Thyrotropin-Releasing Hormone Stimulates Phosphorylation of the Epidermal Growth Factor Receptor in GH3 Pituitary Cells

Ying-Hong Wang, Shall F. Jue and Richard A. Maurer

Department of Cell and Developmental Biology Oregon Health Sciences University Portland, Oregon 97201


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TRH has been found to stimulate tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. A specific EGF receptor kinase inhibitor, tyrphostin AG1478, substantially reduced TRH-stimulated tyrosine phosphorylation of the EGF receptor. TRH-induced EGF receptor phosphorylation was found to lead to the recruitment of the adapter proteins Grb2 and Shc. TRH treatment also led to phosphorylation of the related receptor tyrosine kinase, HER2. HER2 activation likely contributes to downstream signaling events and enhances EGF receptor action. TRH-induced tyrosine phosphorylation of the EGF receptor was reduced by incubation with a protein kinase C (PKC) kinase inhibitor, GF109203X. EGF receptor phosphorylation was required for full TRH-induced activation of mitogen-activated protein kinase (MAPK) and stimulation of specific transcriptional responses.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The TRH receptor is a seven-transmembrane Gq-coupled receptor (1). Activation of the TRH receptor stimulates the activity of phospholipase Cß (2), leading to the production of diacylglycerol and inositol 1,4,5-trisphosphate (3). Increased intracellular levels of diacylglycerol and inositol 1,4,5 trisphosphate result in activation of protein kinase C (PKC) and mobilization of intracellular calcium (4). TRH-induced signaling events include activation of the mitogen-activated protein kinase (MAPK) in both a PKC-dependent and PKC-independent manner (5, 6). While the mechanisms that mediate TRH effects on MAPK activation have not been completely determined, it has been observed that TRH induces rapid tyrosine phosphorylation of Shc leading to activation of Ras (5). This finding suggests that TRH-induced activation of MAPK may involve activation of a tyrosine kinase, which in turn leads to activation of Ras and the MAPK cascade. Thus the signaling pathways that allow the Gq-coupled TRH receptor to activate MAPK may overlap with the well studied pathways through which receptor tyrosine kinases activate Ras and the MAPK cascade (7).

Interestingly, there is evidence that TRH and epidermal growth factor (EGF) have overlapping activities in GH3 cells (8, 9). Both EGF and TRH can stimulate PRL synthesis and inhibit GH synthesis in GH3 cells (9). Long-term incubation with either TRH or EGF can induce similar morphological changes in GH3 cells (8, 9). The similar effects of TRH and EGF in GH3 cells support the possibility that the two signaling pathways may converge at some point.

In the present study we have further examined the possibility that TRH may activate a tyrosine kinase in GH3 cells. We report that TRH induces tyrosine phosphorylation of the EGF receptor. Activation of the EGF receptor by TRH is accompanied by tyrosine phosphorylation of the related receptor tyrosine kinase, HER2, as well as the adapter protein, Shc. Blocking TRH-induced EGF receptor activation was found to reduce activation of the MAPK cascade and specific transcriptional events, demonstrating the necessary role of the EGF receptor in mediating downstream effects of TRH.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TRH-Induced MAPK Activation Requires Ras
As a starting point for further analysis of the signaling pathways mediating TRH effects on MAPK activation, we chose to examine the role of Ras in mediating this response. Previous studies have shown that both TRH and EGF can lead to a 2-fold increase in the percentage of Ras that is bound with GTP (5). While these data strongly support a possible role for Ras in mediating TRH-induced activation of MAPK, they do not determine whether Ras is required for this response. To address this question, GH3 cells were transiently transfected with an expression vector for a dominant-negative, mutant form of Ras (10) and epitope-tagged ERK2. The cells were treated with TRH or EGF, and MAPK activity was determined by an immunocomplex kinase assay. Transfection of the N17Ras mutant substantially reduced both EGF- and TRH-induced MAPK activation (Fig. 1Go). These findings offer evidence that Ras is required for full TRH-induced MAPK activation.



