Activation of MAPK by TRH Requires Clathrin-Dependent Endocytosis and PKC but Not Receptor Interaction with ß-Arrestin or Receptor Endocytosis

Jeffrey Smith, Run Yu and Patricia M. Hinkle

Department of Pharmacology and Physiology and the Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Address all correspondence and requests for reprints to: Dr. Patricia M. Hinkle, Department of Pharmacology and Physiology, Box 711, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642. E-mail: Patricia_Hinkle{at}urmc.rochester.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To determine whether the interaction of the TRH receptor with ß-arrestin is necessary for TRH activation of MAPK, cells expressing either intact or truncated, internalization-defective TRH receptors were transfected with a ß-arrestin-green fluorescent protein conjugate. In cells expressing the wild-type pituitary TRH receptor, TRH caused translocation of the ß-arrestin-green fluorescent protein conjugate from the cytosol to the plasma membrane within 30 sec. After 5 min, the ß-arrestin-green fluorescent protein conjugate was visible in vesicles, where it colocalized with rhodamine-labeled TRH. In hypertonic sucrose, the ß-arrestin-green fluorescent protein conjugate translocated to the plasma membrane after TRH addition but did not internalize. In cells expressing the truncated TRH receptor, TRH did not cause translocation of the ß-arrestin-green fluorescent protein conjugate. TRH activated MAPK strongly in cells expressing intact or truncated TRH receptors, indicating that the receptor does not need to bind ß-arrestin or internalize. MAPK activation by TRH, epidermal growth factor, and phorbol ester was strongly inhibited by hypertonic sucrose and concanavalin A, which block movement of proteins into coated pits and coated pit assembly. Hypertonic sucrose did not affect MAPK activation in cells overexpressing MAPK kinase 1. Dominant negative dynamin, which blocks conversion of coated pits to vesicles, also reduced receptor internalization and TRH activation of MAPK. TRH activation of MAPK required PKC but was insensitive to pertussis toxin and did not require ras, epidermal growth factor receptor kinase, or PI3K. These results show that the TRH receptor itself does not need to bind ß-arrestin or undergo sequestration to activate MAPK but that the endocytic pathway must be intact.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TRH ACTS THROUGH a G protein-coupled receptor (GPCR) to stimulate the release and synthesis of thyrotropin and PRL. The pituitary TRH receptor is coupled to Gq/11, and TRH causes an increase in PLC activity and subsequent mobilization of intracellular calcium and activation of PKC. TRH also stimulates MAPK, both in pituitary cells expressing endogenous receptors and in a number of cell lines expressing transfected receptors (1, 2, 3, 4). MAPK activation is required for TRH stimulation of PRL transcription (4).

Activation of many GPCRs leads to an increase in MAPK activity, but the molecular mechanisms involved are not fully understood (5, 6). ß-Arrestin binds to agonist-activated GPCRs after their phosphorylation by receptor kinases and links the activated GPCRs to clathrin, thereby targeting them to the endocytic pathway (7, 8, 9). It has been proposed that it is actually the receptor-ß-arrestin complex that signals to MAPK for the well studied ß2-adrenergic receptor (10). Formation of the ß-arrestin-receptor complex appears to be essential for MAPK activation. Specifically, it has been suggested that the receptor-ß-arrestin complex acts as a scaffold binding src, a nonreceptor tyrosine kinase, and that src transduces the signal from the GPCR to ras, activating the MAPK cascade (10, 11). Components of the MAPK cascade, including raf, MAPK kinase (MEK), and MAPK, have been identified in isolated endocytic vesicles. Because some GPCRs coupled to all families of G proteins interact with ß-arrestin and subsequently undergo endocytosis, the involvement of ß-arrestin in signaling to MAPK may be of widespread importance (12). Supporting the model are the findings that both dominant negative dynamin and dominant negative ß-arrestin suppress activation of MAPK by the M1 muscarinic receptor, which normally couples via Gq/11 to phospholipid turnover (13), and that dominant negative dynamin partially inhibits MAPK activation by the Gq-coupled GnRH receptor (14). In addition, dominant negative dynamin blocks activation of MAPK by the serotonin 1A and {delta}-opioid receptors (15, 16). The wild-type proteinase-activated receptor 2 also appears to activate MAPK through an internalized receptor-ß-arrestin-raf complex (17). The requirement for receptor endocytosis is not universal, however, since several receptors have been shown to activate MAPK without internalization (18, 19, 20, 21, 22).

