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Endocrinology, doi:10.1210/en.2003-1658
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Endocrinology Vol. 145, No. 7 3297-3306
Copyright © 2004 by The Endocrine Society

Growth Hormone Alters Epidermal Growth Factor Receptor Binding Affinity via Activation of Extracellular Signal-Regulated Kinases in 3T3-F442A Cells

Yao Huang, Yongchang Chang, Xiangdong Wang, Jing Jiang and Stuart J. Frank

Department of Medicine (Y.H., X.W., J.J., S.J.F.), Division of Endocrinology, Diabetes, and Metabolism, and Department of Cell Biology, and Department of Neurobiology (Y.C.), University of Alabama at Birmingham, Birmingham, Alabama 35294;and Endocrinology Section, (S.J.F.) Medical Service, Veterans Affairs Medical Center, Birmingham, Alabama 35233

Address all correspondence and requests for reprints to: Stuart J. Frank, University of Alabama at Birmingham, 1530 3rd Avenue South, BDB 861, Birmingham, Alabama 35294-0012. E-mail: sjfrank{at}uab.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Epidermal growth factor receptor (EGFR) is a transmembrane protein that binds EGF in its extracellular domain and initiates signaling via intrinsic tyrosine kinase activity in its cytoplasmic domain. EGFR is important in development, cellular proliferation, and cancer. GH is a critical growthpromoting and metabolic regulatory hormone that binds the GH receptor, thereby engaging various signaling pathways, including ERKs. Prior studies suggest cross-talk between the GH receptor and EGFR signaling systems. Using the GH- and EGF-responsive 3T3-F442A preadipocyte, we previously observed that GH, in addition to causing EGFR tyrosine phosphorylation, also induced EGFR phosphorylation that was detected by PTP101, an antibody reactive with ERK consensus phosphorylation sites. This latter phosphorylation was prevented by pretreatment with MAPK kinase (MEK)1 inhibitors, suggesting ERK pathway dependence. Furthermore, GH cotreatment with EGF markedly slowed EGF-induced EGFR degradation and down-regulation, thereby potentiating EGF-induced EGFR signaling. These effects were also MEK1 dependent and suggested ERK pathway-dependent influence of GH on EGF-induced EGFR postendocytic trafficking and signaling. We now explore the impact of GH on cell surface binding of EGF in 3T3-F442A cells. We found that GH pretreatment caused transient, but substantial, lessening of 125I-EGF binding. Competitive binding experiments revealed that the decreased binding was primarily due to decreased affinity, rather than a change in the number of EGF binding sites. The effect of GH on EGF binding was concentration dependent and temporally correlated with GH-induced ERK activation and EGFR PTP101-reactive phosphorylation. Blockade of the MEK1/ERK but not the protein kinase C pathway, prevented GH’s effects on EGF binding, and our results indicate that the mechanisms of GH- and phorbol-12-myristate-13-acetateinduced inhibition of EGF binding differ substantially. Overall, our findings suggest that GH can modulate both EGF binding kinetics and the EGFR’s postbinding signaling itinerary in a MEK1/ERK pathway-dependent fashion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EPIDERMAL GROWTH factor receptor (EGFR) is critical in various aspects of development, regulation of normal cell proliferation and behavior, and the genesis and progression of cancers (1, 2, 3). EGFR (also known as ErbB-1) is a member of the ErbB family (ErbB-1–4) of receptors, characterized structurally by the presence of a ligand-binding extracellular domain, a single transmembrane pass, and a cytoplasmic tail that includes a tyrosine kinase domain and several phosphorylation sites (4). Epidermal growth factor (EGF) binding (to either EGFR homodimers or EGFR-ErbB-2 heterodimers) activates the receptor’s tyrosine kinase activity and initiates several phosphorylation-dependent intracellular signaling cascades (4). Also consequent to EGF binding, the EGFR rapidly undergoes internalization and enters postendocytic trafficking pathways that may ultimately lead to the receptor’s down-regulation and degradation. Substantial evidence now suggests that EGFR signaling may in part emanate from not only the cell surface EGFR but also receptors in the process of postendocytic trafficking (5).

In addition to being the key molecule in initiation of EGF action, the EGFR has become appreciated as participating in or interacting with signaling induced by other stimuli. In some instances, the stimulus causes release of soluble EGFR ligands such that they can activate the EGFR on the same or nearby cells by binding the receptor and thereby activating its kinase activity (6, 7, 8, 9, 10). This is referred to as transactivation. Another kind of cross-talk is the use of the EGFR in signaling by another stimulus without activation of the EGFR kinase activity, a phenomenon exemplified by the interaction of GH and EGF signaling. GH treatment causes tyrosine phosphorylation of the EGFR, without the requirement for an intact EGFR kinase domain (11, 12). This results in association of Grb-2 with the EGFR and the use of the EGFR as a docking molecule to enhance GH-induced ERK activation (11).