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Figure 1. TRH-Induced ERK Activation Is Ras Dependent

GH3 cells were transfected with 0.5 µg of an expression vector for ERK2 tagged with a Myc epitope in the absence or presence of 1.5 µg of an expression vector for a dominant negative Ras (RasN17). The cells were untreated (Control) or treated with 100 nM TRH for 2.5 min or 10 nM EGF for 5 min. Myc-tagged ERK2 was immunoprecipitated with the agarose-conjugated anti-Myc antibody and assayed for ERK2 activity using glutathione-S-transferase-Elk1 as a substrate. The expression level of Myc-ERK2 was analyzed by immunoblotting Myc immunoprecipitates with the anti-ERK2 antibody. This experiment has been repeated twice with similar findings.

 
TRH Stimulates Tyrosine Phosphorylation of the EGF Receptor and Recruitment of Adapter Proteins
A number of studies have shown that G protein-coupled receptors can activate Ras and the MAPK cascade through activation of tyrosine kinases (11, 12, 13, 14). As an initial step to explore possible regulation of tyrosine kinase activity by TRH, we compared the effects of TRH and EGF on phosphotyrosine-containing proteins in GH3 cell lysates. As expected, EGF treatment strongly increased the phosphotyrosine content of several proteins (Fig. 2AGo). The effects of TRH treatment were more subtle, but TRH appeared to also stimulate an increase in the phosphotyrosine content of several proteins. In particular, increased phosphotyrosine content was detected for proteins of approximately 185, 170, and 66 kDa. These findings are consistent with an effect of TRH to either activate a tyrosine kinase or inhibit a phosphotyrosine phosphatase.



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Figure 2. TRH Increases Phosphotyrosine Content of Several Proteins in GH3 Cells

A, GH3 cells were serum-starved for 48 h and treated with or without 100 nM TRH for 2.5 min. Cell lysates (100 µg protein) were resolved by denaturing PAGE and transferred to a membrane. Phosphotyrosine-containing proteins were visualized by immunoblotting with an antiphosphotyrosine antibody. B, GH3 cells were treated with TRH at varying times, and cell lysates were prepared and immunoprecipitated with the antirat EGF receptor antibody (provided by Dr. S. H. Earp). The immunoprecipitates were resolved by denaturing PAGE, transferred to a membrane, and immunoblotted with an antiphosphotyrosine antibody (P-Tyr Blot) and then reprobed with anti-EGF receptor antibody (EGFR blot). These experiments have been repeated at least three times with similar findings.

 
The TRH-inducible 170-kDa band comigrated with an EGF-inducible band. As this is the appropriate size for the EGF receptor, this finding suggests that TRH may stimulate phosphorylation of the EGF receptor. To directly test this possibility, GH3 cells were treated with TRH for varying times after which EGF receptor phosphorylation was evaluated by immunoprecipitation of the receptor followed by immunoblotting with antiphosphotyrosine antibody (Fig. 2BGo). TRH treatment was found to increase the phosphotyrosine content of the EGF receptor at the earliest time point examined (2.5 min), and the phosphotyrosine content was elevated for at least 1 h. Although EGF was much more active than TRH in stimulating EGF receptor phosphorylation, TRH stimulated an easily detectable increase in EGF receptor phosphotyrosine content. As expected, EGF, but not TRH, was able to stimulate EGF receptor phosphorylation in the Rat-1 cell line, which does not contain TRH receptors (data not shown).