Once TRH binds, the receptor binds ß-arrestin and internalizes extensively through a clathrin-dependent pathway (23, 24, 25, 26, 27, 28). In this study, we have asked whether ß-arrestin-receptor interaction and subsequent receptor endocytosis are required for activation of MAPK by TRH. We show that ß-arrestin binding and endocytosis of the TRH receptor are not required per se for TRH to activate MAPK, but that a functional endocytic pathway and TRH activation of PKC are necessary.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Interaction of TRH Receptors with ß-Arrestin-Green Fluorescent Protein Conjugate (ß-arr-GFP)
To determine whether an interaction between the activated TRH receptor and ß-arrestin is necessary for the activation of MAPK, we compared the ability of intact and internalization-defective TRH receptors to bind to ß-arrestin and increase MAPK activation. HEK293 cells stably expressing wild-type or truncated TRH receptors were transiently transfected with ß- arr-GFP and then exposed to 1 µM TRH. Cells stably transfected with intact TRH receptors (293TRHR cells) or truncated, internalization-defective TRH receptors terminating at residue 334 (293TRHR-C335Stop cells) bound 4.16 ± 0.13 and 3.69 ± 0.23 pmol [3H]MeTRH/mg protein, respectively, at 10 nM radioligand. Cells expressing these receptors respond to TRH with an increase in IP3 production and a robust calcium signal (24, 29). [3H]MeTRH bound to wild-type receptor was rapidly and extensively internalized after binding, whereas radioligand bound to truncated receptor was not internalized (Fig. 1Go).



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Figure 1. Internalization of [3H]MeTRH

Cells were preincubated at 37 C with: no drug (Control), 0.45 M sucrose for 20 min (Sucrose) or 250 µg/ml concanavalin A for 1 h (Con A); 5 nM [3H]MeTRH was then added in the continued presence of drug and internalized [3H]MeTRH was measured at intervals. Experiments were performed on duplicate dishes, and all errors were within symbol size.

 
We used ß-arr-GFP to monitor the interaction between the receptor and ß-arrestin in live cells (Fig. 2Go). Before stimulation, ß-arr-GFP was evenly distributed in the cytosol. In 293TRHR cells, ß-arr-GFP rapidly translocated from the cytosol to the plasma membrane after exposure to TRH. Translocation was readily apparent within 15 sec. ß-arr-GFP began to appear in cytoplasmic vesicles after 5 min, and it all appeared to be in intracellular vesicles after 20 min of continuous TRH treatment (Fig. 2Go, left panels). TRH was able to translocate ß-arr-GFP at concentrations as low as 1 nM (data not shown). When 293TRHR cells were incubated in HBSS containing 0.45 M sucrose, which inhibits clathrin mobilization, ß-arr-GFP still translocated to the plasma membrane after TRH addition but remained there. In 293TRHR-C335Stop cells, which do not internalize TRH, there was no change in the localization of ß-arrestin after TRH stimulation. These results show that the wild-type TRH receptor interacts strongly with ß-arrestin but the truncated receptor does not.



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Figure 2. Translocation of ß-arr-GFP by TRH in Cells Expressing Intact or Internalization-Defective Receptors

Cells expressing TRH receptors were transiently transfected with ß-arr-GFP. After 24–48 h, cells were placed in HBSS, and the localization of ß-arr-GFP was followed microscopically. Shown are series of images of the same cells before (0), 1, or 20 min after the addition of 1 µM TRH at 37 C. TRHR indicates 293TRHR cells; Truncated TRHR, 293TRHR-C335Stop cells; TRHR+Sucrose, 293TRHR cells in HBSS supplemented with 0.45 M sucrose for 20 min. Similar results have been obtained in multiple experiments, and the results were not affected by overnight serum starvation. TRH had no effect on localization of ß-arr-GFP in HEK293 cells that had not been transfected with the TRH receptor.

 
Internalization of TRH Receptors with ß-arr-GFP
We next asked whether the TRH-receptor complex and ß-arr-GFP are localized in the same vesicles (Fig. 3Go). Rhodamine-labeled TRH (Rhod-TRH), which is a weak agonist for the TRH receptor, was used at a low concentration to minimize nonspecific binding. After 25 min, most of the Rhod-TRH was visible in endocytic vesicles in the 293TRHR cells (top panels). Although much of the ß-arr-GFP remained in the cytosol, some ß-arr-GFP appeared in vesicles, and these vesicles colocalized almost perfectly with the Rhod-TRH vesicles. In the 293TRHR-C335Stop cells, Rhod-TRH did not internalize, and none of the ß-arr-GFP was observed moving to the plasma membrane or in endocytic vesicles (bottom panels).



