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Vol. 19, Issue 7, 2973-2983, July 2008
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Pharmacology Research Center and State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai 200032, China
Submitted October 22, 2007;
Revised March 26, 2008;
Accepted April 28, 2008
Monitoring Editor: Richard Assoian
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Gao et al., 2005). GRKs thus act as a critical modulator of GPCR signal transduction.
GRK2 is a ubiquitous member of the GRK family and its activity and subcellular location appear to be tightly controlled by stimulation of GPCR as well as its subsequent interaction with activated receptors, Gβ
subunits, lipids, anchoring proteins, caveolin, and calmodulin (Penela et al., 2003). The interaction of GRK2 with Gβ subunits through its C-terminal pleckstrin homology (PH) domain helps targeting the kinase to the membrane and enhances its activity toward the receptor (Pitcher et al., 1992). The interaction of GRK2 with phosphatidylinositol 4,5-bisphosphate via the N-terminal portion of its PH domain seems to be necessary for its full activation (Pitcher et al., 1995). Activity of GRK2 may also be regulated by its own phosphorylation. ERK could phosphorylate GRK2 at Ser670 and thus negatively regulate GRK2 activity (Pitcher et al., 1999). Phosphorylation of GRK2 by protein kinase C (PKC) and PKA modulates its membrane-targeting and kinase activity (Chuang et al., 1995; Cong et al., 2001). Another important mechanism of GRK2 activation involves phosphorylation of GRK2 by c-Src. Src-mediated GRK2 phosphorylation at tyrosine in its N-terminal domain has been shown to promote the kinase activity of GRK2 toward both soluble and membrane-bound substrates in vitro (Sarnago et al., 1999). The recruitment of Src to β2-adrenergic receptor (β2AR) and Src-mediated tyrosyl phosphorylation of GRK2 are obligate for agonist-induced desensitization of β2AR (Fan et al., 2001).
Distinct from GPCRs in structure and function, receptor tyrosine kinases (RTKs) constitute another family of transmembrane receptors. As an important member of RTK family, epidermal growth factor receptor (EGFR) plays a critical role in regulation of many cellular processes such as proliferation, differentiation, motility, and survival (Schlessinger, 2000). It has been demonstrated that GRK2 could form a complex with c-Src and PDE to regulate EGF-stimulated activation of p42/p44 MAPKs (Wan et al., 2003). GRK2 is able to phosphorylate EGFR and platelet-derived growth factor receptor (Freedman et al., 2002). These data implicate that GRK2 can also regulate RTK signaling.
Our recent study showed that EGF stimulation induced translocation of GRK2 to the plasma membrane and the formation of GRK2-EGFR complex (Gao et al., 2005). The current study is to explore whether GRK2 could be regulated by EGFR to exert influence on the GPCR signaling. Our results show that the activation of EGFR stimulates its interaction with GRK2 and the EGFR-mediated tyrosyl phosphorylation of GRK2, which activates GRK2 and regulates the internalization of 
-opioid receptor (DOR) and µ-opioid receptor (MOR). Our study thus reveals that GRK2 mediates a novel form of cross-talk between RTK and GPCR signaling pathways.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmid Construction
Plasmids encoding bovine GRK2, N-terminal 185-amino acid deletion mutant of GRK2 (
N-GRK2), and transducin 
were prepared as described previously (Gao et al., 2005). The HA-DOR and M4/5/6 constructs were prepared as described previously (Guo et al., 2000). HA-MOR construct was provided by Dr. H. Loh (University of Minnesota School of Medicine). The Rab5-GFP construct was provided by Dr. D. Pei (Tsinghua University). The GRK2-GFP and GRK2-Flag cDNA constructs were generous gifts from Dr. Marc G. Caron (Duke University Medical Center). The mouse c-Src cDNA was kindly provided by Dr. Joan S. Brugge (Harvard Medical School) and human EGFR cDNA clone kindly provided by Dr. Neil J. Freedman (Duke University Medical Center) were subcloned into pcDNA3 (Invitrogen). The bovine GRK2 mutant GRK2-Y13F/Y86F/Y92F(GRK2-3Y/F) 127 HA-tagged GRK2-kinase domain (amino acid 186-513, HA-GRK2-CD), the GRK2 lacking PH-domain (PH-GRK2), the dominant-negative c-Src mutant (K298R), the HA-MOR 363/370/375A, and the constitutively active c-Src mutant (Y527F) were constructed by PCR mutagenesis. Construction of RNAi plasmid for human GRK2 was performed as described (Sui et al., 2002). The small interfering RNA (siRNA) sequence targeting GRK2 are 5'-GGCCATGAGGAAGACTACGCC-3' corresponding to the positions 1641-1661 relative to the start codon, respectively. The control nonspecific siRNA (5'-GGCCGCAAAGACCTTGTCCTTA-3') was prepared as described previously (Gao et al., 2005).