To investigate the nature of the TRH-induced tyrosine phosphorylation of the EGF receptor, we tested whether a potent and specific inhibitor of the EGF receptor, tyrophostin AG1478 (15, 16), had an effect on TRH-induced EGF receptor tyrosine phosphorylation. AG1478 treatment substantially reduced TRH-induced tyrosine phosphorylation of the EGF receptor (Fig. 3AGo). As AG1478 interacts with the ATP binding site on the receptor (15, 16), this finding suggests that TRH stimulates receptor autophosphorylation. To further investigate whether TRH-induced tyrosine phosphorylation of the EGF receptor reflects activation of the receptor’s intrinsic kinase activity, we performed a kinase assay (17, 18) on EGF receptor immunoprecipitates. TRH stimulated EGF receptor kinase activity, and the TRH-induced increase in kinase activity was blocked by AG1478 treatment (Fig. 3BGo). Similar results were obtained when EGF receptor autophosphorylation was examined (data not shown).



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Figure 3. TRH Stimulates EGF Receptor Intrinsic Kinase Activity

A, GH3 cells were pretreated with 250 nM AG1478 or with the solvent used for AG1478 [dimethylsulfoxide (DMSO)] for 20 min and then treated with 100 nM TRH for varying times. Cell lysates were immunoprecipitated with the antirat EGF receptor antibody. The immunoprecipitates were resolved by denaturing PAGE, transferred to a membrane, and immunoblotted with an antiphosphotyrosine antibody (P-Tyr Blot) and then reprobed with anti-EGF receptor antibody (EGFR blot). B, Analysis of TRH effects on EGF receptor kinase activity. GH3 cells were treated with 100 nM TRH for 2.5 min. The EGF receptor was immunoprecipitated and then assayed for kinase activity using myelin basic protein as a substrate. These experiments have been repeated at least twice with similar findings.

 
Recruitment and phosphorylation of adapter proteins are key events that are essential for mediating the ability of the EGF receptor to activate the Ras/MAPK pathway (19, 20, 21). Treatment of GH3 cells with TRH or EGF resulted in the apparent interaction of the tyrosine-phosphorylated EGF receptor with a Grb2-glutathione-S-transferase fusion protein (Fig. 4AGo). As observed for TRH-induced phosphorylation of the EGF receptor, TRH was much less effective than EGF treatment in stimulating the interaction of Grb2 and the EGF receptor. We also examined phosphorylation of Shc (Fig. 4BGo). After TRH treatment, tyrosine-phosphorylated proteins of 185, 66, and 52 kDa were immunoprecipitated with antisera to Shc (Fig. 4BGo). The 52- and 66-kDa proteins represent isoforms of Shc, consistent with previous studies demonstrating TRH-induced tyrosine phosphorylation of Shc (5). The 185-kDa phosphoprotein that was coimmunoprecipitated likely represents the HER2 receptor tyrosine kinase (see below). The tyrosine phosphorylation of the 185-, 66-, and 52-kDa proteins was greatly reduced by treatment with AG1478.



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Figure 4. TRH Treatment Leads to Recruitment and Phosphorylation of Adapter Proteins

A, TRH induces association between the EGF receptor and Grb2. GH3 cells were treated with 100 nM TRH for 2.5 min or 10 nM EGF for 10 min. Cell lysates were incubated with a resin containing a Grb2-glutathione-S-transferase fusion protein. After washing, the resin was resolved by denaturing gel electrophoresis, transferred to a membrane, and incubated with an antiphosphotyrosine antibody (P-Tyr Blot). A phosphotyrosine-containing protein with the appropriate size for the EGF receptor was detected as shown. B, TRH-induced association with Shc. GH3 cells were treated as above. Cell lysates were immunoprecipitated with 4 µg of anti-Shc antibody and immunoblots probed with the antiphosphotyrosine antibody (P-Tyr blot) and reprobed with the anti-Shc antibody (Shc blot). These experiments have been repeated at least twice with similar findings.

