U.S. patent application number 12/611306 was filed with the patent office on 2010-02-25 for egf receptor transactivation by g-protein-coupled receptors requires metalloproteinase cleavage of prohb-egf.
This patent application is currently assigned to Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.V.. Invention is credited to Henrik Daub, Norbert Prenzel, Axel Ullrich, Esther Zwick-Wallasch.
Application Number | 20100048684 12/611306 |
Document ID | / |
Family ID | 41403236 |
Filed Date | 2010-02-25 |
United States Patent
Application |
20100048684 |
Kind Code |
A1 |
Ullrich; Axel ; et
al. |
February 25, 2010 |
EGF RECEPTOR TRANSACTIVATION BY G-PROTEIN-COUPLED RECEPTORS
REQUIRES METALLOPROTEINASE CLEAVAGE OF proHB-EGF
Abstract
The present invention relates to agents and methods for
growth-factor receptor activation by modulating the G-protein
mediated signal transduction pathway.
Inventors: |
Ullrich; Axel; (Muenchen,
DE) ; Prenzel; Norbert; (Muenchen, DE) ;
Zwick-Wallasch; Esther; (Muenchen, DE) ; Daub;
Henrik; (Regensburg, DE) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W., SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
Max-Planck-Gesellschaft zur
Forderung der Wissenschaften e.V.
Muenchen
DE
|
Family ID: |
41403236 |
Appl. No.: |
12/611306 |
Filed: |
November 3, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09461090 |
Dec 14, 1999 |
7632645 |
|
|
12611306 |
|
|
|
|
Current U.S.
Class: |
514/445 ; 435/29;
435/375 |
Current CPC
Class: |
C07K 16/2863 20130101;
C07K 16/22 20130101; G01N 33/566 20130101; A61P 31/00 20180101;
C07K 2317/76 20130101; A61P 11/06 20180101; C12Q 1/485 20130101;
G01N 2333/96486 20130101; G01N 33/5008 20130101; A61P 35/00
20180101 |
Class at
Publication: |
514/445 ;
435/375; 435/29 |
International
Class: |
A61K 31/381 20060101
A61K031/381; C12N 5/00 20060101 C12N005/00; C12Q 1/02 20060101
C12Q001/02; A61P 35/00 20060101 A61P035/00; A61P 11/06 20060101
A61P011/06 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 16, 2009 |
EP |
99 116 056.5 |
Claims
1. A method for modulating growth-factor activation comprising
contacting a cell or an organism which contains a growth-factor
receptor capable of being activated with a modulator of G-protein
mediated signal transduction.
2. The method of use of claim 1, wherein the activation of the
growth factor receptor is mediated by its extracellular domain.
3. The method of claim 1, wherein the activation of the growth
factor receptor is mediated via an extracellular signal
pathway.
4. The method of claim 1, wherein the growth-factor receptor is
activated by tyrosine phosphorylation.
5. The method of claim 1, wherein said growth-factor receptor is
EGFR.
6. The method of claim 1 wherein the modulator acts on a G-protein,
a G-protein coupled receptor and/or a proteinase.
7. The method of claim 6, wherein the modulator acts on a
proteinase.
8. The method of claim 7, wherein said modulator acts on said
proteinase by directly stimulating or inhibiting the proteinase
activity.
9. The method of claim 7, wherein said proteinase cleaves a
growth-factor ligand precursor.
10. The method of claim 9, wherein said precursor is a membrane
associated molecule.
11. The method of claim 9, wherein said growth factor ligand
precursor is proHB-EGF and said growth-factor receptor is EGFR.
12. The method of claim 7, wherein said proteinase is a
membrane-associated proteinase.
13. The method of claim 7, wherein said proteinase is a
metalloproteinase.
14. The method of claim 13, wherein said metalloproteinase is a
zinc dependent proteinase.
15. The method of claim 7, wherein said proteinase activity is
inhibited by batimastat.
16. The method of claim 1, wherein said modulator acts on cell
which is different from the cell which contains the
growth-factor.
17. The method of claim 1 for the prevention or treatment of
disorders associated with or accompanied by a disturbed, e.g.
pathologically enhanced growth-factor receptor activation.
18. The method of claim 17 for the treatment of cancer or
asthma.
19. The method of claim 1, wherein said modulator is administered
as a pharmaceutically acceptable composition.