Cell Culture and Plasmid Transfection
HEK293 cells and HEK293T cells were cultured in MEM and Dulbecco's MEM containing 10% FBS, respectively. Cells were seeded in 60- or 100-mm tissue culture dishes at 1–3 x 106/dish 20 h before transfection. Transfection of cells with 2–5 µg of each plasmid was performed with calcium phosphate/DNA coprecipitation method. Assays were performed 44–48 h after transfection, and the cells were maintained overnight in FBS-free medium before the assay. A431 cells were cultured in Dulbecco's MEM plus 10% FBS and were transfected using LipofectAMINE 2000 reagent (Invitrogen).
Immunofluorescence Imaging
A431 Cells grown on glass coverslips were transfected with HA-MOR or HA-MOR 363/370/375A plasmids. After 24 h, cells were incubated with mouse anti-HA antibody for 30 min and then treated with EGF (100 ng/ml) at 37°C for 30 min, After washed by phosphate-buffered saline (PBS), cells were fixed immediately and permeabilized with 0.3% (vol/vol) Triton X-100 in PBS. Cells were then incubated with rabbit anti-EGFR antibody overnight. Cells were washed by PBS, then stained with FITC-conjugated goat anti-mouse and Cy3-conjugated goat anti-rabbit antibody at room temperature for 1 h, and washed with PBS. The coverslips were then mounted onto microscope slides with 50% glycerol in PBS. Fluorescence images were taken on a Zeiss 40x oil/1.3 NA Fluar objective under a Zeiss 510 laser confocal microscope (Thornwood, NY). A431 cells transfected with GRK2-GFP alone or Rab5-GFP and GRK2-Flag were treated with EGF (100 ng/ml) at 37°C for the desired time and then were fixed and permeabilized. Cells were incubated with rabbit anti-EGFR antibody alone or with mouse anti-Flag antibody overnight and then stained with Cy3-conjugated goat anti-rabbit secondary antibody alone or with Cy5-conjugated goat anti-mouse antibody at room temperature for 1 h. The coverslips were then treated as described above. HEK293 cells grown on glass coverslips were transfected with HA-EGFR and GRK2-GFP plasmids. After 48 h, cells were incubated with rabbit anti-HA antibody for 1 h at 37°C and then treated with EGF (100 ng/ml) at 37°C for 5 min. Cells were fixed and permeabilized and then stained with Cy3-conjugated goat anti-rabbit secondary antibody at room temperature for 1 h. The coverslips were then treated as described above.