 
TRH Treatment Leads to Tyrosine Phosphorylation of HER2
In general, EGF receptor signaling is rapidly down-regulated due to receptor-mediated endocytosis (22). In contrast, TRH-induced activation of the EGF receptor persists for approximately 1 h, implying that some event modulates the time course of activation. HER2, also known as ErbB2 or neu, is a member of the ErbB family of receptor tyrosine kinases and is most closely related to the EGF receptor (ErbB1) (23). HER2 can form heterodimers with the EGF receptor and enhance EGF-induced tyrosine phosphorylation of the EGF receptor and potentiate and prolong EGF-induced signal transduction (24, 25, 26, 27, 28). To explore the possibility that HER2 may play a role in TRH-induced EGF receptor activation, we examined TRH effects on HER2 phosphorylation. Treatment with TRH resulted in an increase in the phosphotyrosine content of HER2, which persisted for at least 30 min (Fig. 5AGo). To determine whether TRH-induced tyrosine phosphorylation of HER2 was mediated by the EGF receptor, we treated GH3 cells with the specific EGF receptor inhibitor, AG1478. At a concentration of 250 nM, AG1478 strongly inhibited TRH-induced tyrosine phosphorylation of HER2 (Fig. 5BGo). As AG1478 inhibits HER2 only at much higher concentrations (>100 µM) (16), these data suggest that TRH-induced HER2 activation depends on activation of the EGF receptor. These findings provide evidence that TRH induces HER2 phosphorylation in GH3 cells, and the activation of HER2 may contribute to prolonged phosphorylation of the EGF receptor.



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Figure 5. TRH Treatment Stimulates HER2 Phosphophorylation

A, GH3 cells were treated with TRH for different times. Cell lysates were immunoprecipitated with an anti-HER2 antibody, and the proteins were separated by denaturing gel electrophoresis and transferred to a membrane. Phosphotyrosine-containing proteins were detected by immunoblotting with the antiphosphotyrosine antibody (P-Tyr blot) and reprobed with an anti-HER2 antibody. B, TRH-induced HER2 activation is reduced by AG1478. Cells were pretreated with 250 nM AG1478 or the solvent used for AG1478 (DMSO) for 20 min and then treated with 100 nM TRH or 10 nM EGF. Cell lysates (1 mg) were immunoprecipitated with 4 µg of the anti-HER2 antibody. Proteins were separated by denaturing gel electrophoresis and transferred to a membrane. Phosphotyrosine-containing proteins were detected by immunoblotting with the antiphosphotyrosine antibody (P-Tyr blot) and reprobed with an anti-HER2 antibody. These experiments have been repeated at least twice with similar findings.

 
TRH-Stimulated Tyrosine Phosphorylation of the EGF Receptor Requires PKC
Activation of phospholipase Cß by the TRH receptor leads to increased intracellular concentration of diacylglycerol and subsequent activation of PKC. To determine whether PKC lies in the pathway between the TRH receptor and the EGF receptor, we pretreated cells with GF109203X, a specific PKC inhibitor (29). Treatment of GH3 cells with the PKC inhibitor reduced the subsequent TRH-induced tyrosine phosphorylation of the EGF receptor (Fig. 6Go). Thus, PKC activation appears to be necessary for TRH-induced EGF receptor transactivation.



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Figure 6. TRH-Induced Phosphorylation of the EGF Receptor Is PKC Dependent

GH3 cells were pretreated for 20 min with 1 µM GF109203X or the solvent used for the drug (DMSO). The cells were then stimulated with 100 nM TRH for 2.5 min. Tyrosine phosphorylation of the EGF receptor was analyzed by immunoprecipitating with anti-EGF receptor antibody and then immunoblotting with the antiphosphotyrosine antibody (P-Tyr blot) and reprobing with EGF receptor antibody (EGFR blot). This experiment has been repeated twice with similar findings.

 
Previous studies have shown that activation of Src family nonreceptor tyrosine kinases by Gi-coupled receptors can account for tyrosine phosphorylation of both the EGF receptor and Shc (14). To study the mechanism of TRH-induced tyrosine phosphorylation of the EGF receptor, we pretreated the cells with PP1, an inhibitor, which is specific for the Src family tyrosine kinases (30). Although PP1 reduced the phosphotyrosine content of several proteins, it had little effect on tyrosine phosphorylation of the EGF receptor (data not shown). These results suggest that tyrosine phosphorylation of the EGF receptor by TRH is not mediated by PP1-sensitive tyrosine kinases.

