal breast epithelial
cells and a large series of human breast cancer cell lines revealed
that expression was limited predominantly to normal breast cells
and the more highly differentiated, estrogen receptor positive,
breast cancer cell lines. Also, GDU mRNA was overexpressed in the
DU-145 prostate carcinoma cell line relative to the normal prostate
and two other prostate cancer cell lines. GDU may therefore serve
as a prognostic indicator and/or a tumour marker in both breast and
prostate cancer. Furthermore, since altered expression of GDU may
contribute to the abnormal proliferation, invasion and/or migration
of cancer cells, GDU signal transduction may provide a novel
therapeutic target in human cancer. Finally, since GDU is involved
in downstream signalling initiated by the platelet derived growth
factor receptor (PDGFR), it may provide a target in diseases or
conditions in which PDGF plays a regulatory role e.g. wound
healing, fibrotic conditions, atherosclerosis.
[0007] In a first aspect the present invention consists in a
polynucleotide encoding GDU, the polynucleotide having a sequence
which encodes a polypeptide having an amino acid sequence as shown
in FIG. 2 or a sequence which hybridises thereto.
[0008] In a preferred embodiment of the present invention the
polynucleotide has a sequence as shown in FIG. 2.
[0009] In a second aspect the present invention consists in a
polypeptide, the polypeptide having an amino acid sequence as shown
in FIG. 2.
[0010] In a third aspect the present invention consists in an
antibody which binds to the polypeptide of the second aspect of the
present invention.
[0011] The antibody may be monoclonal or polyclonal, however, it is
presently preferred that the antibody is a monoclonal antibody.
[0012] In a fourth aspect, the present invention consists in an
oligonucleotide probe of at least 12 nucleotides, the
oligonucleotide probe having a sequence such that the probe
selectively hybridises to the polynucleotide of the first aspect of
the present invention under stringent conditions.
[0013] In a preferred embodiment of this aspect of the present
invention the oligonucleotide is labelled. In a further preferred
embodiment of the present invention the oligonucleotide is of at
least 18 nucleotides.
[0014] In a fifth aspect the present invention consists in method
of detecting the presence of GDU in a sample, the method comprising
reacting the sample with an antibody of the second aspect of the
present invention or a oligonucleotide probe of the fourth aspect
of the present invention and detecting the binding of the antibody
or the probe.
[0015] In order that the nature of the present invention may be
more clearly understood preferred forms thereof will now be
described with reference to the following examples and Figures in
which:--
[0016] FIG. 1 shows a schematic representation of Grb14 structure
with a restriction map for the Grb14 cDNA and the cDNA clones used
to derive the Grb14 sequence aligned underneath. The initial clone
isolated by CORT screening was designated clone 1. Two other clones
(1-1 and 1-2) were isolated from the 184 cell line library by
screening using clone 1 as a probe. The Grb14 CDNA sequence was
completed using two clones L5 and L6, isolated from a human liver
cDNA library. Abbreviations are as follows: A; Apa I; Av; Avr II,
X; Xho I; E; Eco RI. The numbers refer to distance in bp.
[0017] FIG. 2 shows the nucleotide and amino acid sequence of
Grb14. The PH domain is underlined and the SH2 domain indicated by
bold type. The translation termination codon is shown by an
asterisk in the amino acid sequence. Numbers refer to distances in
bp.
[0018] FIG. 3 shows the sequence homology between Grb14, Grb7,
Grb10 and F10E9.6. As alignment of the amino acid sequences of
Grb14, mouse Grb7, mouse Grb10 and C. elegans F10E9.6 was obtained
using the computer programs Clustal W and SeqVu. Identical residues
are boxed. A highly conserved proline-rich motif is indicated by
the dotted underline, the PH domain by the broken underline and the
SH2 domain by the bold underline. Only the central region of
F10E9.6 exhibiting homology with the Grb7 family is shown. Amino
acid residues for each protein are numbered (from the initiation
methionine) on the right.
[0019] Screening of a Normal Breast Epithelial Cell cDNA Library by
the CORT Technique
[0020] CORT screening of two cDNA libraries prepared from normal
breast epithelial cells led to the isolation of recombinants which
exhibited differential binding to the phosphorylated EGFR
C-terminus. Upon excision of the corresponding pEXlox plasmids and
sequencing of the DNA inserts, two recombinants which bound very
strongly were identified as Grb2 cDNA clones (Lowenstein et al
1992, Cell 70, 431-442, 1992), and a clone exhibiting moderate
binding corresponded to ras-GAP (Trahey et al. Science 242,
1696-1700, 1988). The final clone, designated GDU, bound only
weakly to the EG FR. A database search with the corresponding cDNA
sequence did not detect an exact match but revealed significant
sequence homology with the SH2 domain-containing protein Grb7
(Margolis et al PNAS 89, 8894-8898,1992). The cDNA (GDU Clone 1 in
FIG. 1) encoded a short stretch of amino acids followed by a
C-terminal SH2 domain; homology to Grb7 was apparent over this
entire open reading frame.