20. A method for identifying and providing modulators of G-protein
mediated signal transduction comprising contacting a cell which
contains a growth-factor receptor capable of being activated with a
test compound suspected to be a modulator of G-protein mediated
signal transduction and determining the degree of growth-factor
receptor activation.
Description
[0001] This application is a Continuation of U.S. Ser. No.
09/461,090 filed Dec. 14, 1999, which claims the benefit of
European Patent Application No. 99 116 056.5 filed on Aug. 15,
1999, the disclosure of which is incorporated herein in its
entirety by reference.
[0002] The present invention relates to agents and methods for
modulating growth-factor receptor activation by modulating
G-protein mediated signal transduction.
[0003] Crosstalk between different signalling systems allows the
integration of a great diversity of stimuli that a cell receives
under varying physiological situations. Transactivation of EGF
receptor-dependent signalling pathways upon stimulation of
G-protein-coupled receptors (GPCR) which are critical for the
mitogenic activity of ligands such as LPA, endothelin, thrombin,
bombesin and carbachol represents evidence for such an
interconnected communication network. The mechanism of this
cross-communication is not understood, but based on reported data
it was proposed to be transmitted by intracellular
elements.sup.1-4.
[0004] We report here that activation of growth-factor receptors
such as epidermal growth-factor receptor (EGFR) upon GPCR
stimulation requires the receptor's extracellular domain. As key
element of this mechanism we identify a membrane-spanning
growth-factor ligand precursor, such as proHB-EGF, and a proteinase
activity that is rapidly induced upon GPCR-ligand interaction. We
show that inhibition of growth-factor precursor processing blocks
GPCR-induced growth-factor receptor transactivation and downstream
signals. As evidence for the pathophysiological significance of
this mechanism we demonstrate inhibition of constitutive EGFR
activity upon treatment of human PC-3 prostate carcinoma cells with
the metalloproteinase inhibitor batimastat. Together, these results
establish a new mechanistic concept for crosstalk among different
signalling systems.
[0005] Further, the results demonstrate the importance of
proteinases as targets for the treatment or prevention of diseases
which are associated with pathological growth-factor receptor
overexpression.
[0006] In a first aspect the invention relates to the use of
modulators of G-protein mediated signal transduction for the
manufacture of an agent which modulates growth-factor receptor
activation. Preferably the activation of the growth-factor receptor
is mediated by its extracellular domain and via an extracellular
signal pathway. Thus the modulator may act on cells which are
heterologous to the growth-factor receptor carrying target cells.
The growth-factor receptor activation preferably occurs by tyrosine
phosphorylation, by which an intracellular signal cascade is
mediated. Examples of suitable growth-factor receptors are EGFR,
and other members of the EGFR family such as HER-2, HER-3 or HER-4,
but also other growth-factor receptors such as TNF receptor 1, TNF
receptor 2, CD 30 and IL-6 receptor.
[0007] The modulator of the G-protein mediated signal transduction
may act on one or several compounds of the signal transduction
pathway. Particularly, the modulator may act on a G-protein, a
G-protein coupled receptor, a proteinase and/or a growth-factor
precursor which are key elements of the signal transduction
pathway. Preferably the modulator acts on a proteinase.
[0008] The substrate which is subject to cleavage by the protease
is preferably a growth-factor receptor ligand precursor. This
precursor is preferably a membrane-associated molecule. In a
particularly preferred example the growth-factor ligand precursor
is proHB-EGF which is cleaved to HB-EGF and the growth-factor
receptor is EGFR. Other preferred examples of growth-factor ligands
which are cleaved from precursors are other members of the EGF
family such as TGF.alpha., amphiregulin, epiregulin, EGF,
betacellulin, members of the heregulin/NDF family including
isoforms thereof and TNF.alpha..
[0009] The proteinase which is modulated is usually a
membrane-associated proteinase, preferably a metalloproteinase such
a zinc-dependent proteinase. Examples of these proteinases are
members of the ADAM family. The modulation of proteinase activity
may comprise a stimulation or inhibition. Preferably the proteinase
activity is inhibited which in turn results in an inhibition of
growth-factor receptor activation.
[0010] The modulation of proteinase activity is preferably effected
by adding an acitvator or inhibitor of proteinase activity to the
system which in a particulary preferred embodiment directly
modulates the proteinase activity. A preferred example for such a
modulator for proteinase activity is the proteinase inhibitor
batimastat. Further examples are marimastat (British Biotech), TAPI
(Immunex) and TIMP-1, -2, -3 or -4, particularly TIMP-3.sup.31.