EGF Receptor Activity Is Required for TRH-Induced Activation of MAPK and Specific Transcription
To test the role that EGF receptor phosphorylation plays in mediating TRH effects on MAPK activation, we examined the effects of AG1478 on MAPK activation. As determined by immunoblotting with an anti-phospho-ERK antibody, TRH-induced MAPK activation was reduced by pretreatment with AG1478 (Fig. 7AGo). To further examine the role that the EGF receptor plays, we used an expression vector for a kinase-defective mutant of the EGF receptor (HERK721A) (31). The HERK721A expression vector was transfected into GH3 cells with an expression vector for Myc-tagged ERK2, and ERK2 activity was then determined by an immunocomplex assay (Fig. 7BGo). TRH-induced ERK2 activation was substantially reduced by the HERK721A expression vector.



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Figure 7. EGF Receptor Activity Is Required for TRH-Induced Activation of MAPK

A, The EGF receptor inhibitor, AG1478, reduces TRH-induced ERK activation. GH3 cells were pretreated with 250 nM AG1478 for 20 min and treated with TRH for 2.5 min. ERK activation was analyzed by immunoblotting with antiphospho-ERK antibody. The expression level of ERKs was examined by reprobing the membrane with anti-ERK1 antibody. B, An expression vector for a kinase-defective EGF receptor mutant reduces TRH-induced ERK activation. GH3 cells were transfected with an expression vector for Myc-tagged ERK2 in the absence or presence of a vector for a kinase-defective mutant of the EGF receptor (HERK721A). Cells were treated with TRH or EGF. Cell lysates were immunoprecipitated with antibody to the Myc epitope, and the immunocomplexes were assayed for kinase activity. The expression level of Myc-ERK2 was analyzed by immunoblotting the Myc immunoprecipitates with anti-ERK2 antibody. These experiments have been repeated at least twice with similar findings.

 
To determine the downstream importance of TRH-induced tyrosine phosphorylation of the EGF receptor, we examined the effects of the HERK721A vector on specific transcriptional responses to TRH. Previous studies have shown that activation of the MAPK pathway is essential for TRH effects on the transcriptional activity of a GAL-Elk1 fusion protein as well as full induction of the PRL promoter (6). Transfection of an expression vector for the kinase-defective EGF receptor substantially reduced TRH-induced activation of Gal4-Elk1 activity (Fig. 8AGo) and more modestly reduced induction of the PRL promoter (Fig. 8BGo). As expected, neither TRH nor the expression vector for the mutant EGF receptor affected the activity of thymidine kinase reporter gene (Fig. 8CGo). These data indicate that the EGF receptor’s kinase activity is required for the full induction of transcriptional responses by TRH. The partial inhibition on the PRL promoter also suggests that TRH-induced EGF receptor phosphorylation is not the only pathway that contributes to TRH-induced activation of this transcriptional response.



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Figure 8. EGF Receptor Kinase Activity Is Required for TRH-Induced Transcriptional Events

GH3 cells were transfected with an empty expression vector (control) or an expression vector for a kinase-defective mutant of the EGF receptor (HERK721A). The cells also received an expression vector for GAL4-Elk1 and a reporter gene containing 5 GAL4 binding sites (A) or reporter genes containing the rat PRL promoter (B) or the herpes simplex virus thymidine kinase promoter (C) linked to luciferase. All cells also received a vector directing expression of Renilla luciferase as an internal standard. Forty-eight hours after transfection, the cells were treated with 100 nM TRH or 10 nM EGF and then collected for luciferase assays 6 h later. All values are means ± SEM for three separate transfections, which have been corrected for transfection efficiency. This experiment has been repeated three times with similar findings.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
These studies provide evidence that TRH-induced phosphorylation and activation of the EGF receptor play a role in activation of the MAPK cascade leading to specific transcriptional events. An increasing body of work has shown that tyrosine kinases may play a role as downstream components of some G protein-coupled receptor pathways (32, 33, 34, 35). In the present study, we demonstrate that TRH can stimulate tyrosine phosphorylation of the EGF receptor in a time-dependent manner. The use of specific inhibitors has provided evidence that TRH-induced activation of the EGF receptor is required for several downstream events including phosphorylation of adapter proteins, activation of the MAPK pathway, and stimulation of Elk1 transcriptional activation and full activation of PRL gene transcription. Interestingly, very recent studies have provided evidence that GnRH effects in cells of the gonadotrope lineage also involve the ability of a Gq-coupled receptor to stimulate EGF receptor phosphorylation leading to MAPK activation (36).

