Research
Paper
|
|
Acta Biochim Biophys
Sin 2005,37:525-531
doi:10.1111/j.1745-7270.2005.00076.x
Regulation of EGF-induced ERK/MAPK Activation and EGFR
Internalization by G Protein-coupled Receptor Kinase 2
Jingxia GAO1,2*, Jiali LI1,
and Lan MA1*
1
Pharmacology
Research Center, Shanghai Medical College, Fudan University, Shanghai 200032,
China;
2
Department
of Biochemistry, Medical College, Tongji University, Shanghai 200092, China
Received: May 8,
2005
Accepted: May 20,
2005
This work was
supported by the grants from the National Natural Science Foundation of China
(30230130), the Ministry of Science and Technology (2003CB515405,
2005CB522406), the Ministry of Education (20020246042), and the Shanghai Municipal
Commissions for Science and Technology and Education (02DJ14020, 02GG01)
*Corresponding
authors:
Lan MA: Tel,
86-21-54237522; Fax, 86-21-54237621; E-mail, [email protected]
Jingxia GAO: Tel,
86-21-65984980; E-mail, [email protected]
Abstract G protein-coupled
receptor kinases (GRKs) mediate agonist-induced phosphorylation and
desensitization of various G protein-coupled receptors (GPCRs). We investigate
the role of GRK2 on epidermal growth factor (EGF) receptor signaling, including
EGF-induced extracellular signal-regulated kinase and mitogen-activated protein
kinase (ERK/MAPK) activation and EGFR internalization. Immunoprecipitation and
immunofluorescence experiments show that EGF stimulates GRK2 binding to EGFR
complex and GRK2 translocating from cytoplasm to the plasma membrane in human
embryonic kidney 293 cells. Western blotting assay shows that EGF-induced
ERK/MAPK phosphorylation increases 1.9-fold, 1.1-fold and 1.5-fold (P<0.05) at time point 30, 60 and 120 min, respectively when the cells were transfected with GRK2, suggesting the regulatory role of GRK2 on EGF-induced ERK/MAPK activation. Flow cytometry experiments show that GRK2 overexpression has no effect on EGF-induced EGFR internalization, however, it increases agonist-induced G protein-coupled d opioid receptor internalization by approximately 40% (P<0.01). Overall, these data
suggest that GRK2 has a regulatory role in EGF-induced ERK/MAPK activation, and
that the mechanisms underlying the modulatory role of GRK2 in EGFR and GPCR
signaling pathways are somewhat different at least in receptor internalization.
Key words G protein-coupled
receptor kinase; receptor tyrosine kinase; epidermal growth factor receptor;
ERK/MAPK; internalization
G protein-coupled receptors (GPCRs) constitute a superfamily of
plasma membrane receptors. Members of this family include receptors for many
hormones, neurotransmitters, chemokines and calcium ion, as well as sensory
receptors for various odors, and bitter and sweet tastes, so GPCRs play
important roles in a variety of cellular functions [1]. Repeated agonist
stimulation triggers a negative feedback regulatory mechanism that attenuates
GPCR-mediated signal transduction (desensitization). The initial event of GPCR
desensitization is the phosphorylation of GPCR catalyzed by G protein-coupled
receptor kinases (GRKs). GRKs are a family of Ser/Thr kinases and can
phosphorylate agonist-activated GPCRs and initiate their desensitization and
subsequent down-regulation. Thus GRKs are a key modulator of GPCR signaling
[2].Receptor tyrosine kinases (RTKs) constitute another family of plasma
membrane receptors. RTKs are primary mediators of physiological cell responses,
such as cell proliferation, differentiation, motility and survival [3].