Still a further example is CRM197, a catalytically inactive form of
the diphtheria toxin, which specifically binds to proHB-EGF and
which is capable of blocking the processing of proHB-EGF by
metalloproteinases.
[0011] The modulation of G-protein modulated signal transduction
has great significance for diagnostic and clinical applications.
For example, the modulation of G-protein mediated signal
transduction is a target for the prevention or treatment of
disorders associated with or accompanied by a disturbed e.g.
pathologically enhanced growth-factor receptor acitvation. More
particularly, the present invention provides methods for preventing
or treating, among other diseases, hyperproliferative diseases such
as colon, pancreatic, prostate, gastric, breast, lung, thyroid,
pituitary, adrenal and ovarian tumors, as well as thyroid
hyperplasia, retinitis pigmentosa, precocious puberty, acromegaly
and asthma. More particulary, the growth of human prostate cancer
cells may be inhibited by treatment with proteinase inhibitors such
as batimastat.
[0012] Thus, the present invention provides a method for modulating
growth-factor activation comprising contacting a cell or an
organism which contains a growth-factor receptor capable of being
activated with a modulator of G-protein mediated signal
transduction. The contacting step may occur in vitro, e.g. in a
cell culture or in vivo, e.g. in a subject in the need of medical
treatment, preferably a human. The active agent is added in an
amount sufficient to modulate growth-factor receptor activation,
particularly in an amount sufficient to inhibit growth-factor
receptor activation at least partially. Preferably the active agent
is administered as a pharmaceutically acceptable composition, which
may contain suitable diluents, carriers and auxiliary agents. The
composition may also contain further pharmaceutically active agents
e.g. cytotoxic agents for the treatment of cancer.
[0013] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose. A
therapeutically effective dose refers to that amount of the
compound that results in amelioration of symptoms or a prolongation
of survival in a patient. Toxicity and therapeutic efficacy of such
compounds can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, e.g. for determining the
LD50 (the dose lethal to 50% of the population) and the ED50 (the
dose therapeutically effective in 50% of the population). For any
compound used in the method of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays.
For example, a dose can be formulated in animal models to achieve a
circulating concentration range that includes the IC50 as
determined in cell culture (i.e. the concentration of the test
compound which achieves a half-maximal inhibition of the
growth-factor receptor activity). Such information can be used to
more accurately determine useful doses in humans. The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio between LD50 and ED50. Compounds
which exhibit high therapeutic indices are preferred. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition (see
e.g. Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1, p. 1).
[0014] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are sufficient to
maintain the receptor modulating effects, or minimal effective
concentration (MEC). The MEC will vary for each compound but can be
estimated from in vitro data, e.g. the concentration necessary to
achieve a 50-90% inhibition of the receptor using the assays
described herein. Compounds should be administered using a regimen
which maintains plasma levels above the MEC for 10-90% of the time,
preferably between 30-90% and most preferably between 50-90%.
Dosages necessary to achieve the MEC will depend on individual
characteristics and route of administration. In cases of local
administration or selective uptake, the effective local
concentration of the drug may not be related to plasma
concentration.
[0015] The actual amount of composition administered will, of
course, be dependent on the subject being treated, on the subject's
weight, the severity of the affliction, the manner of
administration and the judgement of the prescribing physician. For
batimastat, and other compounds e.g. a daily dosage of 1 to 200
mg/kg, particularly 10 to 100 mg/kg per day is suitable.
[0016] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections.
[0017] Alternatively, one may administer the compound in a local
rather than a systematic manner, for example, via injection of the
compound directly into a solid tumor, often in a depot or sustained
release formulation.
[0018] Furthermore, one may administer the drug in a targeted drug
delivery system, for example in a liposome coated with a
tumor-specific antibody. The liposomes will be targeted to and
taken up selectively by the tumor.
[0019] Still a further aspect of the present invention is a method
for identifying and providing modulators of G-protein mediated
signal transduction comprising contacting a cell which contains a
growth-factor receptor capable of being activated with a test
compound suspected to be a modulator of G-protein mediated signal
transduction and determining the degree of growth-factor receptor
activation. This method is suitable as a high-throughput screening
procedure for identifying novel compounds or classes of compounds
which are capable of modulating G-protein signal transduction.