The ability of TRH to activate the EGF receptor appears to involve autophosphorylation of the receptor. TRH-induced tyrosine phosphorylation of the EGF receptor and the downstream activation of MAPKs are inhibited by tyrphostin AG1478, which specifically inhibits the EGF receptor’s kinase activity by competing for ATP binding. Similar results were obtained when a kinase-inactive mutant of the EGF receptor was used to block receptor signaling. AG1478 was also found to reduce TRH-induced tyrosine phosphorylation of HER2 and Shc. These findings provide evidence that TRH-induced activation of the EGF receptor and downstream signaling events share many similarities with EGF-induced activation of its receptor (19, 20, 21). Consistent with the view that the TRH signaling pathway involves the EGF receptor positioned upstream of the Ras-dependent MAPK pathway, expression of a dominant-negative Ras mutant reduced TRH-induced MAPK activation.

TRH-induced phosphorylation of the EGF receptor is accompanied by phosphorylation of the related receptor tyrosine kinase, HER2. HER2 is a preferred partner of all members of the ErbB family (37), and HER2 can enhance signaling by the EGF receptor in several ways. HER2 can increase the duration of EGF-induced tyrosine phosphorylation of the EGF receptor by slowing the relatively fast endocytosis rate of the EGF receptor (38). HER2 is very efficient in coupling to the MAPK pathway (38), and HER2 can potentiate EGF-induced MAPK activation by recruiting different SH2-containing substrates to the heterodimer complexes (27, 39). As the EGF receptor inhibitor, AG1478, reduces both TRH-induced HER2 phosphorylation and MAPK activation, it is possible that HER2 contributes to the ability of TRH to regulate MAPK activity.

The molecular events that lead to TRH-induced phosphorylation of the EGF receptor are unclear, although they appear to involve PKC. A requirement for PKC activity has been shown by the ability of the PKC inhibitor, GF109203X, to attenuate TRH-induced tyrosine phosphorylation of the EGF receptor. However, the manner in which PKC contributes to activation of the EGF receptor is unclear. It has been shown that PKC can directly phosphorylate the EGF receptor on threonine 654 (40, 41). However, this does not lead to EGF receptor activation but rather inhibits tyrosine kinase activity of the receptor and reduces high-affinity EGF binding (41, 42, 43). It may be that specific PKC isozymes have different effects on the EGF receptor that account for this apparent discrepancy. It is also possible that PKC may activate the EGF receptor through inactivating a protein tyrosine phosphatase. It has been shown that radiation, oxidants, and alkylating agents can alter the phosphorylation of receptor tyrosine kinases through effects on protein phosphatases (44). Recent studies have provided evidence that the ability of G protein-coupled receptors to stimulate EGF receptor phosphorylation may involve cleavage of heparin-binding EGF-like growth factor (45). Cleavage of the heparin-binding EGF-like growth factor by a metalloproteinase at a juxtamembrane site releases an active form of the molecule (46). PKC (47) may play a role in stimulating cleavage and activation of heparin-binding EGF-like growth factor. We have found that 1,10-phenanthrolene, a metal chelator and general inhibitor of metalloproteinases, reduces the ability of TRH to activate MAPK (S. F. Jue, unpublished observations), suggesting that cleavage of heparin-binding EGF-like growth factor may play a role in mediating TRH-induced phosphorylation of the EGF receptor.