We have been particularly interested in interactions between GH and EGF signaling. GH is an important growth-promoting and metabolic regulatory hormone derived from the anterior pituitary gland (13). It initiates intracellular signaling by interacting with the GH receptor, a transmembrane type 1 glycoprotein, causing the activation of the GH receptor -associated cytoplasmic tyrosine kinase, Janus kinase (JAK)2 (14, 15). GH-induced JAK2 activation causes engagement of three major signaling systems: the signal transducers and activators of transcription, in particular signal transducer and activator of transcription 5b; the phosphatidylinositol 3-kinase pathway; and the MAPKs, most notably ERK1 and ERK2 (16). Whereas GH-induced transcription of certain genes, such as IGF-1, c-fos, serine protease inhibitor 2.1, and hepatic cytochrome P450 enzymes, is linked to GH’s direct activation of some of these pathways (17, 18, 19, 20, 21), the wide array of GH effects is likely also influenced by the interrelationships of GH with other growth factor signaling systems.

In addition to tyrosine phosphorylation, we previously observed that treatment of murine 3T3-F442A preadipocytes with GH caused threonine phosphorylation of the EGFR that is recognized by PTP101, a state-specific antibody reactive with ERK consensus phosphorylation sites (22). This GH-induced PTP101-reactive phosphorylation of the EGFR was prevented by pretreatment with inhibitors of MAPK kinase (MEK)1, suggesting ERK pathway dependence. Interestingly, GH cotreatment with EGF markedly slowed EGF-induced EGFR degradation and down-regulation, thereby potentiating EGF-induced EGFR signaling. Each of these effects of GH were also blocked by MEK1 inhibition. In contrast, the rate of internalization of radioiodinated EGF was unaffected by GH. These findings suggested ERK-dependent effects of GH on EGF-induced EGFR postendocytic trafficking and signaling.

We now explore the impact of GH on the cell surface binding of EGF in 3T3-F442A cells. We find that GH pretreatment caused transient but substantial lessening of 125I-EGF binding. Competitive binding experiments revealed that the decreased binding could be attributed primarily to decreased affinity, rather than a change in the number of EGF binding sites. The effect of GH on EGF binding was concentration dependent and temporally correlated with GH-induced ERK activation. Blockade of MEK activation, but not protein kinase C (PKC) activation, prevented GH’s effects on EGF binding. Overall, our findings suggest that GH can modulate both EGF binding kinetics and the EGFR’s postbinding signaling itinerary in a MEK1/ERK pathway-dependent fashion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human GH was kindly provided by Eli Lilly (Indianapolis, IN). Recombinant human EGF was purchased from Upstate Biotechnology (Lake Placid, NY) and recombinant mouse leukemia inhibitory factor (LIF) was from Chemicon International (Temecula, CA). Murine 125I-EGF (specific activity 150–200 mCi/mg) was purchased from PerkinElmer Life Sciences (Norwalk, CT). Phorbol-12-myristate-13-acetate (PMA) was obtained from Sigma Chemical Co. (St. Louis, MO). The PKC inhibitor GF109203X was purchased from Calbiochem (La Jolla, CA) and the MEK1 inhibitor PD98059 was from Cell Signaling (Beverly, MA).

Antibodies
Polyclonal anti-EGFR antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-phospho-threonineproline antibody PTP101 was purchased from Cell Signaling. Antiactive MAPK affinity-purified rabbit antibody (anti-active ERK, recognizing the dually phosphorylated Thr-183 and Tyr-185 residues corresponding to the active forms of ERK1 and ERK2) was from Promega (Madison, WI), and anti-MAPK affinity-purified rabbit antibody (anti-ERK, recognizing both ERK1 and ERK2) was from Upstate Biotechnology.

Cell culture
3T3-F442A cells (23), kindly provided by Drs. H. Green (Harvard University, Boston, MA) and C. Carter-Su (University of Michigan, Ann Arbor, MI), were cultured in DMEM containing 4.5 g/liter glucose (Cellgro, Inc., Herndon, VA), supplemented with 10% calf serum, 50 µg/ml gentamicin sulfate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Biofluids, Rockville, MD).

Cell starvation, inhibitor pretreatment, cell stimulation, and protein extraction
Serum starvation of 3T3-F442A cells was accomplished by substitution of 0.5% (wt/vol) BSA (fraction V, Roche Molecular Biochemicals, Indianapolis, IN) for calf serum in the culture medium for 16–20 h before experiments. Pretreatments and stimulations were carried out at 37 C in binding buffer [consisting of 25 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 0.1% (wt/vol) BSA, and 1 mM dextrose]. Serum-starved cells were pretreated with PD98059 (50 µM), GF109203X (1 µM), or vehicle (as controls) for 1 h before treatment with GH (500 ng/ml), EGF (1 nM), PMA (1 µg/ml), LIF (20 ng/ml), or vehicle, as specified in each experiment. Stimulations were terminated by washing the cells once with ice-cold PBS supplemented with 0.4 mM sodium orthovanadate (PBS-vanadate) and then harvested by scraping in PBS-vanadate. Cells were collected by brief centrifugation and pelleted cells were solubilized for 15 min at 4 C in lysis buffer [1% (vol/vol) Triton X-100, 150 mM NaCl, 10% (vol/vol) glycerol, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM benzamidine, 5 µg/ml aprotinin, and 5 µg/ml leupeptin]. After centrifugation at 15,000 x g for 15 min at 4 C, the detergent extracts (supernatant) were subjected to immunoprecipitation or directly electrophoresed and immunoblotted, as indicated below.