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Figure 3. Colocalization of Rhod-TRH and ß-arr-GFP

Cells in HBSS were incubated with Rhod-TRH at 37 C for 20–25 min and washed. Rhod-TRH (left panels) and ß-arr-GFP (middle panels) fluorescence images were recorded with appropriate filters. The images were overlaid and orange or yellow color show where the two molecules were colocalized (right panels). There was no significant bleedthrough between these two settings. TRHR indicates 293TRHR cells transfected with ß-arr-GFP; Truncated TRHR, 293TRHR-C335Stop cells transfected with ß- arr-GFP.

 
Activation of MAPK by Intact and Truncated TRH Receptors
MAPK activity was followed by immunoblotting with antibodies against the phosphorylated, active forms of MAPK (ERK1/2). TRH stimulated MAPK phosphorylation in 293TRHR cells in a dose- and time-dependent manner (Fig. 4Go). Activation was evident with as little as 1 nM TRH and apparently maximal at 1 µM TRH. MAPK activation by 1 µM TRH peaked between 2 and 5 min and subsided after 10 min. In subsequent experiments, we used either 1 µM or 10 nM TRH for 5 min at 37 C to activate MAPK. At 5 min, the receptor-ß-arrestin complex was fully formed and largely in intracellular vesicles, although some surface receptor/ß-arrestin complex remained (data not shown). The MAPK response to TRH in 293TRHR cells was comparable to the response to maximally effective concentrations of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which activates PKC, or epidermal growth factor (EGF), which activates the EGF receptor tyrosine kinase (Fig. 5aGo). TRH activated MAPK strongly in cells expressing the truncated receptor, indicating that an interaction with ß-arrestin is not required (Fig. 5aGo).



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Figure 4. Activation of MAPK in 293TRHR Cells

Replicate dishes of 293TRHR cells were serum-starved overnight and then treated with vehicle or TRH at 37 C in HBSS and lysed, and proteins were resolved by SDS-PAGE. Activated MAPK was identified by immunoblotting using phospho-MAPK specific antibody, which labeled 44- and 42-kDa bands, ERK1 and ERK2, respectively. Total MAPK was shown to be the same in all samples on parallel blots that were probed with antibody against total MAPK. a, Cells were incubated with 0 to 10 µM TRH for 5 min. b, cells were incubated with 1 µM TRH for 0–40 min.

 


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Figure 5. Activation of MAPK via an Internalization-Defective TRH Receptor

Cells were serum-starved overnight and then treated as follows. a, Cells were placed at 37 C in either HBSS (left lanes) or HBSS supplemented with 0.45 M sucrose (right lanes); after 20 min, cells were treated for 5 min with no additions, 1 µM TRH, 1 µM TPA, or 10 nM EGF. b, 293TRHR cells were incubated for 1 h without (Control) or with 250 µg/ml concanavalin A (Con A) before treatment with no additions, 1 µM TRH, 1 µM TPA, or 10 nM EGF for 5 min. c, 293TRHR cells were transfected without (TRHR-MEK) or with 1 µg/ml of a plasmid encoding MEK1 (TRHR+MEK) and placed in HBSS. Left lanes, 0.45 M sucrose was added for 0, 10, 30, or 60 min. Right lanes, 50 µM PD98059 was added for 0, 10, 30, or 60 min. Films of the gels grouped with a bracket were exposed identically; a lighter exposure of the gel containing TRHR+MEK is also shown, noted by the asterisk. d, Cells were serum starved overnight in the absence (Control) or presence of 1 µM TPA to deplete PKC (PKC Depl). The cells were then placed in HBSS and treated for 5 min with or without 1 µM TRH. TRHR indicates 293TRHR cells; truncated TRHR, 293TRHR-C335Stop cells.