Immunoprecipitation and Western Blotting
Cells were washed with ice-cold PBS and lysed in 800 µl NP-40 solubilization buffer (50 mM HEPES, pH 8.0, 250 mM NaCl, 0.5% NP-40, 10% glycerol, 2 mM EDTA, 1 mM Na3VO4, plus 10 µg/ml aprotinin, 10 µg/ml benzamidine, and 0.2 mM PMSF) for 1.5 h as described (Gao et al., 2005). The lysate was centrifuged and the supernatant was incubated with 1 µg of anti-FLAG antibody and 15 µl of 50% slurry of protein A-Sepharose beads at 4°C for 4 h. The beads were subsequently washed, and the proteins bound to the beads were eluted in PAGE in the presence of SDS-PAGE sample buffer and separated by SDS-PAGE. The samples were detected in the subsequent Western procedures with the corresponding antibody. To assess DOR phosphorylation, cells were lysed in cell lysis buffer (50 mM Tris, pH 7.4, 0.5% NP-40, 10% glycerol, 150 mM NaCl, 2 mM EDTA, 1 mM NaF, plus 10 µg/ml aprotinin, 10 µg/ml benzamidine, and 0.2 mM PMSF), After centrifugation, receptors were immunoprecipitated with M2-conjugated Sepharose (Sigma) and detected with phospho-DOR (Ser 363) antibody.
For immunoblot analysis, protein bands were visualized by enhanced chemiluminescence. In some experiments, blots were incubated with IRDye 800CW-conjugated or 700CW-conjugated antibody (Rockland Biosciences, Gilbertsville, PA) and infrared fluorescence images were obtained with the Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE).
Fluorescence Flow Cytometry
Receptor internalization was quantitated using fluorescence flow cytometry assay as previously described (Zhang et al., 2005). Cells were incubated in 1 µM DPDPE or 10 µM DAMGO at 37°C under the conditions indicated in figure legend. Then the cells were chilled on ice, and the surface receptors were labeled with mouse against HA antibody for 1 h at 4°C. After sufficient washing, the cells were incubated with FITC-conjugated goat anti-mouse IgG for another 1 h at 4°C. Then the cells were collected and fixed, and the surface receptor staining intensity was analyzed on a fluorescence flow cytometer (FACScan, Becton Dickinson). Basal cell fluorescence intensity was determined with cells stained with the secondary antibody alone.
Cell Surface Biotinylation
Surface biotinylation assay was performed as described (Zhang et al., 2005). Cells were incubated at 4°C with 600 µg/ml Sulfo-NHS-SS-biotin in PBS for 30 min. Unreacted biotin was removed by rinsing cells with Tris-buffered saline (50 mM Tris-HCl, pH 7.4, 150 mM NaCl). Cells were warmed to 37°C for 1 h followed by incubation in the presence or absence of 100 ng/ml EGF for 20 min. Then the cells were stimulated with 1 µM DPDPE for 30 min. The biotin remaining on cell surface was cleaved by incubation in stripping buffer (50 mM glutathione, 100 mM NaCl, 60 mM NaOH, and 1% fetal bovine serum) at 4°C for 15 min. The remaining glutathione was quenched at 4°C for 30 min by 50 mM iodoacetamide resolved in PBS. Cells were extracted in lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 25 mM KCl, 0.5% Triton X-100). After centrifugation, receptors were immunoprecipitated with M2-conjugated Sepharose and detected with HRP-conjugated streptavidin.
In Vitro Kinase Assays
Bovine GRK2-Flag was partially purified from HEK293T cells. Briefly, HEK293T cells overexpressing GRK2-Flag were lysed in lysis buffer (50 mM Tris, pH 7.4, 250 mM NaCl, 1% Triton, 10% glycerol, and 2 mM EDTA, plus 10 µg/ml aprotinin, 10 µg/ml benzamidine, and 0.2 mM PMSF), After centrifugation, proteins were immunoprecipitated with M2-conjugated Sepharose (Sigma). After extensive wash in lysis buffer, beads were rinsed by TBS (50 mM Tris, pH 7.4, 50 mM NaCl) twice. Bound protein was eluted by a competition with 3x Flag peptide (Sigma) as the product instructions suggested. 3x Flag peptide and salt were removed by centrifugation in Amicon Ultra-4 (30-kDa cutoff size; Millipore, Bedford, MA). Protein was concentrated to a final concentration of 3 µg/ml. Protein is >90% pure as determined by 10% SDS-PAGE. Recombinant N-terminal glutathione S-transferase (GST)-tagged human EGFR kinase domain (GST-EGFR) was purchase from Millipore (Lake Placid, NY). In vitro kinase assay was performed as product instruction recommended. Briefly, reactions were performed (30°C, 30 min) in a total volume of 25 µl of kinase buffer [8 mM MOPS, pH 7.0, 0.2 mM EDTA, 10 mM MnCl2, 0.8 M (NH4)SO4] with or without 1 µM GRK2-Flag, 0.1 mM ATP, or the desired concentration of GST-EGFR. Reactions were terminated by the addition of 25 µl of 2x SDS loading buffer and boiled. Half of samples were loaded onto 8% SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody. The remaining samples were subjected to SDS-PAGE for subsequent immunoblotting for GST-EGFR and for GRK2.