Further, the present invention encompasses any novel modulator
identified by the disclosed method.
[0020] In a preferred embodiment of the invention cell lines
expressing G-protein coupled receptors and/or metalloproteinases
may be used to screen for and identify compounds that inhibit the
activity of growth-factor receptors.
[0021] The ability of test compounds to inhibit the activity of
growth-factor receptors extracellulary activated by G-protein
coupled receptor mediated signalling pathways can be determined as
described in the examples.
[0022] Further, the present invention is described in detail by the
following figures and examples:
DESCRIPTION OF FIGURES
[0023] FIG. 1 GPCR-induced EP-R transactivation redefines
endogenous EGFR-mediated signalling to PDGFR-specific signals.
Proteins were immunoblotted with .alpha.PY antibody (4G10).
[0024] a) Rat-1/EP-R cells were 3 minutes treated with ET-1 (200
nM), thrombin (2 U/ml) and EGF (2 ng/ml) or
[0025] b) preincubated with tyrphostins as indicated prior to
thrombin stimulation and EP-R was selectively precipitated with mAb
108.1.
[0026] c) Different stable Rat-1 cell lines were untreated or
[0027] d) 1 h preincubated with EGFR-E Ab ICR-3R (20 .mu.g/ml),
stimulated for 3 minutes with GPCR agonists, EGF or PDGF-BB (25
ng/ml) as indicated and SHP-2 was precipitated.
[0028] e) Rat-1/EP-R were treated as in b) and SHC was
immunoprecipitated.
[0029] FIG. 2 Carbachol-induced intercellular transactivation of
the EGF receptor. Stable Rat-1 cell lines either expressing M1R or
HERc and control cells were mixed in 1:3 ratio. In
[0030] a) after stimulation with carbachol (1 mM), HERc was
precipitated and immunoblotted with .alpha.PY antibody.
[0031] b) Co-cultures of Rat-1/M1R and Rat-1/HERc cells were
planted in different densities, preincubated with EGFR-E blocking
Ab ICR-3R (20 .mu.g/ml) and HERc was precipitated following
carbachol-stimulation.
[0032] c) High density co-cultures of Rat-1/M1R and Rat-1/HERc
cells were incubated with heparitinase or chlorate and HERc was
precipitated following carbacol- or EGF-stimulation.
[0033] FIG. 3 GPCR-induced EGFR transactivation and adapter protein
tyrosine phosphorylation is dependent on HB-EGF function. a), c),
d) COS-7 and b) HEK 293 cells, transfected with the M1R or ET-R,
respectively, untreated or CRM197 preincubated, were stimulated for
3 minutes with the GPCR agonists LPA (10 .mu.M) or Carbachol (1
mM), EGF (2 ng/ml) or 1 .mu.M TPA (5 min) as indicated.
Subsequently EGFR (a,b), SHC (c) or Gab 1(d) was immunoprecipitated
and proteins were immunoblotted with .alpha.PY antibody (4G10).
[0034] FIG. 4 GPCR-induced proteolytic processing of proHB-EGF and
EGFR transactivation are critically dependent on metalloproteinase
function.
[0035] a) COS-7 cells were co-transfected with either M1R or BombR
(0.5 .mu.g each) and VSV-proHB-EGF (0.7 .mu.g) and stimulated with
carbachol (1 mM), bombesin (200 nM), TPA (1 .mu.M) or EGF (2
ng/ml). ProHB-EGF was analysed with .alpha.HB-EGF Ab (upper part),
cleaved VSV-HB-EGF was monitored by anti VSV immunoblotting (lower
part).
[0036] b) COS-7 cells transfected as in a) were preincubated with
batimastat (5 .mu.M, 30 min), stimulated as indicated and anti-VSV
immunoprecipitates were subjected to .alpha.HB-EGF
immunoblotting.
[0037] c) Flow cytometric analyses of proHB-EGF in COS-7 cells
treated for 10 minutes with LPA, TPA, EGF or batimastat
preincubation following LPA stimulation.
[0038] d,e) COS-7 cells, transfected with the M1R, untreated or
BB-94 preincubated, were stimulated as in FIG. 3a) and EGFR (d) or
SHC (e) were immunoprecipitated. Proteins were immunoblotted with
.alpha.PY antibody (4G10).