 
Role of the Endocytic Pathway in MAPK Activation
We next measured MAPK activation in the presence of hypertonic sucrose, which permits the interaction between the TRH receptor and ß-arrestin (Fig. 2Go) but prevents clathrin mobilization and endocytosis (23, 30). Hypertonic sucrose delayed internalization of the TRH receptor, measured by the conversion of receptor-bound [3H]MeTRH to an acid/salt-resistant form (Fig. 1Go) and fluorescence microscopy using Rhod-TRH (23). Sucrose does not impair the ability of TRH to stimulate phosphoinositide turnover or a calcium response (24). In 293TRHR cells, hypertonic sucrose treatment increased basal MAPK activity slightly by itself but severely inhibited activation of MAPK by TRH, EGF, or TPA (Fig. 5aGo). Sucrose also abrogated activation of MAPK by diverse signals in cells expressing the truncated receptor. We also used concanavalin A, a lectin, to block the endocytic pathway. Concanavalin A slowed [3H]MeTRH internalization (Fig. 1Go), although it was not as effective as sucrose, and it inhibited MAPK responses to TRH, EGF, and TPA (Fig. 5bGo).

Dominant negative forms of dynamin (K44A) or ß- arrestin (319–418) were also tested for their effects on TRH-induced receptor internalization and MAPK activation (Fig. 6Go). To assess the effectiveness of the dominant negative proteins, we cotransfected HEK293 cells stably expressing an epitope-tagged TRH receptor with plasmids encoding GFP and either K44A dynamin or 319–418 ß-arrestin. The hemagglutinin (HA) epitope was at the amino terminus of the receptor where it would not be expected to interfere with ß- arrestin interactions. We then exposed cells to vehicle or TRH for 5 min, and fixed and localized receptors immunocytochemically with antibody against the HA epitope. Receptor localization was measured in GFP-positive cells, since these had been successfully transfected. In cells transfected with the control plasmid, receptor was predominantly on the plasma membrane of 82% of untreated cells but only 35% of cells treated with TRH for 5 min (Fig. 6aGo). In cells transfected with either K44A dynamin or 319–418 ß-arrestin, TRH did not change the fraction of cells with predominantly cell surface receptor, indicating that the dominant negative proteins had blocked ligand-driven receptor internalization. When internalization was assessed by conversion of receptor-bound [3H]MeTRH to an acid/salt resistant form (Fig. 6bGo), dominant negative dynamin and ß-arrestin could still be seen to inhibit internalization (Fig. 6bGo), but their effects were less pronounced. Much of the receptor-bound [3H]MeTRH was acid/salt resistant in cells expressing K44A dynamin and 319–418 ß-arrestin even though by microscopy the receptor appeared to be on the plasma membrane. This suggests that the hormone-receptor complex forms a stable, acid/salt-resistant complex before it moves into an endosome, and that dominant negative dynamin and ß-arrestin block at a late step after the receptor has become acid stable.



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Figure 6. Effect of Dominant Negative Dynamin and ß-Arrestin on TRH Internalization and MAPK Activation

A, HEK293 cells stably expressing an epitope-tagged TRH receptor were transiently transfected with 0.6 µg GFP and with 3 µg of either pcDNA3, K44A dynamin, or 319–418 ß-arrestin and grown for 24 h. Cells were serum-starved overnight and then treated with vehicle or 1 µM TRH for 5 min, fixed, and stained with monoclonal antibody against the HA epitope. Slides were coded and then evaluated by an observer unaware of treatment group. Cells positive for GFP fluorescence (~half of the cells) were divided into those with receptor clearly localized on the plasma membrane and those with receptor localized partially or entirely intracellularly. B, HEK293 cells were transiently transfected with 1 µg TRH receptor and 3 µg pcDNA3, K44A dynamin, or 319–418 ß-arrestin. Dishes were incubated with 5 nM [3H]MeTRH for 1 h at 0 C in HBSS and washed. The fraction of specifically bound [3H]MeTRH internalized was determined before and after a subsequent 5-min incubation at 37 C based on resistance to an acid/salt wash. C, HEK293 cells were transfected with 1 µg TRH receptor and 3 µg pcDNA3 (lanes 1 and 2), or with 1 µg TRH receptor and 3 µg K44A dynamin (lanes 3 and 4) or 3 µg 319–418 ß-arrestin (lanes 5 and 6). The next day, cells were changed to medium lacking serum 16 h before stimulation. Dishes were rinsed with HBSS and treated with or without 10 nM TRH for 5 min and assayed for activated MAPK (upper blot) or total MAPK (lower blot).