Statistical Analysis
Data were analyzed using either Student's t test or two-way ANOVA for comparison of independent means with pooled estimates of common variances.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), suggesting that GRK-catalyzed DOR phosphorylation is essential for the enhancement of DOR internalization by EGF.
EGF Stimulates GRK2 Translocation to the Plasma Membrane and Association with EGFR through Its Catalytic Domain
Immunoprecipitation experiment showed that stimulation of 100 ng/ml EGF induced an increase in GRK associated with EGFR in HEK293 cells coexpressing GRK2 and Flag-EGFR. The maximum association of GRK2 and EGFR was observed around 5–10 min of EGF stimulation (Figure 2, A and B). Confocal microscopy data obtained in HEK 293 cells transfected with GRK2-GFP and HA-tagged EGFR revealed that in response to EGF treatment, GRK2 translocated to the membrane and became colocalized with EGFR (Figure 2C). These data indicate that activation of EGFR stimulates GRK and induces the formation of GRK-EGFR complex on the membrane. We also examined GRK2 distribution in A431 cells, which express endogenous EGFR. After a 5-min exposure to EGF, a portion of the GRK2 appeared in plasma membrane and colocalized with the distribution of the endogenous EGFR (Figure 2D). These data indicate that GRK2 can be recruited to the membrane and form a complex with EGFR in an EGFR agonist–dependent manner.
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q (Pitcher et al., 1998), and the PH domain and its -helix extension (residues 561-670) can bind acidic lipids and Gβ (Pitcher et al., 1992, 1995; Carman et al., 2000), indicating that these domains are essential for the membrane targeting of GRK2 and phosphorylation of GPCR. Thus we first examined the interaction of N-GRK2 (the N-terminal deletion mutant) and PH-GRK2 (the PH domain and its -helix extension deletion mutant; Figure 3A). As shown in Figure 3B, both N-GRK2 and PH-GRK2 could be detected in the Flag-EGFR immunoprecipitates. N-GRK2 and PH-GRK2 appeared to be associated with EGFR more efficiently than with the wild-type GRK2. This result suggests that neither the N-terminal domain nor the PH domain of GRK2 is essential for the association of GRK2 with EGFR. To explore if the catalytic domain of GRK2 is responsible for the binding of EGFR with GRK2, an HA-tagged peptide encoding GRK2 catalytic domain (residues 185–514, HA-GRK2-CD) was constructed (Figure 3A). A large amount of HA-GRK2-CD was detected in the Flag-EGFR immunoprecipitates but not in the control one (Figure 3C), confirming that GRK2 associates with EGFR through its catalytic domain, and indicating the catalytic domain is sufficient for GRK interaction with EGFR. We examined the EGF-stimulated interaction of EGFR with GRK2 truncation mutants, and the results showed that EGF stimulation increased the association of EGFR with these GRK2 truncation mutants (Figure 3, D and E), suggesting that the increased association of EGFR with GRK2 in response to EGF treatment may be largely attributed to the elevated affinity of GRK2 catalytic domain to the activated EGFR.