Epidermal growth factor receptor (EGFR) belongs to the RTK family. Binding of
EGFR with its ligand induces dimerization of EGFR, resulting in
autophosphorylation of their cytoplasmic domains, thus recruiting the Src homology
2 and phosphotyrosine binding domain-containing proteins, which subsequently
activates multiple signaling cascades and ultimately induces altered gene
expression in the nucleus [4]. Disregulation of EGFR by overexpression,
mutation or continuous activation of its intrinsic tyrosine kinase is
frequently linked to hyperproliferative diseases such as cancer [5]. Thus EGFR
signaling must be under stringent control. Previous studies have demonstrated
that EGFR signaling is modulated by tyrosine dephosphorylation [6], receptor
internalization and degradation [7,8]. Previous studies have shown that overexpression of GRK2, a member of
GRK family, could attenuate phosphoinositide hydrolysis, cell chemotaxis and
proliferation evoked via platelet-derived growth factor receptor b (PDGFRb) [9,10], thus
expanding our understanding of the roles GRKs may play. A recent study has
shown that EGF stimulation induces GRK2-EGFR complex formation via Gbg– and
Src-dependent mechanisms [11]. But the modulatory role of GRKs on RTK signaling
pathways is limited to signals mediated by PDGFRb to date, moreover, the
mechanisms underlying the modulatory role of GRK2 remains unknown. In the present study, we investigated the regulatory role of GRK2 on
EGF-induced extracellular signal-regulated kinase and mitogen-activated protein
kinase (ERK/MAPK) activation and EGFR internalization.
Materials and Methods
Materials
Human EGF and [D-Pen2,D-Pen5]enkephalin
(DPDPE) were obtained from Sigma Chemical Co. (St. Louis, USA). Modified
Eagle’s medium (MEM) and fetal bovine serum (FBS) were purchased from Life
Technologies Incorporated (Grand Island, USA). Protein A-Sepharose was
obtained from Amersham Pharmacia Biotech (Piscataway, USA). Rabbit
anti-phospho and total ERK1/2 were supplied by New England Biolabs (Beverley,
USA). Mouse monoclonal antibody against GRK2 was kindly provided by Dr. Martin
OPPERMANN (Georg-August University, G?ttingen, Germany). Mouse monoclonal
antibody against DYKDDDDK octapeptide (FLAG) epitope and mouse monoclonal
antibody 12CA5 recognizing influenza hemagglutinin (HA) epitope were supplied
by Roche Molecular Biochemicals (Indianapolis, USA). Fluorescein
isothiocyanate (FITC)-conjugated goat anti-mouse IgG was purchased from Jackson
Immunoresearch (West Grove, USA).
Cell culture and plasmid transfection
Human embryonic kidney 293 (HEK293) cells were obtained from
American Type Culture Collection (Manassas, USA). HEK293 cells cultured in MEM
containing 10% FBS were seeded in 35 mm or 60 mm tissue culture dishes at 0.2–1?106 cells/dish 20
h before transfection. Plasmids encoding bovine GRK2, GRK2-GFP (green
fluorescence protein), human FLAG-tagged EGFR and mouse HA-tagged d opioid receptor
(DOR, one kind of GPCR) were prepared as described previously. Plasmids (1–3 mg each) were transfected into the HEK293 cells using the calcium
phosphate/DNA co-precipitation method as described previously. Experiments were
performed 44–48 h after transfection and
the cells were maintained overnight in FBS-free medium before the experiments.Human embryonic kidney 293 (HEK293) cells were obtained from
American Type Culture Collection (Manassas, USA). HEK293 cells cultured in MEM
containing 10% FBS were seeded in 35 mm or 60 mm tissue culture dishes at 0.2–1?106 cells/dish 20
h before transfection. Plasmids encoding bovine GRK2, GRK2-GFP (green
fluorescence protein), human FLAG-tagged EGFR and mouse HA-tagged d opioid receptor
(DOR, one kind of GPCR) were prepared as described previously. Plasmids (1–3 mg each) were transfected into the HEK293 cells using the calcium
phosphate/DNA co-precipitation method as described previously. Experiments were
performed 44–48 h after transfection and
the cells were maintained overnight in FBS-free medium before the experiments.