 
Dominant negative dynamin and dominant negative ß-arrestin both blunted the MAPK response to 10 nM TRH (Fig. 6cGo). Neither dominant negative protein decreased total MAPK, which is shown in the lower blot. Based on densitometry, 10 nM TRH stimulated an average 2.4-fold increase in phosphorylation of ERK1/2 in these experiments (n = 4). K44A dynamin and 319–418 ß-arrestin both reduced basal levels of activated MAPK. K44A dynamin reduced the TRH-stimulated increase in phosphorylated MAPK to an average of 39 ± 14% of control and 319–418 ß-arrestin to 82 ± 19% of control.

Pathways leading from EGF, TPA, and TRH to MAPK activation are believed to converge at raf-1, where PKC acts directly. Because hypertonic sucrose blocks MAPK activation by all three agents, it presumably inhibits at raf-1 or a site distal to it. To pinpoint the site, we determined whether hypertonic sucrose prevented MEK from activating MAPK. In control 293TRHR cells, basal MAPK activity was low and was slightly increased by hypertonic sucrose (Fig. 5cGo). When MEK1 was overexpressed, basal MAPK activity was very high, as expected (Fig. 5cGo), and was not inhibited by addition of hypertonic sucrose over 60 min. In contrast, the MEK inhibitor PD98059 inhibited MAPK rapidly and significantly in cells overexpressing MEK (Fig. 5cGo). These results indicate that hypertonic sucrose does not prevent MEK from activating MAPK and imply that sucrose instead inhibits raf-1 activation of MEK.

Importance of PKC, PI3K, and EGF Receptor Kinase
Because ß-arrestin interaction and receptor endocytosis were not required for MAPK activation by TRH, we examined the requirement for PKC, which has been shown to be responsible for 50–60% of MAPK activation in pituitary and COS cells (2, 3, 4). Overnight treatment with a high concentration of a phorbol ester, which down-regulates some isoforms of PKC, prevented activation of MAPK by 10 nM TRH in cells expressing intact or truncated TRH receptors (Fig. 5dGo). Down-regulation of PKC had no effect on the TRH-induced translocation of ß-arr-GFP (data not shown). Staurosporine and GF109203X (bisindolylmaleimide I), two PKC inhibitors, blocked MAPK activation by 10 nM TRH and partially inhibited MAPK activation by 1 µM TRH in 293TRHR cells (data not shown).

PD98059, a selective MEK inhibitor, effectively blocked MAPK activation by TRH, TPA, and EGF (Fig. 7aGo). To assess the requirement for ras in TRH activation of MAPK, we expressed a dominant negative form of ras, N17 ras. N17 ras markedly reduced EGF activation of MAPK but had relatively little effect on the TRH response (Fig. 7bGo). Wortmannin and LY294002, both inhibitors of PI3K, block MAPK activation by some GPCRs (31). Wortmannin has been reported to inhibit TRH activation of MAPK in pituitary GH3 cells (1). These inhibitors had no effect on TRH activation of MAPK but significantly reduced stimulation by serum (Fig. 7cGo). The specific tyrphostin AG1478, an inhibitor of the tyrosine kinase activity of the EGF receptor (32), has been reported to prevent MAPK activation by some receptors coupled to Gq (33). AG1478 inhibited EGF but not TRH stimulation of MAPK activity (Fig. 7dGo). Likewise, the general tyrosine kinase inhibitor, genistein, inhibited EGF but not TRH activation. TRH activation of MAPK was completely insensitive to pertussis toxin (Fig. 7eGo).



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Figure 7. Involvement of ras, PI3K, and the EGF Receptor in TRH Activation of MAPK in 293TRHR Cells