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). To determine whether EGFR could phosphorylate GRK2, HEK293 cells coexpressing EGFR and GRK2-Flag were incubated with EGF, and phosphorylation of GRK at tyrosine was assessed with tyrosyl-phospho–specific antibody after immunoprecipitation. As shown in Figure 4A, after EGF treatment, GRK2 was strongly tyrosyl-phosphorylated and GRK phosphorylation reached a plateau (
20-fold) during 15–30 min of stimulation. AG 1478, a specific inhibitor of EGFR tyrosine kinase, completely blocked EGF-induced tyrosyl phosphorylation of GRK2 (Figure 4B). Significant tyrosyl phosphorylation of GRK2 was not detected in HEK293 cells after EGF treatment, likely due to low abundance of endogenous GRK2 and endogenous EGFR in these cells. In SK-BR3 cells, which express abundant endogenous EGFR, significant tyrosyl phosphorylation of endogenous GRK2 was detected after EGF treatment (Figure 4C). These results suggest that EGFR-mediated tyrosyl phosphorylation of GRK2 requires tyrosine kinase activity of EGFR, and it could occur in cells under physiological conditions.
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N-GRK2 was not observed (data not shown), suggesting that EGFR-mediated GRK2 phosphorylation occurs in the N-terminal tyrosyl residues of GRK2. It has been shown that GRK2 can be phosphorylated by c-Src at N-terminal Tyr13, Tyr 86, and Tyr 92 residues (Penela et al., 2001). Further experiment showed that EGF-induced tyrosyl phosphorylation of GRK2–3Y/F-Flag, a mutant with Tyr13, Tyr 86, and Tyr 92 substituted to Phe residues, was reduced more than 80% compared with that of its wild-type counterpart (Figure 4, D and E). The residual phosphorylation signal of GRK2-3Y/F-FLAG suggests the presence of additional, minor tyrosyl phosphorylation sites in GRK2. Data obtained from mutant GRK2 carrying single substitution mutation at Tyr13, Tyr 86, or Tyr 92 (not shown) indicate that Tyr13, Tyr 86, and Tyr 92 residues located at the N-terminal domain of GRK2 are EGFR-mediated primary phosphorylation sites.
EGFR-mediated GRK2 Tyrosyl Phosphorylation Occurs on the Plasma Membrane
Some of EGFR signaling events such as full tyrosine phosphorylation of EGFR and the activation of MAPKs occur during the endocytosis process of the receptor (Vieira et al., 1996). EGFR is endocytosed through both the clathrin-dependent and lipid raft-dependent route (Sigismund et al., 2005). We used hypertonic sucrose, which inhibits clathrin-dependent endocytosis, and dominant negative dynamin mutant K44A, which inhibits both clathrin-dependent and lipid raft-dependent endocytosis, to explore whether EGFR-mediated GRK2 phosphorylation also occurs on the endocytic vesicles. As shown in Figure 5, A and B, treatments of 0.4 M sucrose has no effect on EGF-induced GRK2 phosphorylation, and dynamin K44A significantly elevated the phosphorylation of GRK2. Confocal microscopy data obtained in A431 cells coexpressing Rab5-GFP and GRK2 revealed that 30-min EGF stimulation resulted in redistribution of EGFR in the Rab5 positive early endosomes. However, no obvious accumulation of GRK2 at these positions could be observed (Figure 5C). These data suggest that endocytosis of EGFR is not required for EGFR-mediated GRK2 tyrosyl phosphorylation, and EGF induced tyrosyl phosphorylation of GRK2 may occur on the plasma membrane, not on the endocytic vesicles. These data are in agreement with that GRK2 colocalized with EGFR on the membrane, what we observed in Figure 2, C and D. The elevated phosphorylation of GRK2 in cells expressing dominant negative dynamin mutant K44A is likely a result from accumulation of the activated EGFR on the plasma membrane. It also supports our hypothesis that EGF-induced tyrosyl phosphorylation of GRK2 occurs on the plasma membrane.