[0039] f) PC-3 cells were serum-starved for 36 hours, preincubated
with batimastat and stimulated for 3 minutes with bombesin, TPA or
EGF (7 ng/ml) as indicated. EGFR was immunoprecipitated and
immunoblotted with .alpha.PY antibody.
[0040] g) Unstarved PC-3 cells were treated for indicated times
with DMSO or batimastat and EGFR tyrosine phosphorylation was
monitored with .alpha.PY immunoblot.
EXAMPLES
1. Methods
[0041] Cloning and Plasmids
[0042] The following plasmids have been described: pcDNA1-BombR and
pcDNA3-M1R.sup.1. For stable expression of the M1R in Rat-1 cells
the receptor was subcloned into pLXSN. pro-HB-EGF and the
Endothelin receptor were amplified by PCR from a MCF-7 or Rat-1
cDNA library and subcloned into pcDNA3-VSV or pcDNA3,
respectively.
[0043] Cells and Transfections
[0044] Rat-1 cells and COS-7 cells were grown and infected or
transfected, respectively, as described.sup.1,2. Rat-1 HERc cells
have been described elsewhere.sup.1. HEK 293 cells were grown in
DMEM containing 10% fetal calf serum (FCS) and transfections were
carried out using the Ca-phosphate method. CRM197 (10 .mu.g/ml,
Sigma) or batimastat (BB-94), (5 .mu.M, British Biotech) were added
20 minutes before the respective growth-factor. Tyrphostin AG1478
(250 nM, Calbiochem) and AG1295 (1 .mu.M, Calbiochem) were added 15
minutes before stimulation.
[0045] Immunoprecipitation and Western Blotting
[0046] The antibodies against human EGFR (108.1), SHP-2, Shc and
Gab1 have been characterized.sup.1,12,19,2. Western blotting
against the EP-R chimera was performed using rabbit polyclonal
.alpha.-hPDGFR.beta. antibody (Upstate Biotechnology). Cells were
lysed and proteins were subsequently immunoprecipitated as
described.sup.1. To precipitate the VSV-tagged HB-EGF a monoclonal
VSV antibody (P5D4, Boehringer) in combination With Protein
G-Sepharose was used, HB-EGF was detected with antibody C-18
(Santa-Cruz). Due to the small size of pro-HB-EGF and the processed
form of HB-EGF we used the Tricine SDS-PAGE system established by
Schlagger as described.sup.30.
[0047] Flow Cytometry Analysis
[0048] COS-7 cells were seeded in 6 cm-dishes; 20 h later cells
were washed and cultured for a further 24 h in serum-free medium
until treatment with growth factors as indicated. After collection
cells were incubated with goat .alpha.HB-EGF antibody (R&D
Systems) for 30 minutes on ice. After washing with PBS, cells were
incubated with FITC-conjugated rabbit anti-goat antibody (Sigma)
for 20 minutes on ice. Cells were analysed with FACSCalibur (Becton
Dickinson).
2. Results
[0049] Epidermal growth-factor receptor (EGFR) transactivation was
identified as a critical element in mitogenic signalling.sup.1,5,6
induced by G-protein-coupled receptors (GPCR), regulation of
chloride channels.sup.7, as well as modulation of potassium channel
activity.sup.8. Since the process was found to be very
rapid.sup.1,7,9, and GPCR-induced release of EGFR ligands into the
cell culture medium could not be detected.sup.5,8, EGFR
transactivation has been generally assumed to be exclusively
mediated via intracellular signals.sup.3,4.
[0050] Surprisingly, however, even though PDGF receptors are not
transactivated upon treatment of Rat-1 cells with GPCR
ligands.sup.2, this was the case for a chimera EP-R consisting of
an EGFR extracellular and the platelet-derived growth-factor
receptor (PDGFR) transmembrane and cytoplasmic signalling
domain.sup.10 (FIG. 1a). This receptor chimera immunoprecipitates
with monoclonal antibody 108.1 which recognizes the extracellular
portion of human but not rat EGFR. Treatment of Rat-1/EP-R cells
with the PDGFR inhibitor AG1295.sup.11, but not with the EGFR
kinase antagonist AG1478.sup.1, blocked thrombin-induced tyrosine
phosphorylation of the chimeric receptor (FIG. 1b), which clearly
demonstrated a critical function of the EGFR extracellular domain
for GPCR-mediated transactivation. As shown in FIG. 1c, this EP-R
transactivation results in a PDGF-characteristic downstream signal,
since the SH2 domain-containing phosphatase 2 (SHP-2), a preferred
mediator of PDGFR signalling.sup.12, was tyrosine phosphorylated
upon endothelin (ET-1) and thrombin stimulation of Rat-1/EP-R
cells, while exposure to the same ligands did not induce SHP-2
tyrosine phosphoryation in Rat-1 cells overexpressing the PDGFR or
control cells. Pretreatment of Rat-1/EP-R cells with monoclonal
antibody ICR-3R.sup.13 that blocks ligand binding to the human EGFR
resulted in complete inhibition of ET-1 and EGF-induced SHP-2
tyrosine phosphorylation, whereas the PDGF-mediated response was
not affected (FIG. 1d), confirming that GPCR-induced
transactivation of the EP-R chimera depends on the extracellular
EGFR domain. In contrast to the results obtained for SHP-2 (FIG.