a, Cells were incubated with vehicle, 1 µM TRH, 1 µM TPA, or 10 nM EGF for 5 min; in the right lanes, 50 µM PD98059 was added 30 min before other drugs. b, Cells were mock transfected or transfected with N17ras and then incubated in serum-free medium overnight, placed in HBSS at 37 C, and treated with vehicle, 0.5 nM EGF (EGF 0.5), 1 nM EGF (EGF 1), or 10 nM TRH (TRH) for 5 min. Similar results were obtained in experiments where 10 nM EGF was used. c and d, Cells were serum starved overnight and then placed in HBSS and incubated for 10 min with no drug, 100 nM wortmannin, 25 µM LY294002, or 100 nM AG1478, as shown. Cells were then treated for 5 min with vehicle, 10 nM TRH, 10 nM EGF, or 30% FCS. In the lower blot, cells were incubated with 100 µM genistein for 30 min and then exposed to 10 nM EGF or 1 µM TRH for 5 min. e, Cells were incubated with 50 ng/ml pertussis toxin overnight, placed in HBSS at 37 C, and treated with vehicle or 1 µM TRH for 5 min.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GPCRs have been classified according to their ability to bind various arrestins, a characteristic dictated by the cytoplasmic carboxy-terminal region of the receptor (34). The pituitary TRH receptor falls into class B, the group of receptors able to bind all three arrestins, ß-arrestin1, ß-arrestin2, and visual arrestin. Groarke and co-workers (25) showed that ß-arrestin cointernalizes with TRH receptors in HEK293 cells, and we found that it cointernalizes with rhodamine-labeled TRH (Fig. 3Go). Taken together, these results indicate that it is a complex containing at least hormone, TRH receptor, and ß-arrestin that internalizes in HEK293 cells. We have found that TRH also causes translocation of ß-arr-GFP to the membrane and then to vesicles in GH3 cells, which express endogenous TRH receptors.1 A large number of GPCRs have been shown to interact with ß-arrestin; in some cases (ß2-adrenergic, dopamine D1A, and endothelin ETA receptors), the ß-arrestin does not internalize with the receptor, whereas in others (neurotensin and angiotensin AT1A receptors) it does (34, 35). In contrast, ß-arrestin remains on the plasma membrane and does not move into endocytic vesicles with rhodamine-labeled TRH when the TRH receptor is expressed in Fq/11 fibroblasts from mice lacking G{alpha}q and G{alpha}11 (36). The difference between Fq/11 and pituitary and HEK293 cells probably results from either the low level of receptor expression in the Fq/11 cells or differences in the pools of endogenous ß-arrestin.

TRH is capable of activating MAPK just as effectively through a truncated receptor, which does not bind ß-arrestin or internalize, as through the full-length receptor, proving that receptor-ß-arrestin interaction is not necessary for the activation of MAPK by TRH. Similar results have been reported for several other GPCRs (18, 19, 20, 21, 22). Interaction of activated receptor with ß-arrestin is also not sufficient for MAPK activation by TRH, because the wild-type TRH receptor could not activate MAPK after PKC was down-regulated even though ß-arrestin translocation was intact. We cannot formally eliminate the possibility that the wild-type TRH receptor activates MAPK through a complex containing ß-arrestin and the truncated TRH receptor activates MAPK by a different, arrestin-independent mechanism. In fact, this appears to be the case for a mutated form of the proteinase-activated receptor 2 (17). However, the finding that intact and truncated TRH receptors both require active PKC suggests that they activate MAPK by the same pathway. Our results on MAPK activation by the TRH receptor differ from the results reported for the ß2-adrenergic receptor, wild-type proteinase-activated receptor 2, and M1 muscarinic receptor, which must interact with ß-arrestin to activate src and subsequently MAPK (13, 17, 37). Based on currently available data, it appears that the role of receptor-ß-arrestin interaction in MAPK is receptor- and perhaps cell type-specific.

Hypertonic sucrose prevents the recruitment of clathrin and interferes with normal coated pit formation and endocytosis (30). Because hypertonic sucrose and concanavalin A, another inhibitor of endocytosis, inhibited MAPK activation by internalization-defective as well as intact TRH receptors, these inhibitors did not block MAPK activation by inhibiting TRH receptor internalization per se. Instead, our results suggest that even though endocytosis of the TRH receptor itself is not essential for TRH activation of MAPK, early steps in the endocytic pathway may be required. Hypertonic sucrose and concanavalin A would both be expected to prevent the assembly of proteins in coated pits, and both treatments caused a global inhibition of MAPK activation, reducing the responses to activators of a GPCR, a receptor tyrosine kinase and PKC. Dominant negative dynamin would be expected to block conversion of coated pits to coated vesicles, and it too inhibited TRH activation of MAPK. Because sucrose, concanavalin A, and K44A dynamin inhibit the endocytic pathway by very different mechanisms, it seems likely that they inhibited MAPK activation as a result of their effects on clathrin-dependent endocytosis and not because of other actions. The results are consistent with the idea that proteins in the MAPK cascade need to assemble at coated pits or endocytic vesicles for effective MAPK activation (19, 38), although the TRH receptor itself does not need to bind ß-arrestin or undergo internalization. Such a model is a variant on schemes that envision assembly of GPCR, ß-arrestin, src, and proteins in the MAPK cascade in endosomes.