Immunoprecipitation and immunoblotting
For immunoprecipitation, cell extracts (500 µg) were mixed with 5 µl polyclonal anti-EGFR antibody (1 µg) and incubated at 4 C for 2 h with continuous agitation. Protein A-Sepharose (20 µl) (Amersham Pharmacia Biotech, Piscataway, NJ) was added and incubated at 4 C for an additional 1 h. The beads were washed four times with lysis buffer. Laemmli sample buffer eluates were resolved by SDS-PAGE and immunoblotted as indicated below.

Proteins resolved by SDS-PAGE were transferred to Hybond ECL Nitrocellulose membranes (Amersham Pharmacia Biotech). The membranes were blocked with TBST buffer [20 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% (vol/vol) Tween 20] containing 2% BSA and incubated with primary antibodies (0.5–1 µg/ml) as specified in each experiment. After three washes with TBST, the membranes were incubated with appropriate secondary antibodies (1:10,000 dilution) and washed. The bound antibodies were detected with SuperSignal chemiluminescent substrate (Pierce Chemical Co., Rockford, IL). Membrane stripping was performed according to the manufacturer’s suggestions (Amersham Pharmacia Biotech).

Densitometric analysis
Immunoblots were scanned using a high-resolution scanner (Hewlett Packard, Portland, OR). Densitomeric quantification of images was performed using a Macintosh II-based image analysis program (Image J 1.30, developed by W. S. Rasband, Research Services Branch, National Institute of Mental Health, Bethesda, MD).

Ligand binding experiments
In general, 3T3-F442A cells (duplicates for each treatment) were grown in 6-well plates in culture medium until they formed monolayers. The cells were starved and pretreated with GH (500 ng/ml), LIF (20 ng/ml), PMA (1 µg/ml), or vehicle in binding medium [DMEM containing 4.5 g/liter glucose, supplemented with 20 mM HEPES and 0.5% (wt/vol) BSA] at 37 C for 10 min. The cells were then incubated with 0.1 nM of 125I-EGF (specific activity 150–200 mCi/mg) in binding medium in the presence or absence of the above stimuli at 4 C for 2 h. At the end of incubation, the medium was aspirated, and the monolayers were rapidly washed three times with ice-cold PBS containing 0.1% (wt/vol) BSA to remove unbound ligand. The cells were lysed in 1 ml of lysis solution [100 mM NaOH and 0.1% (wt/vol) sodium dodecyl sulfate] at room temperature for 1 h and used to determine the total 125I-EGF binding. Radioactivity was counted with a {gamma}-counter. Nonspecific binding was assessed in the presence of 50-fold molar excess of unlabeled EGF and was not more than 5% of the total counts. Specific 125I-EGF binding was obtained by subtracting the nonspecific binding from the total radioligand binding.

For competitive binding assays to determine the radioligand equilibrium dissociation constant (Kd) and the maximal receptor binding sites (Bmax), 3T3-F442A cells (duplicates for each treatment) were starved and pretreated with GH (500 ng/ml) or vehicle at 37 C for 10 min. The cells were incubated with a single 0.05 nM concentration of 125I-EGF (specific activity 150–200 mCi/mg) at 4 C for 2 h, in the presence of increasing concentrations of unlabeled EGF (0–50 nM) to obtain a displacement curve. Binding data were analyzed using GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA; www.graphpad.com). To determine the Kd and Bmax values, nonlinear regression was used to fit the data based on the one-site model. The significance (P value) of difference of pooled results was estimated using unpaired t tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH acutely reduces 125I-EGF cell surface binding in 3T3-F442A cells
The initial step in EGF signaling is its specific interaction with the cell surface EGFR. Using 3T3-F442A preadipocytes, we and others (11, 12, 22) have previously demonstrated interactions between GH and EGF signaling. Because they endogenously display receptors for both ligands, 3T3-F442A cells provide a useful system to study such interactions without the need for overexpression of any of the necessary components (11, 12, 14, 19, 22, 24, 25, 26). To better understand effects of GH on EGFR function, we examined whether GH treatment influenced the degree of 125I-EGF binding. Serum-starved cells were pretreated for 10 min at 37 C with GH (500 ng/ml) or vehicle to allow activation of GH signaling. Intact cell monolayers were then incubated with 125I-EGF either in the presence or absence of a 50-fold molar excess of unlabeled EGF at 4 C for 2 h to assess specific cell surface EGF binding. As shown in Fig. 1Go, pretreatment of the cells with GH resulted in nearly 50% reduction, on average, in specific EGF binding when compared with cells not treated with GH.



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  		FIG. 1. GH reduces 125I-EGF surface binding in 3T3-F442A cells. Serum-starved 3T3-F442A cells were pretreated with GH (500 ng/ml; gray bar) or vehicle (black bar) at 37 C for 10 min and were then incubated with 125I-EGF (0.1 nM) at 4 C for 2 h. The specific 125I-EGF binding without GH pretreatment (control) is considered as 100%. Data are mean ± SD (error bars) of eight independent experiments (P < 0.005).

 
To further characterize this effect of GH on EGF binding, we performed GH time-course experiments (Fig. 2AGo). To determine the time course, cells were pretreated with GH (500 ng/ml) for 0–60 min before assessment of 125I-EGF binding. Inhibition of EGF binding was detectable with as little as 5 min of GH pretreatment, maximal after 10 min, and still evident after 30 min. At 500 ng/ml, GH’s effect was no longer evident when it was present for 60 min before assessment of EGF binding. These data indicate that the GH-induced reduction of EGF cell surface binding is acute and transient.