1c), tyrosine phosphorylation of the adaptor protein SHC following
thrombin stimulation was completely blocked by pretreatment of
Rat-1/EP-R cells with AG1478, but remained unaffected by
preincubation with the PDGFR antagonist AG1295 (FIG. 1e). This
confirms that thrombin transactivates endogenuos rat EGFR in
Rat-1/EP-R cells resulting in SHC tyrosine phosphorylation, whereas
activation of the EP-R chimera redefines thrombin stimulation to
generate a PDGFR-characteristic SHP-2 signal.
[0051] To address the question whether the extracellular signal
which activates the EP-R chimera acts via an autocrine or paracrine
mode, we performed a co-culture experiment with Rat-1 cells either
stably overexpressing the M1 muscarinic acetylcholine receptor
(M1R) or the human EGFR (HERc) at a ratio of one to one.
Stimulation of the Rat-1/M1R+Rat-1/HERc co-culture with the M1R
agonist carbachol prior to immunoprecipitation with human
EGFR-specific antibody 108.1, rapidly induced tyrosine
phosphorylation of HERc (FIG. 2a). Since neither of the control
cells responded to carbachol, this result clearly demonstrated the
possibility of transactivation between two cells. To investigate
the influence of cell density on this paracrine process, HERc was
immunoprecipitated from subconfluent versus confluent co-cultures
of Rat-1/M1R and Rat-1/HERc cells following stimulation with
carbachol. As shown in FIGS. 2b and 2c, EGFR tyrosine
phosphorylation in response to M1R agonist only occurred in
confluent co-cultures and was completely inhibited by preincubation
with ICR-3R antibody, heparitinase or chlorate. This further
demonstrated the requirement of the EGFR ligand binding function
for intercellular signal transmission and the necessity of close
cell-cell contact. Together, these results lead us to conclude that
EGF-like ligands, synthesized as transmembrane precursors and
converted to the mature form by proteolytic cleavage.sup.14, may be
involved in GPCR-mediated transactivation. The discrepancy between
previous results obtained from medium-transfer experiments.sup.5,8
in which EGF-like ligands could not be detected upon GPCR
activation and our finding of density-dependent intercellular
crosstalk might be due to a scenario in which upon proteolytic
processing EGF-like ligands remain with the heparin sulfate
proteoglycan matrix prior to interaction with their high-affinity
receptors as shown for fibroblast growth-factors.sup.15.
[0052] Ectodomain shedding has been shown to be induced by stimuli
such as activators of heterotrimeric G-proteins, AlF.sub.4.sup.-
and GTP.gamma.S.sup.16, as well as the PKC activator
tetradecanoyl-phorbol-13-acetate (TPA) and the Ca.sup.2+-ionophore
ionomycin.sup.17,18. The latter, which induces HB-EGF release in
prostate epithelial cells.sup.18, has recently been shown to be a
potent activator of EGFR transactivation in PC12 cells.sup.19, and
TPA has been reported to induce EGFR tyrosine phosphorylation in
HEK 293 cells.sup.8. HB-EGF, a member of the EGF family, has the
ability to bind to cell surface heparan sulfate
proteoglycans.sup.20, which prevents the immediate release of the
growth-factor and increases the local growth factor concentration
in the cellular microenvironment. Based on these properties the
proHB-EGF precursor matched our proposed requirement for
GPCR-induced EGFR transactivation. Besides its function as a
growth-factor precursor, proHB-EGF serves as a high-affinity
receptor for diphteria toxin (DT).sup.21. CRM197, a non toxic
mutant of DT, was shown to inhibit strongly and specifically the
mitogenic activity of HB-EGF.sup.22. Therefore, we tested the
influence of CRM197 on GPCR-mediated EGFR transactivation. We found
that CRM197 pretreatment completely inhibits tyrosine
phosphorylation of the EGFR induced by the GPCR agonists
lysophosphatidic acid (LPA) or carbachol as well as TPA in COS-7
cells (FIG. 3a). Inhibition was also observed for ET-1 or
TPA-stimulated HEK 293 cells transiently transfected with the
endothelin receptor (FIG. 3b). In contrast, EGF-induced receptor
tyrosine phosphorylation was unaltered demonstrating CRM197
specificity. Furthermore, complete abrogation of LPA- and
carbachol-induced receptor tyrosine phosphorylation suggested that
HB-EGF is the only growth-factor mediating EGFR transactivation in
the cell lines presented here.