[0021] Characterisation of GDU
[0022] In order to obtain the full length cDNA sequence for GDU;
two cDNA library screens were performed. In the first, the cDNA
insert from Clone 1 was used to screen the breast cDNA library.
Screening of 5.times.10.sup.5 recombinants isolated 2 cDNAs,
designated 1-1 and 1-2, of 1.6 and 1.4 kb, respectively (FIG. 1).
In the second, a 213 bp EcoRI-Xho I restriction fragment derived
from 1-1 (FIG. 1) was used to screen a human liver cDNA library.
Screening of 1.times.10.sup.6 recombinants isolated 2 cDNAs,
designated L5 and L6, of 1.3 and 1.7 kb, respectively (FIG. 1).
Clones 1-1, 1-2, L5 and L6 were sequenced in their entirety on both
strands to obtain the cDNA sequence shown in FIG. 2. The 2.4 kb of
DNA sequence derived from these overlapping clones corresponds
closely to the size of the three most abundant mRNA species
detected upon Northern blot analysis.
[0023] Analysis of the cDNA sequence identified an open reading
frame of 540 amino acids. The initiation codon is preceded by an
in-frame termination codon and is surrounded by a consensus
sequence for strong translational initiation. The encoded protein
is similar both in molecular architecture and amino acid sequence
to Grb7 (Margolis et al, Proc. Natl. Acad. Sci. USA 89, 8894-8898,
1992) and the recently identified Grb10 (Ooi et al, Oncogene 10,
1621-1630, 1995), consisting of a N-terminal region containing at
least one proline-rich motif, a central region which exhibits
significant homology to the putative C. elegans protein F10E9.6
(Stein et al EMBO J, 13, 1331-1340, 1994) and which also
encompasses a PH domain, and a C-terminal SH2 domain. An alignment
of the amino acid sequences of GDU, Grb7, Grb10 and F10E9.6 is
shown in FIG. 3.
[0024] GDU is similar in size to Grb7, Grb10 possessing a more
extended N-terminus. The N-terminal region exhibits low sequence
homology between GDU, Grb7 and Grb10 apart from a highly conserved
amino acid motif PS/AIPNPFPEL. Also of note is the presence of two
clusters of basic residues which flank this motif. Overall the
N-terminal region of GDU displays a lower proline content than that
of Grb7 and Grb10 (GDU amino acids 1-110; 11% proline, Grb10 amino
acids 1-113; 15%, Grb7 amino acids 1-103; 23%).
[0025] GDU, Grb7 and Grb10 share a central, conserved region of
approximately 320 amino acids which exhibits significant homology
to a domain found in the C. elegans protein F10E9.6. Over this
region, GDU bears 48, 55 and 28% amino acid identify respectively
with Grb7, Grb10 and F10E9.6 (FIG. 3). The core of this region is
provided by a PH domain (FIGS. 1, 2 and 3), over which GDU exhibits
56, 61 and 35% amino acid identity, respectively, with Grb7, Grb10
and F10E9.6. However, as noted by Ooi et al, (Oncogene 10,
1621-1630, 1995) another region of particularly marked homology
spanning approximately 100 amino acids exists amino-terminal to the
PH domain (FIG. 3).
[0026] The most highly conserved region amongst Grb7 family members
is the SH2 domain (FIG. 3). The GDU SH2 domain displays 67 and 74%
amino acid identity, respectively, with the corresponding domain in
Grb7 and Grb10.
[0027] Northern Blot Analysis of GDU Gene Expression
[0028] The tissue specificity of GDU gene expression was
investigated by hybridizing Northern blots of poly A.sup.+ RNA
isolated from a variety of human tissues to a GDU specific cDNA
probe. GDU gene expression was highest in the testis, ovary, heart,
liver, skeletal muscle, kidney and pancreas. Moderate expression
was detected in the small intestine, colon, peripheral blood
leukocytes, brain and placenta, whilst expression in the spleen,
thymus, prostate and lung was low or undetectable. Several mRNA
transcripts were detected which displayed tissue-specific variation
in their relative abundance. The three most prominent transcripts
were approximately 2.3, 2.4 and 2.5 kb. Often co-expressed with one
or two of these transcripts was a transcript of approximately 9.5
kb. In the ovary a still larger transcript of undetermined size was
also expressed.
[0029] Since the Grb14 cDNA was originally isolated from a cDNA
library prepared from normal human breast epithelial cells, we were
interested in determining the expression profile of Grb14 mRNA in a
panel of human breast cancer cell lines. Upon Northern blot
analysis of total RNA isolated from 3 normal human breast
epithelial cell strains and 19 human breast cancer cell lines,
Grb14 gene expression could be detected in HMEC 184 and HMEC-219-4
cells, 6/7 ER+human breast cancer cell lines and 2/12 ER-cell lines
(Table 1). Thus Grb14 gene expression appears largely restricted to
normal breast epithelial and ER+ breast cancer cells. Differential
expression of Grb14 was also observed amongst human prostate cancer
cell lines. Although Grb14 mRNA expression was undetectable in the
normal prostate, low expression could be detected in the PC3 and
LnCaP prostate cancer cell lines and high expression in the DU145
line (Table 1).