Co-immunoprecipitation and Western blotting
HEK293 cells were incubated at 37 ?C in the presence or absence of
100 ng/ml EGF for 5 min, then the cells were washed twice with ice-cold
phosphate buffered saline and lysed in 800 ml ice-cold NP-40
solubilization buffer (250 mM NaCl, 50 mM HEPES, 0.5% NP-40, 10% glycerol, 2 mM
EDTA, pH 8.0, 1 mM Na3VO4, 10 mg/ml aprotinin,
10 mg/ml
benzamidine and 0.2 mM PMSF) as described previously [12] for 1.5 h. The lysate
was centrifuged, and the supernatant was incubated with 1 mg of anti-FLAG
antibody and 15 ml of 50% slurry of protein A-Sepharose beads at 4 ?C for 16 h. The
beads were subsequently washed three times with NP-40 solubilization buffer.
The proteins bound to the beads were eluted using the SDS-PAGE sample buffer
and separated by SDS-PAGE. The presence of EGFR and GRK2 in the immunocomplexes
was detected in the subsequent Western blotting with antibody specifically
against FLAG epitopes and GRK2 respectively. The immunoblots were visualized
using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences) following
the manufacturer’s suggested protocol. An aliquot (2.5%) of the cell lysate was
analyzed by Western blotting to quantify the expression level of the protein
studied.
Laser confocal fluorescence microscopy
HEK293 cells were transfected with plasmid encoding GRK2-GFP. For
real-time fluorescence analysis of GRK2-GFP in living cells, the fluorescence
was observed under a microscope equipped with a temperature controller at 37
?C. The EGF was applied directly over the selected cells. The image scanned
before EGF application represented GRK2-GFP distribution in cells. After EGF
treatment, the same cells were scanned again in a time series. Scanning images
were recorded with a TCS NT laser confocal microscope (Leica Microsystems,
Bensheim, Germany).
Quantitation of receptor internalization by fluorescence flow
cytometry assay
Receptor internalization was quantitated using fluorescence flow
cytometry assay. Briefly, stably transfected HEK293 cells were chilled on ice
after agonist stimulation and the surface receptors were labeled with
corresponding antibody for 1 h at 4 ?C. After sufficient washing, the cells
were incubated with FITC-conjugated goat anti-mouse antibody for 1 h at 4 ?C.
The cells were then collected and fixed and the surface receptor staining
intensity was analyzed using FACScalibur flow cytometry (Becton Dickenson,
Mountain View, USA). Basal cell fluorescence intensity was determined with
cells stained with the secondary antibody alone.
Statistical analysis
Data were analyzed using Student’s t-test for comparison of
independent means with pooled estimates of common variances.
Results
EGF stimulates GRK2-EGFR complex formation in HEK293 cells
HEK293 cells expressing FLAG-tagged EGFR and GRK2 or GRK2 alone were
incubated in the presence or absence of 100 ng/ml EGF, then FLAG-EGFR was
immunoprecipitated with specific anti-FLAG antibody. As shown in Fig. 1(A),
there was little GRK2 in the EGFR immunoprecipitation complex before EGF
stimulation. After EGF stimulation there was a large amount of GRK2 detected in
the EGFR immunoprecipitaion complex using GRK-specific antibody. In the cells
expressing GRK2 alone there was no GRK2 detected in the EGFR immunoprecipitation
complex upon EGF stimulation [Fig. 1(A), upper panel]. This result
indicates that the detected GRK2 in the EGFR immunoprecipitation complex was
specific. Direct Western blot analysis of the total cell lysate detecting
FLAG-tagged EGFR and GRK2 expression [Fig. 1(A), lower panel] was shown
to ensure similar expression levels. These results are in accordance with our
previous study [11], and clearly demonstrate that EGF stimulates GRK2-EGFR
complex formation in HEK293 cells overexpressing GRK2 and EGFR.
To further demonstrate that EGF stimulates GRK2-EGFR complex
formation, we observed the subcellular redistribution of GRK upon EGF
stimulation in HEK293 cells transiently expressing GRK2-GFP using a laser
confocal microscope. As shown in Fig. 1(B), the green fluorescence
representing GRK2 mainly resided in the cytoplasm before EGF stimulation. The
real-time recording of GRK2-GFP fluorescence images in living cells showed that
after EGF stimulation the GRK2-GFP fluorescence increased quickly on the
membrane. At the same time GRK2-GFP fluorescence decreased in the cytoplasm,
and this redistribution was accompanied by changes in membrane shape [Fig.