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  		FIG. 2. Characterization of GH-induced reduction of 125I-EGF cell surface binding. A, Time course. Serum-starved 3T3-F442A cells were pretreated with GH (500 ng/ml) at 37 C for indicated durations before 125I-EGF binding assays. B, GH concentration dependence. Serum-starved 3T3-F442A cells were pretreated with the indicated concentrations of GH at 37 C for 10 min before 125I-EGF binding assays. The specific 125I-EGF binding without GH pretreatment is considered 100%. Data are mean ± range of duplicate determinations. The experiments shown are representative of two such experiments.

 
The GH concentration dependence for inhibition of EGF binding was also examined (Fig. 2BGo). In this experiment, 3T3-F442A cells were pretreated for 10 min with 0–500 ng/ml GH before the 125I-EGF binding assay. Reduction of EGF binding was detected with 20 ng/ml of GH and the reduction was greater than 30% with 100 ng/ml of GH. When added at 500 ng/ml, GH even further inhibited EGF binding. These GH concentrations are in the range observed with GH pulses in intact rodents (27) and constitute well-established conditions for cell stimulation used by several laboratories to study biologically relevant signaling phenomena in 3T3-F442A and other cell systems (14, 19, 28, 29). In other experiments (not shown), concentrations of GH greater than 500 ng/ml failed to yield greater EGF binding inhibition; interestingly, however, at 50 µg/ml (a supraphysiologic dose), GH pretreatment did not reduce EGF binding, suggesting a bell-shaped GH concentration dependence, as previously observed for other GH-dependent effects in cell-based experiments (30, 31, 32). The data in Figs. 1Go and 2Go suggest that GH pretreatment substantially reduces EGF binding in 3T3-F442A cells and that maximal inhibition occurs with 10 min treatment of 500 ng/ml GH.

GH-induced reduction in 125I-EGF binding is accounted for by reduction in binding affinity
The experiments in Figs. 1Go and 2Go revealed a marked decrease in EGF binding capacity in response to GH, but did not indicate whether this reflects a change in EGF Kd vs. the number of EGF Bmax. To better understand the effects of GH on EGF binding kinetics, we conducted a series of competitive binding assays. In these experiments, cells were either pretreated with GH (500 ng/ml for 10 min at 37 C, referred to as GH) or not (control), after which they were incubated with 125I-EGF (0.05 nM; specific activity 150–200 mCi/mg) in the presence of increasing concentrations (0.1–50 nM) of unlabeled EGF and equilibrium binding of the radiolabeled EGF was measured. The displacement curve obtained from a typical experiment are shown in Fig. 3AGo, in which 125I-EGF binding (counts per minute) is plotted against the concentration of unlabeled EGF (nanomoles). As anticipated, radiolabeled EGF binding was lessened in the presence of increasing concentrations of unlabeled EGF in the 0.1–50 nM range independent of whether the cells were pretreated with GH (dashed line) or not (solid line).



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  		FIG. 3. GH reduces EGF binding affinity in 3T3-F442A cells. A, Displacement curves. Serum-starved 3T3-F442A cells were pretreated with GH (500 ng/ml) or vehicle (control) at 37 C for 10 min and then incubated with 125I-EGF (0.05 nM) in the presence of increasing concentrations of unlabeled EGF (0–50 nM) at 4 C for 2 h, as described in Materials and Methods. 125I-EGF binding (counts per minute) is plotted against the concentration of unlabeled EGF (nanomoles) and the curves (dashed line for GH pretreatment and solid line for control) are fits of binding data by nonlinear regression based on a one-site model with GraphPad Prism version 4.0. Data are mean ± range of duplicate determinations. The experiment shown is representative of four such experiments. B, GH reduces the EGF binding affinity but not the number of binding sites. Competitive binding data shown in A, along with those obtained from three other experiments, were further analyzed using GraphPad Prism version 4.0 to derive the Kd (nanomoles) and Bmax (counts per minute per 103 cells) values as described in Materials and Methods. P < 0.01 for Kd comparison is indicated.

 
These binding data were further analyzed using GraphPad Prism software (as in Materials and Methods) to derive the Kd and Bmax for EGF binding. As previously observed for murine 3T3 fibroblasts (33), this analysis suggested the presence of a single class of EGF binding sites. In the absence of GH pretreatment (control), Kd and Bmax values were 0.80 nM and 41.8 cpm/103 cells (roughly 2 x 104 binding sites per cell), respectively. For cells pretreated with GH, the Kd was 1.95 nM and the Bmax was 48.4 cpm/103 cells. Similar analysis was performed for four such independent experiments, and the pooled data are displayed in Fig. 3BGo. These data revealed that the average Kd rose from 0.90 ± 0.20 nM to 2.17 ± 0.25 nM when cells were pretreated with GH (P < 0.01), whereas there was no significant change in the Bmax (40.8 ± 3.7 vs. 46.2 ± 2.8 cpm/103 cells). Thus, GH pretreatment led to an approximately 2.4-fold reduction in EGF binding affinity without changing the number of EGF binding sites. We note that a reduction in binding affinity of this degree is likely to be functionally relevant. For example, decreases in ligand binding affinity of similar magnitude have been associated in certain instances with hormone insensitivity syndromes, such as insulin and IGF-1 resistance (34, 35).