[0053] Tyrosine phosphorylation of the adaptor protein SHC is
considered to be a critical step in the coupling of GPCR activation
to Ras-dependent signalling pathways.sup.23. In order to
investigate the role of HB-EGF in this process, we examined the
effect of the diphteria toxin mutant CRM197 on GPCR ligand and
TPA-mediated SHC tyrosine phosphorylation. As shown in FIG. 3c, in
COS-7 cells, LPA-, carbachol-and TPA-induced SHC tyrosine
phosphorylation was dramatically reduced by CRM197 pretreatment,
while the EGF-mediated response was not affected. The same
inhibitory effect of CRM197 was observed in HEK 293 cells (data not
shown). Similarly, in COS-7 cells, tyrosine phosphorylation of the
multidocking protein Gab1 in response to LPA or thrombin was not
detected in the presence of CRM197 (FIG. 3d) confirming its
signalling position downstream of the EGFR.sup.2.
[0054] Next, in order to examine whether proHB-EGF is
proteilytically processed upon stimulation of GPCRs, we transfected
plasmids containing VSV-tagged proHB-EGF in COS-7 cells together
with the M1R or the bombesin receptor (BombR) and stimulated with
respective ligands for different times. TPA, a potent inducer of
proHB-EGF processing, or EGF were added as positive and negative
controls, respectively. FIG. 4a shows that as previously described
proHB-EGF is expressed in form of heterogenous translation products
of 20 to 30 KDa.sup.17, which can be detected with antibodies
against the C-terminus of the precursor (upper panel) or the
VSV-tag (lower panel). Stimulation with carbachol or bombesin led
to a rapid breakdown of the membrane-anchored growth-factor
precursor and proteolytic cleavage was concommitant with the
appearance of the 9 KDa VSV-tagged HB-EGF fragment containing the
transmembrane anchor. Interestingly, under these conditions the
GPCR signal induced proteolytic proHB-EGF proccessing as fast and
potently as TPA. As for TPA.sup.17, GPCR-induced conversion of
proHB-EGF is an extremely rapid process that generates mature
HB-EGF. In contrast to GPCR-induced tyrosine phosphorylation of
endogenous EGFR which is fast and transient.sup.1,7,9,
overexpression of the protease substrate VSV-proHB-EGF led to a
rapid but more sustained ectodomain cleavage of proHB-EGF.
[0055] Since zinc-dependent metalloproteinases have been implicated
in pro-HB-EGF shedding by TPA.sup.24, we analysed carbachol-induced
processing in the presence of batimastat (BB-94).sup.25, a protease
inhibitor which has recently been shown to block proteolytic
maturation of human amphiregulin.sup.26. As shown in FIG. 4b, BB-94
treatment significantly reduced HB-EGF processing in response to
carbachol supporting our conclusion that metalloproteinases are
critical elements in GPCR-induced HB-EGF generation and EGFR
activation. In contrast thereto, PGL-hydroxamate, an MMP-specific
inhibitor has no effect on LPA- or carbachol-induced
transactivation (not shown).
[0056] To confirm GPCR-induced proHB-EGF processing, we used an
ectodomain-specific antibody and flow cytometry upon treatment of
non-transfected COS-7 cells with LPA, TPA or EGF. Within 10 minutes
after addition of LPA and TPA, the content of cell surface
proHB-EGF was reduced while EGF stimulation showed no effect (FIG.