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). Therefore, c-Src is likely the candidate kinase that catalyzes EGFR-mediated GRK2 phosphorylation. However, overexpressing K298R, a dominant negative mutant of c-Src (Fan et al., 2001), failed to inhibit EGF-induced GRK2 tyrosyl phosphorylation (Figure 6A). Furthermore, dose–effect experiments showed that EGF-stimulated GRK2 tyrosyl phosphorylation was not sensitive to pretreatment of PP2, a Src-seletive tyrosine kinase inhibitor, in contrast to what observed with GRK2 phosphorylated by constitutively active c-Src (Y527F; Figure 6, B–D). High concentration PP2 (5 µM) not only completely blocked tyrosyl phosphorylation of GRK2 by c-Src Y527F but also greatly inhibited EGF-stimulated tyrosyl phosphorylation of EGFR and GRK2 (Figure 6, B–D). This result is consistent with a previous report that PP2 at high concentrations inhibits EGFR tyrosine kinase activity (Sorkina et al., 2002). The loss of EGFR-mediated tyrosyl phosphorylation of GRK2 after 5 µM PP2 pretreatment could be attributed to the inhibition of EGFR kinase activity by PP2. The above data thus suggests that c-Src activity is not required for EGFR-mediated phosphorylation of GRK2.
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As shown in Figure 7F, PH-GRK2-Flag, which lacks the PH domain, was significantly phosphorylated after EGF treatment. This implies that neither Gβ nor PH domain of GRK2 is required for EGFR-mediated GRK2 tyrosyl phosphorylation. Consistent with the above result, transducin , Gβ scavenger, showed no significant effect on the level of EGFR-mediated tyrosyl phosphorylation of GRK2 (data not shown).
EGFR-mediated Tyrosyl Phosphorylation of GRK2 Enhances the Catalytic Activity of GRK2 in Intact Cells
To correlate EGFR-mediated tyrosyl phosphorylation of GRK2 with GRK2 activity directly, the change in DOR phosphorylation level after EGF treatment was assessed using an antibody specifically recognize phospho-Ser 363, a major GRK2 phosphorylation site of DOR (Guo et al., 2000). As shown in Figure 8A, DPDPE stimulation induced phosphorylation of DOR in cells expressing endogenous GRK2, and overexpression of GRK2 enhanced basal and DPDPE-induced phosphorylation of DOR. However, M4/5/6, the DOR mutant lacking GRK2 phosphorylation sites, failed to undergo DPDPE-induced phosphorylation, even in cells overexpressing GRK2 (Figure 8B). The effect of EGF stimulation on DOR phosphorylation was tested next in cells coexpressing GRK2 or GRK2–3Y/F with DOR and EGFR. Previous study showed that neither the subcellular localization pattern nor the kinase activity of GRK2-3Y/F toward GPCR or soluble substrates was significantly altered when compared with the wild-type GRK2 (Penela et al., 2001). Our results showed that in the absence of EGF, the phosphorylation levels of DOR in cells expressing the wild-type GRK2 and GRK2–3Y/F were equivalent. After EGF treatment, the phosphorylation of DOR was significantly elevated in cells expressing the wild-type GRK2 (Figure 8, C and F), and this effect could be inhibited by AG1478 (Figure 8E). However, EGF-induced phosphorylation of DOR was greatly decreased in cells expressing GRK2–3Y/F compared with those expressing the wild-type GRK2 (Figure 8, C and F). Notably EGF stimulation also caused an increase of phosphorylation of DOR in cells expressing GRK2–3Y/F, likely resulted from EGF-induced membrane translocation of GRK2. As expected, EGF treatment failed to induce phosphorylation of M4/5/6 (Figure 8D). Taken together, these data strongly suggests that EGF stimulation enhances activity of GRK2, and that EGFR-mediated tyrosyl phosphorylation of GRK2 contributes to the increased catalytic activity of GRK2 toward GPCR substrates.