1TABLE 1 Expression of Grb14 mRNA in different human cell lines.
Total cellular RNA was extracted from the indicated cell lines and
subjected to Northern blot analysis using a Grb14 cDNA probe. The
relative expression levels of Grb14 mRNA were then scored on a
scale from + (low) to ++++ (high). -; undetectable expression.
Origin Cell Line Expression Normal human HMEC 184 +++ breast
epithelial HMEC-219-4 + HMEC-1001-7 - Human breast T-47D +++
cancer, ER+ ZR-75-1 ++ MCF-7 + BT-483 + MDA-MB-134 + MDA-MB-361 +
BT-474 - Human breast MDA-MB-330 + cancer, ER- MDA-MB-468 + BT-20 -
SK-BR-3 - BT-549 - H3578T - DU-4475 - MDA-MB-157 - MDA-MB-175 -
MDA-MB-231 - MDA-MB-436 - MDA-MB-453 - Human prostate cancer PC3 +
LnCaP + DU-145 ++++ Human epidermoid carcinoma A431 - Human
embryonic kidney HEK 293 ++++
[0030] Expression of Grb14 Protein
[0031] In order to characterize the Grb14 protein a polyclonal
antiserum was raised against a GST-Grb14 SH2 domain fusion protein.
Following affinity purification, this antiserum was used to Western
blot cell lysates derived from cell lines in which Grb14 MRNA was
either expressed at high levels (DU145 and HEK 293) or was
undetectable (A431 and SK-BR-3) (Table 1). The antiserum recognized
a protein of approximately 58 kDa in DU145 cells, whilst in HEK 293
cells a tight doublet of this mobility was detected. These bands
were not observed upon Western blotting with pre-immune serum or in
the cell lines which do not express Grb14 mRNA. This estimated size
of Grb14 upon SDS-PAGE is in accordance with the predicted size of
the translation product of the Grb14 cDNA (60 kDa).
[0032] Since DU145 cells overexpress Grb14 mRNA relative to the two
other prostate carcinoma cell lines examined (Table 1), we
investigated whether this was accompanied by an upregulation of
Grb14 protein expression. Upon Western blot analysis, Grb14 was
clearly detectable in DU145, but not PC3 or LnCaP, cell lysates,
indicating that Grb14 protein is overexpressed in this cell
line.
[0033] Phosphorylation of Grb14
[0034] In order to characterize further the role of Grb14 in
receptor tyrosine kinase signalling, the phosphorylation state of
Grb14 was investigated before and after growth factor stimulation.
Since the anti-Grb14 antiserum 264 did not immunoprecipitate Grb14
under either native or denaturing conditions, we utilized an
expression construct (pRcCMV.sub.Flag) which tagged Grb14 with the
8 amino acid Flag epitope at the C-terminus. This construct was
stably transfected into HEK 293 cells, leading to the isolation of
stable clones of cells expressing an epitope-tagged Grb14 which
could be immunoprecipitated with the M2 anti-Flag monoclonal
antibody and Western blotted with either this antibody or
anti-Grb14 antiserum 264. Immunoprecipitation of Grb14 from serum
starved cells which were metabolically labelled with
.sup.32P-orthophosphate demonstrated that Grb14 was phosphorylated
in this basal state. Phosphoamino acid analysis of the isolated
protein demonstrated that phosphorylation was on serine
residues.
[0035] Treatment of the cells with EGF did not significantly
increase this level of phosphorylation, although activation of
native EGFRs could be demonstrated by anti-phosphotyrosine blotting
of the cell lysates. However, stimulation with PGDF BB resulted in
an approximately 1.5 fold increase within 5 min of administration,
and transient transfection of a cDNA encoding .beta.-PDGFRs into
the cells further amplified this response to approximately 2-fold.
The small increase in phosphorylation which occurred when this
construct was present in the absence of PGDF BB was presumably due
to the constitutive activation of RTKs often observed with this
system. Phosphoamino acid analysis demonstrated that the
PDGF-induced increases in Grb14 phosphorylation also occurred on
serine residues.
[0036] As will be recognised by persons skilled in this field the
present inventors have identified a novel signalling molecule which
they have designated GDU or Grb14. GDU has the potential to be used
as a prognostic indicator/tumour marker in both breast and prostate
cancer. In addition, as GDU may influence invasive/metastatic
behaviour it may also serve as a marker of invasive/metastatic
disease in these and other cancers. Finally, the involvement of GDU
in signalling by the PDGFR suggests that it may represent a
therapeutic target in diseases or conditions in which PDGF plays a
regulatory role.
[0037] Signalling via GDU could be targeted by competitive peptides
or dominant negative mutants, or restored by gene therapy. The
information provided herein will clearly assist in the rational
design of a GDU antagonist.
[0038] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
* * * * *