Reduction of EGF binding correlates with GH-induced ERK activation
GH treatment of 3T3-F442A cells induces at least two types of phosphorylation of the EGFR. Tyrosine phosphorylation of EGFR in response to GH is independent of EGFR tyrosine kinase activation and can be detected with phosphospecific antibodies mainly at EGFR tyrosine residues Y-845 and Y-1068 (11, 12, 22). We (22) have also detected GH-induced EGFR phosphorylation by use of a state-specific antibody, PTP101, that recognizes phosphorylated threonine and/or serine residues that reside within consensus sites for phosphorylation by proline-directed kinases such as ERKs (36, 37, 38). The ability of GH to induce this PTP101-reactive EGFR phosphorylation correlates to GH-induced modulation of EGF-induced EGFR trafficking and signaling (22). Thus, we sought to determine whether GH-induced ERK activation and/or ERK-dependent EGFR phosphorylation might be involved in mediating GH’s effects on EGF binding.

In Fig. 4Go, we examined the time courses of GH (500 ng/ml)-induced EGFR PTP101 reactivity and ERK activation. Serum-starved cells were treated with GH for varying duration over a 0- to 2-h period. Detergent extracts were prepared and a portion of each sample was immunoprecipitated with anti-EGFR. Eluates were resolved by SDS-PAGE and immunoblotted with the PTP101 monoclonal antibody (Fig. 4AGo). GH induced PTP101-reactive EGFR phosphorylation was detected within 5 min of GH exposure (upper panel, lane 2 vs. 1). This phosphorylation peaked at roughly 10–15 min and began to decline after 30 min of GH stimulation (lanes 3–7). Reprobing of this immunoblot with anti-EGFR revealed similar abundance of EGFR in all samples (Fig. 4AGo, lower panel). Portions of the same cell extracts were directly electrophoresed and immunoblotted with anti-active ERK antibody (Fig. 4BGo, upper panel). ERK activation was substantial within 5 min (lane 2) of GH exposure and peaked at 10 min (lane 3), declining at 15 min (lane 4) and precipitously thereafter (lanes 5–7). This is similar to time courses of ERK activation detected by others in the same cell line (24, 39, 40).



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  		FIG. 4. Kinetics of GH-induced PTP101-reactive phosphorylation of EGFR and ERK activation in 3T3-F442A cells. A, Time course of GH-induced PTP101-reactive phosphorylation of EGFR. Serum-starved 3T3-F442A cells were stimulated with GH (500 ng/ml) for indicated durations. Detergent extracts (500 µg) were immunoprecipitated with an anti-EGFR antibody. Eluted proteins were analyzed by immunoblotting with PTP101 (upper panel) and anti-EGFR (lower panel), respectively. B, Time course of GH-induced ERK activation. Detergent extracts (30 µg) as in A were resolved by SDS-PAGE and immunoblotted with antiactive ERK (upper panel) and anti-ERK (lower panel), respectively. The experiments shown in A and B are representative of three such experiments.

 
The relative time courses of GH-induced PTP101-reactive EGFR phosphorylation and GH-induced ERK activation were then compared by estimating the intensity of both signals by densitometry. In Fig. 5Go, each is plotted relative to the maximal signals achieved over the 2-h time course. In addition, the time course of GH’s effect on radiolabeled EGF binding over this period is expressed on the same graph as the degree of reduction relative to the maximal reduction at 10 min. The time courses of the onset of the three parameters (ERK activation, PTP101-reactive EGFR phosphorylation, and reduction of 125I-EGF binding) were remarkably similar, with ERK activation slightly preceding the other two. PTP101-reactive EGFR phosphorylation, however, decayed less rapidly than the other two phenomena, suggesting that EGFR phosphorylation may not alone be responsible for the binding effects.



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  		FIG. 5. Reduction of EGF binding correlates with GH-induced ERK. To compare the kinetics of GH-induced reduction of EGF binding, ERK activation, and PTP101-reactive phosphorylation of EGFR, data shown in Fig. 4Go, A and B, were subjected to densitometric analysis. The relative intensity at each time point is normalized to the maximal signals achieved over the 2-h time course and plotted. The time course of GH’s effect on the 125I-EGF binding over this period is expressed on the same graph as the degree of reduction relative to the maximal reduction occurring at 10 min. These three parameters are indicated as dotted line (GH-induced EGFR PTP101 reactivity), solid line (GH-induced ERK activation), and dashed line (GH-induced reduction of 125I-EGF binding), respectively.

 
To further explore the correlation between GH-induced ERK activation and effects on EGF binding, we examined the time course of stimulation with a lower GH concentration (50 ng/ml) (Fig. 6Go). Both the timing and degree of loss of EGF binding induced by this lower GH concentration (Fig. 6AGo) differed from that observed with 500 ng/ml GH (Fig. 2AGo). With 50 ng/ml GH, maximal reduction of EGF binding was seen between 10–30 min after GH addition (in contrast to 10 min with 500 ng/ml) and was less substantial (roughly 35%, rather than the approximately 50% observed with 500 ng/ml). Interestingly, the profile of ERK activation induced by 50 ng/ml GH was similarly slower and less robust (Fig. 6BGo, upper panel) than that seen with 500 ng/ml GH (Figs. 4BGo and 5Go). The prolonged, but dampened, pattern of ERK activation at 50 ng/ml vs. 500 ng/ml GH is similar to that previously observed in other GH-responsive cells (29).