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; Schulz et al., 2004). We next examined the effect of EGF on the internalization of MOR phosphorylation–deficient mutant, HA-MOR363/370/375A. EGF stimulation failed to promote internalization of HA-MOR363/370/375A (Figure 9A). We next used confocal immunofluorescence microscope to examine the localization of MOR after EGF treatment. In unstimulated A431 cells transfected with HA-MOR and HA-MOR 363/370/375A, HA-MOR was localized primarily on the cell membrane. EGF treatment led to redistribution of cell surface HA-MOR to the intracellular structures. However, redistribution of HA-MOR 363/370/375A was not observed after EGF stimulation (Figure 9B). These results suggest that EGF-induced internalization of MOR critically depends on the phosphorylation of MOR. As shown in panels C and D of Figure 9, GRK2 siRNA significantly reduced EGF-induced MOR internalization. Taken together, these results further demonstrate that EGF stimulation results in internalization of MOR and this is mediated by GRK2-induced MOR phosphorylation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-opioid receptor. Our preliminary data showed that EGF also regulated internalization of other GPCRs including MOR. These data suggest that the transregulation of GPCR by EGFR is not restricted to -opioid receptor. Our results propose that GRK as a mediator of cross-talk from RTK to GPCR signaling pathway.
Changes in subcellular distribution and functionality of GRKs greatly affect GPCR functions, as reported under several pathological conditions such as hypertension (Gros et al., 1997), congestive heart failure (Ungerer et al., 1993) and rheumatoid arthritis (Lombardi et al., 2001). In addition to the well-documented roles of GRKs in GPCR signaling, GRKs have recently been recognized as a potential regulator of signal transduction mediated by certain receptor tyrosine kinases. GRK2 is capable to form a signaling complex with c-Src and PDE to regulate EGF-stimulated p42/p44 MAPK activation. GRK2 can interact with PDGFR and EGFR in vivo (Freedman et al., 2002; Gao et al., 2005) and phosphorylate PDGFR and EGFR (Freedman et al., 2002). GRK2-mediated seryl phosphorylation of PDGFR inhibits tyrosyl phosphorylation of PDGFR, PDGFR-evoked phosphoinositide hydrolysis, and Akt activation (Hildreth et al., 2004). Our results propose a novel mechanism for regulation of the GPCR signaling by receptor tyrosyl kinases via growth factor–mediated membrane translocation and phosphorylation of GRK2. We also demonstrated that tyrosyl phosphorylation of GRK2 induced by the activation of receptor tyrosine kinase stimulates the serine/threonine kinase activity of GRK2 and thus results in altered responsiveness of GPCR signaling.
C-Src has been implicated in EGFR signal transduction. We show that the activated EGFR phosphorylates GRK2 at the same tyrosyl sites as c-Src does. However, our data also demonstrate that Src contributes little, if any, in EGFR-mediated GRK2 tyrosyl-phosphorylation (Figure 6). Our in vitro pulldown and kinase assays further confirm that GRK2 is a direct binding partner and substrate of EGFR (Figure 7). These results are in consistent with those of Wu et al. (2005), who reported recently that PDGFRβ can tyrosyl phosphorylate GRK2 directly. However, EGFR and PDGFR may employ distinct mechanisms to phosphorylate GRK2. Wu et al. postulate that PDGFRβ localizes in caveolae after its activation and phosphorylates GRK2, which is localized to the caveolae by interaction with caveolin. It is more likely that the activated and autophosphorylated EGFR forms a docking site for GRK2 by binding its catalytic domain and then phosphorylates the N-terminal tyrosine residues of GRK2. Support for this model comes from two lines of evidences: first, overexpression of the peptide encoding the catalytic domain of GRK2, which is responsible for the interaction with EGFR, greatly inhibited EGFR-mediated GRK2 tyrosyl phosphorylation. Second, the caveolin-binding domain containing peptide encoding N-terminal 1–185 amino acid residues of GRK2 (Carman et al., 1999) could be phosphorylated by a constitutive active form of c-Src, but not the activated EGFR. These data demonstrate that GRK2 serves as a substrate for both receptor tyrosine kinase and nonreceptor tyrosine kinase. It further supports our hypothesis that GRK2 acts as a mediator of cross-talk from RTK to the GPCR signaling pathway.