1(B), 3 min and 5 min]. The redistribution of GRK2-GFP was restored to the
basal state at 10 min of EGF stimulation [Fig. 1(B), 10 min]. These results demonstrated that EGF stimulation could induce
translocation of GRK2 from cytoplasm to the plasma membrane and form a complex
with EGFR on the membrane in HEK293 cells overexpressing GRK2 and EGFR.
Overexpression of GRK2 enhances EGF-stimulated ERK/MAPK activation
EGFR activation leads to activation of the ERK/MAPK pathway. To demonstrate
whether GRK-EGFR complex formation upon EGF stimulation leads to modulation of
EGFR signaling, we observed the effect of GRK2 on EGF-stimulated ERK/MAPK
phosphorylation. HEK293 cells overexpressing GRK2 or b-Gal were incubated in the
presence or absence of 10 ng/ml EGF for a period ranging from 0 min to 120
min. Phospho-ERK/MAPK and total ERK/MAPK were probed employing phospho-specific
and total ERK/MAPK antibody. Western blotting analysis showed that
phospho-ERK/MAPK was detected at 2 min of EGF stimulation and reached its
maximum at 5 min, then it gradually decreased. Total ERK/MAPK did not show
detectable change before or after EGF stimulation. Phospho-ERK/MAPK in GRK2
transfected cells was significantly increased compared with b-Gal transfected
controls, although the time course of phospho-ERK/MAPK was similar [Fig.
2(A)]. The resulting phospho-ERK/MAPK levels from four independent sets of
experiments were quantified normalizing with total ERK as a loading control.
The mean values are presented graphically in Fig. 1(B).
Phospho-ERK/MAPK was enhanced 1.9-fold, 1.1-fold and 1.5-fold respectively (P<0.05) in GRK2 transfected cells compared with b-Gal transfected controls
at time point 30 min, 60 min and 120 min [Fig. 2(B)].
The effect of GRK2 overexpression on EGF-induced EGFR
internalization and DPDPE-induced DOR internalization
Agonist-induced EGFR internalization is a critical regulatory
mechanism in EGFR signaling. EGFR internalization induces receptor
down-regulation by decreasing the amount of EGFR present on the plasma
membrane. To determine whether the modulatory role of GRK2 on EGFR signaling is
through affecting EGFR internalization, the following experiments were carried
out. First we constructed a stable HEK293 cell line expressing FLAG-tagged
EGFR, then we used flow cytometry to quantitatively determine the
characteristics of EGF-induced EGFR internalization and the effect of GRK2
overexpression on the receptor internalization. Cell surface EGFR declined
gradually after EGF stimulation. At 30 min of EGF stimulation there was about
70% of EGFR left on the cell surface compared with the cells left untreated,
indicating that about 30% of cell surface EGFR was internalized into cytoplasm
(data not shown). This data was in accordance with a previous study [13],
suggesting EGFR was sequestered from plasma membrane in response to EGF
stimulation. Then, overexpression of GRK2 on EGFR and G protein-coupled DOR
internalization was examined. Fig. 3(A) shows representative results
from a set of experiments. Stimulation of cells with indicated agonist led to
the reduction of both the percentage of the positive cells and the mean
fluorescence density. Quantitatively, overexpression of GRK2 in HEK293 cells
stably expressing FLAG-EGFR did not have any significant effect on EGF-induced
EGFR internalization (P=0.96) compared with control cells overexpressing
b-Gal
[Fig. 3(B)]. But in an HEK293 cell line stably expressing DOR, DOR
internalization induced by its agonist DPDPE was enhanced by approximately 40%
(P<0.01) when the cells were transfected with GRK2 compared with control cells transfected with b-Gal [Fig. 3(B)]. To exclude the possible effect of high
density of plasma membrane EGFR on EGFR internalization and the possible
saturability of the EGFR internalization pathway [14], we measured the
internalization of endogenously expressed EGFR in HEK293 cells and HeLa cells.
GRK2 overexpression had no significant effect on EGF-induced EGFR
internalisation either, although the internalization of endogenously expressed
EGFR upon EGF stimulation was more rapid and to a greater degree (data not
shown).