Although TRH and EGF share many signaling events in common, there appears to be a substantial quantitative difference in the activation of specific signaling steps. For instance, EGF has a much greater effect than TRH on phosphorylation of the EGF receptor, HER2, Shc, and Grb2. Nonetheless, TRH effects on EGF receptor phosphorylation are clearly important as the dominant negative kinase-defective EGF receptor, HERK721A, substantially reduced TRH-induced MAPK activation as well as transcriptional responses. What accounts for this apparent discrepancy between weak signal activation and an important functional role? One aspect may involve an additive or synergistic effect of multiple TRH-induced signaling events. For instance, TRH treatment results in Ca2+ influx through voltage-gated Ca2+ channels (48). Our previous studies have provided evidence that Ca2+ influx contributes to MAPK activation in GH3 cells (6). It is possible that TRH initiates multiple signaling pathways including EGF receptor phosphorylation and Ca2+ influx that converge on MAPK activation.

The present findings provide new insights into the overlapping biological activities of EGF and TRH in GH3 cells. Both EGF and TRH stimulate PRL synthesis (8, 9) and PRL gene transcription (49, 50). TRH and EGF effects on expression of the PRL gene are mediated via multiple, common DNA elements (51). Previous studies have shown that both EGF and TRH can induce tyrosine phosphorylation of Shc and activate the MAPK pathway (5). The present study reveals a new level of signal convergence, which occurs at the level of the EGF receptor. As both TRH and EGF activate the EGF receptor and several common downstream signaling steps, it is not surprising that TRH and EGF share some biological actions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Cell culture media and supplies were purchased from Life Technologies, Inc. (Gaithersburg, MD). Radioisotopes and enhanced chemiluminescent reagents were purchased from DuPont New England Nuclear (Boston, MA). Anti-ERK polyclonal antibodies, anti-phospho-ERK monoclonal antibody, antiphosphotyrosine monoclonal antibody, antihuman EGF receptor polyclonal antibody, anti-neu polyclonal antibody, agarose-conjugated anti-Myc antibody, agarose-conjugated glutathione-S-transferase-Grb2, and horseradish peroxidase-coupled secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Shc polyclonal antibody was purchased from Transduction Laboratories, Inc. (Lexington, KY). Antirat EGF receptor antibodies were kindly provided by Dr Shelton Earp (University of North Carolina at Chapel Hill) or purchased from Calbiochem (La Jolla, CA). The PKC inhibitor, GF109203X, and Src-like tyrosine kinase inhibitor, PP1, were purchased from Calbiochem. AG1478 was from Research Biochemicals International (Natick, MA); EGF was obtained from Roche Molecular Biochemicals (Indianapolis, IN). TRH was purchased from Peninsula Laboratories, Inc. (Belmont, CA).

Cell Culture and Transfections
GH3 cells were cultured in DMEM supplemented with 15% horse serum and 2.5% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. For transient transfection assays, 2.5 or 5 x 105 cells per well were subcultured in six-well plates 1 day before the transfection. DNA was transfected into these cells using lipofectamine (Life Technologies, Inc.) according to a protocol provided by the manufacturer. In each experiment, the total amount of transfected DNA per well was maintained as a constant (usually 2 µg) by addition of empty expression vector (either pCDNA3a or pRK5). After 5 h of treatment with the lipofectamine/DNA mixture, an equal volume of serum-containing culture medium was added. After an additional 18 h, the cells were transferred to serum-free medium for a further 24 h before lysis and analysis.