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  		FIG. 6. Kinetics of ERK activation and reduction of 125I-EGF cell surface binding induced by GH (50 ng/ml). A, Time course of GH-induced reduction of 125I-EGF cell surface binding. Serum-starved 3T3-F442A cells were pretreated with GH (50 ng/ml) at 37 C for indicated durations before 125I-EGF binding assays. B, Time course of GH-induced ERK activation. Serum-starved 3T3-F442A cells were stimulated with GH (50 ng/ml) for indicated durations. Detergent extracts (15 µg) were resolved by SDS-PAGE and immunoblotted with antiactive ERK (upper panel) and anti-ERK (lower panel), respectively. The experiments shown in A and B are representative of two such experiments.

 
GH-induced reduction of EGF binding is MEK1/ERK dependent
The time courses displayed in Figs. 5Go and 6Go suggested that the effect of GH on EGF binding could be related to GH-induced ERK activation. To test this possibility, we employed PD98059, an inhibitor of MEK1, the upstream activating kinase of ERKs 1 and 2. We previously demonstrated that this drug prevented GH-induced ERK activation and GH-induced PTP101-reactive phosphorylation of the EGFR in 3T3-F442A cells (22). In the experiment shown in Fig. 7AGo, we tested the effect of MEK1 inhibition on GH-induced reduction of 125I-EGF binding. As expected, GH (500 ng/ml) treatment for 10 min substantially reduced EGF cell surface binding (GH bar vs. control bar). PD98059 alone (control + PD) did not affect EGF binding. However, in the presence of PD98059, the effect of GH on EGF binding was prevented (GH + PD vs. GH), suggesting ERK activation-dependence for this effect of GH. As a control, we employed the PKC inhibitor, GF109203X (41), which we previously found does not inhibit GH-induced ERK activation in these cells (22). Notably, GF109203X did not undo the inhibitory effect of GH on EGF binding (GH + GF vs. GH).



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  		FIG. 7. GH-induced reduction of EGF binding is MEK1/ERK dependent. A, GH-induced reduction of EGF binding is blocked by MEK1 inhibitor PD98059. Serum-starved 3T3-F442A cells were preincubated with 50 µM PD98059, 1 µM GF109203X, or vehicle at 37 C for 1 h before stimulation with GH (500 ng/ml) for 10 min. The cells were then subjected to 125I-EGF binding assays. Specific 125I-EGF binding in the absence of GH, PD98059, and GF109203X is considered 100%. Data are mean ± range of duplicate determinations. The experiment shown is representative of three such experiments. B, PMA-induced reduction of EGF binding is blocked by PKC inhibitor GF109203X. Serum-starved 3T3-F442A cells were preincubated with 1 µM GF109203X, 50 µM PD98059, or vehicle at 37 C for 1 h before stimulation with PMA (1 µg/ml) for 10 min. The cells were then subjected to 125I-EGF binding assays. Specific 125I-EGF binding in the absence of PMA is considered 100%. Data are mean ± range of duplicate determinations. The experiment shown is representative of three such experiments.

 
Surface EGF binding is known to be inhibited by treatment with pharmacologic activators of PKC such as phorbol esters, although the mechanisms of this effect are uncertain (42, 43, 44, 45, 46). As a comparison with GH’s effects, we tested the effect of acute treatment with the phorbol ester, PMA, on the binding of 125I-EGF to 3T3-F442A cells (Fig. 7BGo). As anticipated, treatment with PMA greatly reduced EGF binding (PMA vs. control). In contrast to its lack of effect on GH inhibition of EGF binding, inclusion of GF109203X markedly reversed PMA’s inhibition of EGF binding (PMA + GF vs. PMA). By virtue of blocking PMA-induced PKC activation, GF109203X also inhibits PMA-induced ERK activation in these cells (22); therefore, the use of this inhibitor does not allow distinction of whether PKC vs. ERK activation underlies PMA’s inhibition of EGF binding. To address this, we also tested PD98059, which is known to inhibit PMA-induced ERK activation (Ref.46 and data not shown). As has been previously observed in other cells (46), PMA-induced reduction of EGF binding was not affected by PD98059 in 3T3-F442A cells. Thus, the pattern of sensitivity to GF109203X and PD98059 was quite distinct for PMA-induced vs. GH-induced reduction of EGF binding, strongly suggesting that the mechanisms by which these two stimuli change EGF binding kinetics differ.

LIF inhibits EGF binding in a MEK1/ERK-dependent fashion
Combined with our previous data (22), the results shown above indicate that the effects of GH on EGFR PTP101reactive phosphorylation and EGF binding are dependent on activation of the ERK pathway. We sought to determine whether another cytokine might display similar properties. For this, we used LIF, a multifunctional cytokine that is known to activate JAK2 in 3T3-F442A cells (12, 14, 47, 48). Cells were treated with either GH or LIF and compared for their abilities to activate ERKs and EGFR phosphorylation (Fig. 8Go, A and B). Immunoblotting of detergent extracted proteins after SDS-PAGE resolution with antiactive ERK revealed that both stimuli caused acute ERK 1/2 activation (Fig. 8AGo, lanes 3 and 5 vs. 1), which was inhibited by treatment in the presence of PD98059 (Fig. 8AGo, lanes 4 vs. 3 and 6 vs. 5), consistent with previous results (12). Portions of the same cell extracts were immunoprecipitated with anti-EGFR. Eluates were resolved and immunoblotted with PTP101 and reprobed with anti-EGFR as a loading control (Fig. 8BGo, upper and lower panels, respectively). This indicated that LIF, like GH, induced EGFR PTP101-reactive phosphorylation, which is PD98059-sensitive.