Discussion
Previous studies have shown that GRK2 plays a role in the negative regulation
of signaling pathways mediated by PDGFRb, besides its classical
role in phosphorylating and desensitizing agonist-activated GPCRs. In the
current study we investigated the role of GRK2 in the EGFR signaling pathway,
including ERK/MAPK activation induced by EGFR activation and the role of GRK2
in EGF-induced EGFR internalization, as well as the possible mechanisms
underlying it. Our results have shown that overexpression of GRK2 enhances
ERK/MAPK activation induced by EGF stimulation. EGF stimulation induces GRK2
translocation from cytoplasm to the plasma membrane and GRK-EGFR complex
formation. But overexpression of GRK2 had no significant effect on EGF-induced
EGFR internalization; however, agonist-induced DOR internalization increased
significantly.Our present and previous studies have shown that EGF stimulates
GRK2-EGFR complex formation. There are several lines of evidence to support
this. First, co-immunoprecipitation experiment showed that there was a large
amount of GRK2 in EGFR immunoprecipitation complex upon EGF stimulation.
Second, confocal laser microscopy experiment showed that GRK2-GFP translocated
from cytoplasm to plasma membrane in a real-time confocal fluorescence record
for living cells. The mechanisms by which GRK activity is regulated can be
divided into three categories: subcellular localization, alterations in
intrinsic kinase activity and alterations in GRK expression level [15,16]. Our
results showed that GRK2 translocated to plasma membrane upon EGF stimulation,
indicating that upon EGFR activation GRK2 was also activated. Previous studies
have demonstrated that GRK2 exhibits a primarily cytosolic distribution in
unstimulated cells and appear to translocate to the plasma membrane upon GPCR
activation [15,16]. Our results are in accordance with this, suggesting that
GRK2 may exert its modulatory role in EGFR signaling via mechanisms similar to
GPCRs.EGFR activation leads to its dimerization and autophosphorylation of
the cytoplasmic domains of EGFR. Adaptor proteins such as SHC bind to the
phospho-tyrosine residues and subsequent formation of an SHC-Grb2-Sos complex
and induction of Raf function, thus, the Ras/MAPK pathway was activated. This
cascade couples agonist stimulation to gene transcription [4]. The current
study has shown that overexpression of GRK2 enhances EGF-induced ERK/MAPK
activation, suggesting that, in contrast to its negative regulation of GRK2 on
PDGFRb signaling, GRK2 may exert a positive regulation on EGF-induced
ERK/MAPK phosphorylation. Phospho-ERK/MAPK can enter the nucleus and
phosphorylate transcription factors such as Elk-1 [17], so enhanced ERK/MAPK
activation by GRK2 overexpression may also lead to changes in gene
transcription mediated by EGFR activation.GRK-catalyzed GPCR phosphorylation leads to GPCR desensitization and
subsequent internalization and degradation, thus GRK plays an important role in
GPCR internalization. The present study showed that overexpression of GRK2 had
no effect on EGF-induced EGFR internalization. However, overexpression of GRK2
significantly enhanced agonist-induced DOR internalization. Ligand-induced EGFR
internalization requires intrinsic receptor tyrosine kinase activity [18] and
specific sequences in the carboxyl-terminus of the receptor distal to the
kinase domain. Some adaptors, such as the m2 subunit of the AP2
protein recognize the endocytic signals thus involved in receptor endocytosis
and recycling [19]. The GRK2 binding domain on EGFR and the possible
phosphorylation sites on EGFR catalyzed by GRK2 remain elusive. We presume
that EGF-induced GRK2 binding to EGFR does not affect the specific sequences
involved in EGFR endocytosis due to the long distance between the binding
domain or phosphorylation sites and the endocytic sequences, thus, EGF-induced
EGFR internalization was unaffected by GRK2 overexpression. Taken together, these data demonstrate in HEK293 cells
overexpressing GRK2 and EGFR that GRK2 has the regulatory role in EGF-induced
ERK/MAPK activation. However, EGF-induced EGFR internalization is not affected,
suggesting that the mechanisms underlying the modulatory role of GRK2 in EGFR
and in GPCR signaling pathways is somewhat different, at least in receptor
internalization.