Since the pathways leading to MAPK activation by EGF and phorbol esters converge on raf-1, and TRH activation of MAPK requires PKC, which acts at the level of raf-1, hypertonic sucrose appears to inhibit at raf-1 or a site distal to it. Daaka and co-workers (37) showed that raf-1 activation is not prevented by inhibiting clathrin-dependent endocytosis, and we showed that MAPK activation caused by overexpression of MEK is not prevented by hypertonic sucrose. These findings suggest that hypertonic sucrose blocks the MAPK cascade at the level of raf activation of MEK. This conclusion appears to conflict with results of Kranenburg and co-workers (19), who reported that dominant negative dynamin abrogates activation of MAPK by multiple signals but does not prevent activation of ras, raf, or MEK. Instead, they find that dominant negative dynamin blocks the transfer of phosphorylated, active MEK from the plasma membrane to the cytosol in endocytic vesicles. Our results are compatible with this model if overexpression of MEK eliminates the need for clathrin-dependent translocation of activated MEK. Some GPCRs signal to MAPK by causing activation of the EGF receptor, which must undergo endocytosis for MAPK stimulation (33, 39), and others via src (40, 41, 42). However, TRH does not signal by these pathways because MAPK responses are completely insensitive to the tyrphostin AG1478 and to the broad specificity tyrosine kinase inhibitor genistein. It remains unclear why endocytosis is essential for MAPK activation in some systems but not in others (13, 16, 18, 19, 21, 22, 37).

TRH acts via Gq/11 to activate PLC and increase the concentration of diacylglycerol, which activates PKC, and IP3, which releases calcium. The fact that prolonged phorbol ester pretreatment nearly eliminated the MAPK response to TRH in HEK 293 cells indicates that PKC isoforms subject to down-regulation are critical for the MAPK response in this cell type. PKC has been reported to account for about two-thirds of MAPK activation by TRH in pituitary cells (2) and COS-7 cells (3). In COS-7 cells, the PKC-independent activation of MAPK by TRH appears to be insensitive to pertussis toxin, independent of ras activation, but dependent on G protein ß{gamma}-subunits (3). We have found that the truncated TRH receptor can activate MAPK in COS-7 cells (data not shown), indicating that an interaction with ß-arrestin is again not necessary.

In summary, we have shown that TRH activates MAPK in a manner that does not require interaction between the receptor and ß-arrestin but does require PKC. Early steps in the endocytic pathway appear to be important for MAPK activation by multiple signal pathways. Our findings emphasize the diversity in signal transduction pathways used by members of the GPCR superfamily to activate the MAPK cascade.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
HBSS and LipofectAmine were purchased from Life Technologies, Inc. (Gaithersburg, MD). Plasmids encoding a conjugate pß-arr-GFP protein (12) and K44A dynamin were gifts from Dr. Marc Caron (Duke University, Durham, NC). Other plasmids were generously provided by the following: N17ras from Dr. Ian Macara (University of Virginia, Charlottesville, VA), intact and truncated TRH receptors from Marvin C. Gershengorn (Weill Medical College of Cornell University, New York, NY), and 318–419 ß-arrestin from Dr. Jeffrey L. Benovic (Thomas Jefferson University, Philadelphia, PA). A plasmid encoding an HA-tagged TRH receptor was constructed by PCR, adding two HA sequences separated by a Gly after the initial Met, and inserted in pcDNA3.2 Plasmid encoding MEK1 was purchased from Stratagene (La Jolla, CA) and plasmid encoding GFP was from CLONTECH Laboratories, Inc. (Palo Alto, CA). Rhod-TRH was synthesized as previously described (23). Antibodies against phospho-MAPK and total MAPK were from Cell Signaling Technology (Beverly, MA). TRH, GF1095203X, and PD98059 were from Calbiochem (San Diego, CA); cell attachment matrix was from Upstate Biotechnology, Inc. (Lake Placid, NY); and pertussis toxin, genistein, EGF, concanavalin A, staurosporine, and TPA were from Sigma (St. Louis, MO). LY294002 and wortmannin were purchased from BioMol (Plymouth Meeting, PA). Horseradish peroxidase- labeled antibody to rabbit IgG and [3H]MeTRH (64 Ci/mmol) were obtained from Dupont/NEN Life Science Products (Boston, MA); and enhanced chemiluminescence reagent was from Amersham Pharmacia Biotech (Arlington Heights, IL).