4c). In contrast to the experiments with transfected cells shown in
FIGS. 4a and b, activation of endogenous LPA receptors was not as
potent as TPA to induce proteolytic cleavage of proHB-EGF.
Nonetheless, consistent with FIG. 4b, the modest LPA-induced effect
was completely inhibited by batimastat.
[0057] Our results demonstrate that metalloproteinase-dependent
cleavage of proHB-EGF is rapidly induced upon activation of GPCRs
and consequently suggest a critical and general role of this
process in EGFR transactivation. We therefore investigated the
effect of the metalloproteinase inhibitor batimastat in GPCR--as
well as TPA-induced EGFR transactivation. In COS-7 cells, BB-94
pretreatment completely abrogated LPA- and carbachol-induced
tyrosine phosphorylation of the EGFR, as well as TPA-mediated
receptor activation (FIG. 4d). Since TPA--but not GPCR-mediated
EGFR tyrosine phosphorylation is sensitive to PKC inhibition in
COS-7 cells (data not shown), it appears that at least two distinct
metalloproteinase-dependent transactivation pathways exist.
Analogous results were obtained for ET-1-induced transactivation in
HEK 293 cells and bradykinin-stimulated EGFR tyrosine
phosphorylation in PC12 cells (data not shown). Finally, the
general implication of proteolytic processing in EGFR
transactivation and downstream signal transmission is demonstrated
by the complete abrogation of GPCR- and TPA-induced SHC tyrosine
phosphorylation by batimastat (FIG. 4e).
[0058] Because of the well established role of EGFR family members
in the pathogenesis of a variety of cancers and the physiological
abundance of GPCR ligands such as LPA, we addressed the
pathophysiological significance of transactivation with the human
prostate cancer cell line PC-3 which has been reported to utilize
EGFR-dependent pathways for growth promotion and is also responsive
to the GPCR ligand bombesin.sup.27,28. FIG. 4f shows that in PC-3
cells that were starved for 36 hours, bombesin, TPA and EGF induce
tyrosine phosphorylation of the EGFR which is completely blocked by
batimastat-treatment. Moreover, even high constitutive
phosphotyrosine content of the EGFR in unstarved PC-3 cells is
reduced by long-term treatment with BB-94 (FIG. 4g). All in all,
our results allow the conclusion that metalloproteinase-mediated
precursor cleavage represents a direct link between BombR
activation, constitutive tyrosine phosphorylation of the EGFR and
proliferation of human prostate cancer cells. Recently, ADAM9, a
member of the metalloproteinase-disintagrin family has been
reported to process proHB-EGF upon TPA treatment of Vero-H
cells.sup.24. We were unable, however, to block EGFR
transactivation with dominant-negative ADAM9 mutants in COS-7 and
HEK 293 cells (data not shown) leaving the identity of the
precursor processing protease unresolved.
[0059] Our findings identify the ubiquitously expressed HB-EGF
precursor and a metalloproteinase activity as critical pathway
elements between GPCR signals and activation of the EGFR and extend
our understanding of the mechanisms that underly the multiple
biological processes known to be regulated by heterotrimeric
G-proteins. Based on our current state of understanding,
GPCR-induced EGFR signal transactivation represents a new paradigm
because it entails three different transmembrane signal
transmission events: First, a ligand activates heterotrimeric
G-proteins by interaction with a GPCR which results in an
intracellular signal that induces the extracellular activity of a
transmembrane metalloproteinase. This then results in extracellular
processing of a transmembrane growth-factor precursor and release
of the mature factor which, directly or via the proteoglycan
matrix, interacts with the ectodomain of the EGFR leading to
intracellular autophosphorylation and signal generation. Our
previous findings indicate that this pathway may be utilized by a
variety of GPCRs in diverse cell types and that the preferred
transactivation target is the EGFR and its relatives.sup.1-4. The
demonstration of the pathophysiological relevance of this novel
mechanism in prostrate cancer cells leads us to propose that EGFR
transactivation via G-protein-mediated proteolytic growth precursor
processing represents a general mechanism with broad significance.
Moreover, since a great variety of bioactive polypeptides as
diverse as TNF-.alpha., FAS-ligand or L-selectin are processing
products of transmembrane precursors.sup.29 that have been
connected to pathophysiological disorders, our findings shed new
light on the importance of membrane-associated proteinases as
targets for disease intervention strategies.
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