Acknowledgements
We thank Yanqin GAO, Hui GAO and Yalin HUANG for their technical
assistance. We also thank Xiaoqing ZHANG and Min ZHU for their helpful
discussion.
References
1 Ji TH, Grossmann M, Ji I. G
protein-coupled receptors. I. Diversity of receptor-ligand interactions. J Biol
Chem 1998, 273: 17299–17302
2 Penela P, Ribas C, Mayor F Jr.
Mechanisms of regulation of the expression and function of G protein-coupled
receptor kinases. Cell Signal 2003, 15: 973–981
3 Schlessinger J. Cell signaling by
receptor tyrosine kinases. Cell 2000, 103: 211–225
4 Zwick E, Hackel PO, Prenzel N,
Ullrich A. The EGF receptor as central transducer of heterologous signalling
systems. Trends Pharmacol Sci 1999, 20: 408–412
5 Holbro T, Civenni G, Hynes NE. The
ErbB receptors and their role in cancer progression. Exp Cell Res 2003, 284: 99–110
6 Ostman A, Bohmer FD. Regulation of
receptor tyrosine kinase signaling by protein tyrosine phosphatases. Trends
Cell Biol 2001, 11: 258–266
7 Vieira AV, Lamaze C, Schmid SL.
Control of EGF receptor signaling by clathrin-mediated endocytosis. Science
1996, 274: 2086–2089
8 Wiley HS.Trafficking of the ErbB
receptors and its influence on signaling. Exp Cell Res 2003, 284: 78–88
9 Peppel K, Jacobson A, Huang X,
Murray JP, Oppermann M, Freedman NJ. Overexpression of G protein-coupled
receptor kinase-2 in smooth muscle cells attenuates mitogenic signaling via G
protein-coupled and platelet-derived growth factor receptors. Circulation 2000,
102: 793–799
10 Peppel K, Zhang L, Huynh TT, Huang X,
Jacobson A, Brian L, Exum ST et al. Overexpression of G protein-coupled
receptor kinase-2 in smooth muscle cells reduces neointimal hyperplasia. J Mol
Cell Cardiol 2002, 34: 1399–1409
11 Gao J, Li J, Chen Y, Ma L. Activation of
tyrosine kinase of EGFR induces Gbg-dependent GRK-EGFR complex formation.
FEBS Lett 2005, 579: 122–126
12 Maudsley S, Pierce KL, Zamah AM, Miller
WE, Ahn S, Daaka Y, Lefkowitz RJ et al. The b2-adrenergic
receptor mediates extracellular signal-regulated kinase activation via assembly
of a multi-receptor complex with the epidermal growth factor receptor. J Biol
Chem 2000, 275: 9572–9580
13 Kim J, Ahn S, Guo R, Daaka Y. Regulation
of epidermal growth factor receptor internalization by G protein-coupled
receptors. Biochemistry 2003, 42: 2887–2894
14 Wiley HS. Anomalous binding of epidermal
growth factor to A431 cells is due to the effect of high receptor densities and
a saturable endocytic system. J Cell Biol 1988, 107: 801–810
15 Pitcher JA, Freedman NJ, Lefkowitz RJ. G
protein-coupled receptor kinases. Annu Rev Biochem 1998, 67: 653–692
16 Penn RB, Pronin AN, Benovic JL.
Regulation of G protein-coupled receptor kinases. Trends Cardiovasc Med 2000,
10: 81–89
17 Papavassiliou AG. The role of regulated
phosphorylation in the biological activity of transcription factors SRF and
Elk-1/SAP-1. Anticancer Res 1994, 14: 1923–1926
18 Wiley HS, Herbst JJ, Walsh BJ,
Lauffenburger DA, Rosenfeld MG, Gill GN. The role of tyrosine kinase activity
in endocytosis, compartmentation, and down-regulation of the epidermal growth
factor receptor. J Biol Chem 1991, 266: 11083–11094
19 Wiley HS. Trafficking of the ErbB receptors
and its influence on signaling. Exp Cell Res 2003, 284: 78–88