For reporter gene assays, two different reporters that can be assayed independently were used in each experiment. Firefly luciferase constructs (52) were used to assess transcriptional activation. A reporter gene containing five Gal4 binding sites upstream of a minimal promoter (53) was used to examine transcriptional activation of a GAL4-Elk1 fusion protein (54). Firefly luciferase reporter genes containing the proximal 255 bp of the 5'-flanking region of the PRL gene (6) or a minimal herpes simplex virus thymidine kinase promoter (55) have been reported previously. To correct for transfection efficiency, the cells were also transfected with pRL, which expresses Renilla luciferase. Renilla luciferase requires a different substrate than the firefly luciferase and can be assayed independently. The activities of firefly and Renilla luciferase were determined using a protocol and reagents from Promega Corp. (Madison, WI). Total firefly luciferase light units were normalized to total Renilla luciferase activity.

Immunoprecipitation and Immunoblotting
Cells were lysed for 10 min on ice in lysis buffer [150 mM NaCl, 20 mM Tris, pH 7.5, 1% Triton X-100, 1 mM EDTA, 50 mM ß-glycerolphosphate, 10 mM NaF and 10% (vol/vol) glycerol] with freshly added 1 mM Na3VO4,and 1x Complete proteinase inhibitor (Roche Molecular Biochemicals). The cell lysates were centrifuged for 10 min at 12,000 x g. For immunoprecipitation experiments, 1 mg of cell lysate was immunoprecipitated by incubation with the appropriate antibody overnight at 4 C. Twenty microliters of protein A/G agarose beads were then added for an additional 45 min. Immune complexes were washed three times with the lysis buffer and resuspended in SDS sample buffer [62.5 mM Tris-HCl, pH 6.8, 2% (wt/vol) sodium dodecyl sulfate, 10% (vol/vol) glycerol, 5% (vol/vol) ß-mercaptoethanol, and 0.05% (wt/vol) bromophenol blue]. For immunoblotting, samples were resolved by denaturing-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and incubated with the selected antibody. Immunoblots were developed with enhanced chemiluminescent reagents (DuPont New England Nuclear). For reprobing of immunoblots, the membrane was stripped in 62.5 mM Tris, pH 6.8, 2% SDS, and 100 mM ß-mercaptoethanol at 50 C for 30 min.

Immunocomplex Kinase Assay
Immunoprecipitates were prepared as described above. For the ERK kinase assay, immunoprecipitates were washed once in the ERK kinase assay buffer (20 mM Tris pH 7.5, 10 mM MgCl2, 0.1% Triton X-100, and 2 mM EGTA). The kinase assays were carried out for 25 min at 30 C in 20 µl of ERK kinase assay buffer supplemented with 1 µCi of [{gamma}-32P] ATP and 5 µg glutathione-S-transferase fusion with the carboxy-terminal region of Elk1 (6). For the EGF receptor kinase assay, the immunoprecipitates were washed once with EGF receptor kinase buffer (20 mM HEPES, pH 7.3, 10 mM MnCl2, 1 mM dithiothreitol, 0.2 mM Na3VO4). The kinase assays were carried out for 10 min at 30 C in 20 µl of the EGFR kinase assay buffer in the presence of 5 µCi of [{gamma}-32P] ATP. For measuring the EGF receptor’s kinase activity by using an exogenous substrate, 5 µg of myelin basic protein for each sample were added to the reaction mixture. The kinase reactions were stopped by adding SDS sample buffer, and the reactions were resolved on a denaturing polyacrylamide gel. Phosphorylated proteins were detected by autoradiography and quantitated using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).


    ACKNOWLEDGMENTS
 
We thank Dr. Shelton H. Earp for the antirat EGF receptor polyclonal antibody, Dr. Mihail Iordanov for the kinase-defective EGF receptor construct, Dr. Geoffrey Cooper for the dominant-negative Ras construct, and Dr. Christopher Marshall for the myc-ERK2 construct. We also thank Bobbi Maurer for assistance and aid in preparing this manuscript.


    FOOTNOTES
 
Address requests for reprints to: Richard A. Maurer, Department of Cell and Developmental Biology, L215 Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97201.

This research was supported by NIH Grant DK-40339 to R.A.M.

Received for publication March 24, 2000. Revision received May 12, 2000. Accepted for publication May 23, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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