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  		FIG. 8. LIF reduces the 125I-EGF surface binding of 3T3-F442A cells in a MEK1/ERK-dependent fashion. A, LIF induces ERK activation in 3T3-F442A cells, which is inhibited by PD98059. Serum-starved 3T3-F442A cells were preincubated with 50 µM PD98059 (lanes 2, 4, and 6) or vehicle (lanes 1, 3, and 5) at 37 C for 1 h before stimulation with 500 ng/ml GH (lanes 3 and 4) or 20 ng/ml LIF (lanes 5 and 6) for 10 min. Detergent extracts (30 µg) were resolved by SDS-PAGE and immunoblotted with antiactive ERK. B, LIF induces EGFR PTP101 reactivity in a MEK1/ERK-dependent fashion. Detergent extracts (500 µg) as in A were immunoprecipitated with an anti-EGFR antibody. Eluted proteins were analyzed by immunoblotting with PTP101 (upper panel) and anti-EGFR (lower panel), respectively. C, LIF induces the reduction of EGF binding, which is blocked by the MEK1 inhibitor PD98059. Cells were pretreated with PD98059 as in A before stimulation with LIF (20 ng/ml) for 10 min. The cells were then subjected to 125I-EGF binding assays. Specific 125I-EGF binding in the absence of LIF is considered 100%. Data are mean ± range of duplicate determinations. The experiment shown is representative of three such experiments.

 
The effects of LIF on EGF binding were tested in the same fashion as GH was tested (Fig. 8CGo). Treatment with LIF for 10 min reduced 125I-EGF binding substantially (LIF vs. control). As with GH treatment, inclusion of PD98059 during the LIF treatment largely undid the inhibitory effect of LIF on EGF binding (LIF + PD vs. LIF). These results suggested that ERK activation by both GH and LIF is responsible for mediation of the inhibitory effect of each stimulus on EGF binding in 3T3-F442A cells. In other experiments (not shown), we also examined the effects of interferon-{gamma} (IFN{gamma}), which activates JAK2, but not ERKs 1/2, in these cells (12). Notably, IFN{gamma} failed to inhibit EGF binding, suggesting that JAK2 activation alone (without ERK activation) is not sufficient for this effect.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we continued our examination of cross-talk between the GH and EGF signaling systems. We previously demonstrated in 3T3-F442A cells that GH promotes both tyrosine and PTP101-reactive (likely threonine) phosphorylation of the EGFR and that inhibition of the MEK1/ERK pathway prevents the GH-induced PTP101-reactive, but not tyrosine, phosphorylation (12, 22). Furthermore, GH treatment, in a MEK1-dependent fashion, markedly diminished EGF-induced EGFR down-regulation and potentiated early aspects of EGF signal transduction (22). In that study, we noted that the rate of internalization of surface-bound 125I-EGF during the 10 min after initial binding was not affected by pretreatment with GH before adding the radiolabeled EGF; that is, in either circumstance, roughly 80% of the initially bound EGF was resistant to acid stripping after 10 min. This was interpreted to indicate that the rate of ligandmediated endocytosis of EGF binding sites did not differ after GH exposure.

We have now investigated the impact of GH on the degree of surface EGF binding and have uncovered intriguing effects. GH caused a concentration- and time-dependent reduction of EGF binding to 3T3-F442A cells. This effect was substantial (reaching roughly 50% reduction), although transient, with the maximal reduction observed after 10 min of GH (500 ng/ml) treatment and the reduction no longer detectable after 1 h of GH exposure. Analysis of the binding kinetics by competitive binding assays indicated that GH pretreatment did not change the number of EGF binding sites but rather reduced the binding affinity by roughly 2.4-fold, on average. The importance of GH-induced ERK activation in this effect on EGF binding was suggested by two sets of experimental findings. First, the time courses of GH (500 ng/ml)-induced ERK activation, PTP101-reactive phosphorylation, and reduction of EGF binding were compared and found to be quite similar; notably, GH-induced ERK activation slightly preceded the reduction of EGF binding. In addition, the GH concentration dependence for reduction of EGF binding was similar to that observed for activation of ERKs in these cells (40) and at a lower concentration (50 ng/ml), the time courses of ERK activation and loss of EGF binding were correlated. Second, a MEK1 inhibitor, PD98059, which blocked GH-induced ERK activation, also reversed GH’s inhibitory effect on EGF binding. As further evidence of a role for ERK activation in mediation of inhibition of EGF binding, another JAK2- and ERK-activating cytokine, LIF, also reduced surface EGF binding in a MEK1 inhibitordependent fashion and IFN{gamma}, which activates JAK2 but not ERKs in these cells, failed to inhibit EGF binding.