Cell Culture and Transfection
Properties of HEK293 cells stably expressing the wild-type mouse TRH receptor (293TRHR cells) and HEK293 cells stably expressing an internalization-defective, truncated mouse TRH receptor (Cys 335 converted to a stop codon) (293TRHR-C335Stop cells) have been described previously (36). HEK293 cell lines were grown in DMEM supplemented with 5% FBS and transfected with LipofectAmine according to the manufacturer’s instructions. After transient transfection, between 20 and 60% of cells expressed detectable GFP or ß-arr-GFP. We also carried out several experiments to estimate the efficiency of cotransfection. When cells were cotransfected with plasmids encoding epitope-tagged receptors and GFP at ratios of >=3:1, 95% of the GFP-positive cells also stained for the receptor by immunocytochemistry.

Microscopy
293TRHR and 293TRHR-C335Stop cells were transfected with pß-arr-GFP (1 µg/6 cm dish). Cells were then split onto ECL-coated coverslips. On the second or third day after transfection, cells were stimulated at 37 C for up to 20 min with 1 µM TRH in normal HBSS buffered with 15 mM HEPES to pH 7.4 (HBSS) or buffered HBSS supplemented with 0.45 M sucrose and localization of ß-arr-GFP was followed microscopically. In some experiments, cells were coincubated with Rhod-TRH (1:100, ~400 nM, in HBSS) at 37 C for 25 min. Cells expressing an HA-tagged TRH receptor were fixed with paraformaldehyde and incubated with a 1:1,000 dilution of monoclonal antibody to the HA epitope (Covance Laboratories, Inc., Princeton, NJ) followed by a 1:500 dilution of goat rhodamine-labeled antimouse IgG (American Qualex, LaMiranda, CA), as previously described (23).

GFP fluorescence was detected with fluorescein filters and Rhod-TRH and secondary antibody with rhodamine filters, using a 40x objective on a Nikon inverted microscope (Nikon, Melville, NY) with a Micromax camera (Princeton Instruments, Trenton, NJ). Bleedthrough was negligible under the conditions used. Images were analyzed using Metamorph software (Universal Imaging, Media, PA).

Activation of MAPK
293TRHR and 293TRHR-C335Stop cells plated on coated 35- or 60-mm dishes were serum-starved for 24 h before stimulation. In other experiments HEK293 cells were transiently transfected with receptor and/or dominant negative constructs, grown in complete medium for 1 d and then serum-starved overnight before use. Cells were then washed with HBSS and incubated with TRH or other drugs in HBSS at 37 C, usually for 5 min, placed on ice, and immediately lysed with SDS-PAGE sample buffer (150 µl/6 cm dish). Equal amounts of sample (10 to 20 µl) were loaded into each lane of 10% SDS-PAGE gels. After electrophoresis, proteins were transferred to nitrocellulose membranes, and the membranes were blocked and probed with phospho-MAPK- specific polyclonal antibody (1:1,000) and subsequently with goat antirabbit IgG horseradish peroxidase (1:5,000) and visualized with enhanced chemiluminescence reagents. Samples of the cell extracts were run on separate gels and blotted with antibody against total MAPK to ensure that the amounts of MAPK protein were equal within each treatment group. All experiments have been replicated two to ten times with comparable results.

TRH Binding
To measure receptor density, cells plated on replicate 35-mm dishes were incubated for 1 h in HBSS containing 10 nM [3H]MeTRH, and specific binding was determined as previously described (26). Protein was determined by the Lowry method using BSA as standard. Radioligand internalization was determined by measuring the fraction of specifically bound [3H]MeTRH resistant to washing with ice-cold 0.2 N acetic acid/0.5 M NaCl (26). Nonspecific binding, measured in the presence of a 1,000-fold excess of unlabeled hormone, has been subtracted from all points.


    ACKNOWLEDGMENTS
 
We are grateful to Caroline Perkowski and John Puskas for excellent technical assistance.


    FOOTNOTES
 
This work was supported in part by NIH Grants DK-19974 and Cancer Center Core Research Grant CA-11098. R.Y. was supported by a Wilmot Fellowship.

Abbreviations: ß-arr-GFP, ß-arrestin-green fluorescent protein conjugate; EGF, epidermal growth factor; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; MEK, MAPK kinase; Rhod-TRH, rhodamine-labeled TRH; TPA, 12-O-tetradecanoylphorbol 13-acetate.

1 Yu, R., and P. M. Hinkle, unpublished results. Back

2 Zhu, C.-C., and P. M. Hinkle, manuscript in preparation. Back

Received for publication January 28, 2000. Accepted for publication May 29, 2001.


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