We also postulate that EGFR-mediated GRK2 phosphorylation may occur on the membrane, several lines of evidence support this model: First, GRK2 was colocalized with EGFR on the membrane but not in the endocytic vesicles (Figures 2, C and D, and 5C). Second, inhibition of endocytosis of EGFR by dynamin K44A enhanced EGF-induced phosphorylation of GRK2 (Figure 5, A and B).
Tyrosyl-phosphorylation of GRK2 has been shown to play an important role in regulation of GRK2 activity. Tyrosyl-phosphorylation of GRK2 by c-Src promotes the kinase activity of GRK2 toward both soluble and membrane-bound substrates in vitro (Sarnago et al., 1999). Fan et al. (2001) demonstrated that tyrosyl-phosphorylation and activation of GRK2 participate in agonist-induced desensitization of β2AR agonist triggers tyrosyl phosphorylation of β2AR and creates a canonical SH2-binding domain on the receptor that recruits and activates Src. Subsequently, Src phosphorylates and activates GRK2 (Fan et al., 2001). PDGFR-mediated GRK2 tyrosyl phosphorylation activates GRK2 and enhances GRK2-mediated seryl phosphorylation of the PDGFR, an event that reduces PDGFR signaling (Wu et al., 2005). In this study, we showed that EGF treatment regulated internalization of both - and µ-opioid receptors through EGFR-mediated GRK2 phosphorylation and activation. These data suggest that tyrosyl-phosphorylation of GRK2 may act as a common and important mechanism of GRK2 activation when cells mobilize GRK2 toward non-GPCR targeting extracellular stimulus to generate integrate cellular response that coordinates with GPCR signaling.
In the current study, we have used opioid receptors to explore the transregulation of GPCR by EGFR. Activation of opioid receptors results in a multitude of effects including analgesia, respiratory depression, and euphoria, feeding the release of hormones, inhibition of gastrointestinal transit, and effects on anxiety. Opioid receptors are widely expressed in many tissues such as brain, spinal cord, intestine, adrenal, kidney, lung, spleen, testis, ovary, and uterus, where EGFR is also expressed. It has long been known that EGFR is overexpressed in many tumors. Deletion mutations and point mutations resulting in constitutive activation of EGFR have been discovered in some malignancies, such as gliomas and non–small-cell lung cancers (Yun et al., 2007). All three subtypes of opioid receptors , µ, and 
are expressed in non–small-cell lung cancers and other tumor cells (Campa et al., 1996). Expression of opioid receptors correlated inversely with tumor growth in vivo, and administration of opioid agonists resulted in increased survival rates of tumor-bearing mice (Gomez-Flores et al., 2005). Currently, opioid-based anticancer drugs are in early clinical trials (Smith et al., 2004). Studies support that transregulation of opioid receptors by EGFR may play a role in tumorigenesis. However, we must note that our results were obtained from model cellular systems, and the potential physiological significance of these findings remains to be demonstrated further.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work. 
Address correspondence to: Lan Ma (lanma{at}shmu.edu.cn)
Abbreviations used: DPDPE, D-Pen2, D-Pen5 enkephalin; GRKs, G protein–coupled receptor kinases; DOR, -opioid receptor; M4/5/6, a DOR mutant lacks GRK2 phosphorylation sites; MOR, µ-opioid receptor; EGFR, epidermal growth factor receptor; GST-EGFR, GST-tagged EGFR kinase domain; HEK293, human embryonic kidney 293 cells.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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