Our results are the first of which we are aware that implicate ERK activation in modulation of EGF binding. In this context, we find particularly interesting the differences we observed between stimulation with GH (or LIF) and phorbol ester. It is well known that PMA causes down-regulation of EGF binding in other cell types, and this effect is inhibited by PKC inhibitors (42, 43, 44, 45, 46). Our current data in 3T3-F442A cells are in concordance with these previous findings and furthermore indicate that inhibition of PMA-induced MEK1/ERK activation does not reverse the effect of PMA on EGF binding. Thus, we conclude that the PMA-induced down-regulation of EGF binding, unlike the effect of GH on EGF binding, requires activation of PKC activity(ies) but not ERK activation.

Although we do not yet know the mechanism by which GH decreases EGF binding or the basis for this distinct difference in the effects of GH and PMA, several intriguing issues are raised. We note that our prior studies indicated that GH does not itself affect the mass of EGFRs detected by immunoblotting in these cells (22). Whereas reports vary concerning PMA’s effects, acute PMA-induced decrease in EGFR abundance has been noted in 3T3-F442A cells (49). Thus, activation by PMA (via PKC), but not GH, of pathways that lessen EGFR protein levels could underlie some of the differences we observed.

In principle, phosphorylation of the EGFR could account for changes in its ability to bind EGF. Importantly, however, previous studies have shown that mutagenesis of EGFR cytoplasmic domain residues known to be phosphorylated in response to PMA did not prevent PMA-induced inhibition of EGF binding (45). These included the two major juxtamembrane threonine phosphorylation sites, T-654 and T-669 (50, 51, 52, 53). It is thought that PMA-induced T-654 phosphorylation is mediated via PKC, whereas the phosphorylation of T-669 (which resides in the only ERK phosphorylation consensus site in the EGFR cytoplasmic domain) is targeted by ERKs (54, 55). Our findings that PD98059, although it inhibited PMA-induced ERK activation and EGFR PTP101-reactive phosphorylation, failed to reverse inhibition of EGF binding further argue against a direct influence of EGFR threonine phosphorylation on PMA-induced lessening of EGF binding. However, we cannot yet draw the same conclusions for the effect of GH on EGF binding, which clearly differs mechanistically from that of PMA. This will await further studies in which the GH-induced EGFR PTP101-reactive phosphorylation site(s) is mapped and the effects of mutagenesis of it and potentially other phosphorylation sites are tested.

Recent structural studies of the soluble EGFR extracellular domain alone and in association with its ligands have provided important insights (56, 57, 58, 59). EGF causes EGFR extracellular domains to dimerize with a stoichiometry of 2:2 EGF/EGFR. Interestingly, this is accomplished by each EGF molecule promoting dramatic structural rearrangements within the extracellular domain of an EGFR monomer such that the monomers achieve conformations that allow dimerization. The influence of the receptor cytoplasmic domain in these changes is unknown. It is tempting to speculate that EGF-independent changes in the EGFR cytoplasmic domain, attributable to either its own phosphorylation or phosphorylation-dependent alteration in its association with other proteins, could affect the kinetics of EGF-EGFR extracellular domain interaction and/or impact events consequent to that interaction. Indeed, others have also noted in a cell-free membrane system that the reversible interaction of the EGFR cytoplasmic domain with a set of inducibly phosphorylated membrane proteins may influence the affinity with which EGF binds to the receptor extracellular domain (60). Furthermore, a very recent study (61) suggests that the high-affinity binding of EGF by EGFR may be accounted for by an additional binding event in which occupied EGFR dimers bind to an additional external site (e.g. a cellular component involved in EGFR turnover). This leads us to question whether the GH-induced EGF binding affinity changes we observe could reflect GH-induced, ERK-dependent alterations in the nature of the association of the EGF-engaged EGFR with such an external site.

We note that we have not yet detected changes in the degree of acute EGF-induced EGFR tyrosine phosphorylation after GH pretreatment, despite the substantial reduction in EGF binding affinity [(22) and data not shown]. However, we have observed clear changes in the postendocytic fate of the EGF-engaged EGFR and in the degree of EGF signaling in the setting of GH pretreatment (22). At this point, we are intrigued that GH, via MEK1/ERK activation protects EGFR from EGF-induced degradation; enhances aspects of acute EGF-induced signaling; and transiently reduces EGF binding affinity. Whether the first two GH effects happen because of, despite of, or are unrelated to the third effect is not yet known. However, given that ERKs are activated by numerous hormones and cytokines, deciphering these potentially complicated effects will be important in future studies. In fact, preliminary data (not shown) suggest that treatment of the T47-D human breast carcinoma cell with prolactin results in a MEK1-dependent decrease in EGF binding, much like we report herein for GH in 3T3-F442A cells. Further studies in this vein may shed light on the generalizability of our findings and their implications for modulation of EGF signaling in human cells.


   Acknowledgments
 
The authors appreciate helpful conversations with Dr. K. He, K. Loesch, J. Cowan, N. Yang, and X. Li and the generous provision of reagents by those named in the text. We thank Dr. Paul J. Bertics for critically reviewing the manuscript.


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Grant DK46395 (to S.J.F.) and in part by NIH Grant DK58259 (to S.J.F.).

Abbreviations: Bmax, Maximal receptor binding sites; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; IFN{gamma}, interferon-{gamma}; JAK, Janus kinase; Kd, radioligand equilibrium dissociation constant; LIF, leukemia inhibitory factor; MEK, MAPK kinase; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate.

Received December 8, 2003.

Accepted for publication March 29, 2004.


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