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Originally published In Press as doi:10.1074/jbc.M413393200 on December 17, 2004

J. Biol. Chem., Vol. 280, Issue 12, 11281-11288, March 25, 2005
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Parathyroid Hormone Receptor Trafficking Contributes to the Activation of Extracellular Signal-regulated Kinases but Is Not Required for Regulation of cAMP Signaling*

Colin A. Syme{ddagger}, Peter A. Friedman§, and Alessandro Bisello{ddagger}§

From the {ddagger}Division of Endocrinology and Metabolism, Department of Medicine and the §Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Received for publication, November 29, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Agonist-mediated activation of the type 1 parathyroid hormone receptor (PTH1R) results in several signaling events and receptor endocytosis. It is well documented that arrestins contribute to desensitization of both Gs- and Gq-mediated signaling and mediate PTH1R internalization. However, whether PTH1R trafficking directly contributes to signaling remains unclear. To address this question, we investigated the role of PTH1R trafficking in cAMP signaling and activation of extracellular signal-regulated kinases ERK1/2 in HEK-293 cells. Dominant negative forms of dynamin (K44A-dynamin) and {beta}-arrestin1 ({beta}-arrestin1-(319–418)) abrogated PTH1R internalization but had no effect on cAMP signaling; neither acute cAMP production by PTH nor desensitization and resensitization of cAMP signaling were affected. Therefore, PTH1R trafficking is not necessary for regulation of cAMP signaling. PTH-(1–34) induced rapid and robust activation of ERK1/2. A PTHrP-based analog ([p-benzoylphenylalanine1, Ile5,Arg11,13,Tyr36]PTHrP-(1–36)NH2), which selectively activates the Gs/cAMP pathway without inducing PTH1R endocytosis, failed to stimulate ERK1/2 activity. Inhibition of PTH1R endocytosis by K44A-dynamin dampened ERK1/2 activation in response to PTH-(1–34) by 69%. Incubation with the epidermal growth factor receptor inhibitor AG1478 reduced ERK1/2 phosphorylation further. In addition, ERK1/2 phosphorylation occurred following internalization of a PTH1R mutant induced by PTH-(7–34) in the absence of G protein signaling. Collectively, these data indicate that PTH1R trafficking and Gq (but not Gs) signaling independently contribute to ERK1/2 activation, predominantly via transactivation of the epidermal growth factor receptor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Parathyroid hormone (PTH)1 is the primary regulator of serum calcium homeostasis and bone metabolism. In response to low blood calcium levels, PTH is released into the circulation and acts primarily on the receptor for the PTH/PTH-related peptide (PTHrP) in bone and kidney, PTH1R (1). PTHrP was first described as the hormone responsible for hypercalcemia of malignancy (2–4) and has subsequently been described as having a role in both cell proliferation and differentiation (5).

The identification of the PTH1R as a seven-transmembrane class II G protein-coupled receptor (GPCR) (6) was followed shortly thereafter by the elucidation of associated signaling mechanisms (7, 8). Upon stimulation with either PTH or PTHrP, a G protein-mediated cascade of events occurs via Gs and Gq, resulting in activation of the cAMP/protein kinase A and phospholipase C/protein kinase C pathways, respectively. In turn, these events lead to receptor phosphorylation (9–11) and {beta}-arrestin recruitment coupled to receptor desensitization (12–14). Agonist-receptor complex internalization then occurs via clathrin-coated pits in a {beta}-arrestin-dependent manner (12, 15).

Many of the actions of PTH are mediated by cAMP, including its well established skeletal anabolic action (16). We have demonstrated that regulation of cAMP production by the PTH1R is predominantly under the control of {beta}-arrestins (12, 15). PTH1R activation is rapidly followed by arrestin recruitment to the cell membrane and the consequent receptor desensitization (12, 15, 17). Although arrestin-mediated PTH1R desensitization is probably independent of receptor internalization, this has not been firmly demonstrated. In fact, whether PTH1R trafficking contributes at all to the ability of the receptor to generate cAMP is at present unknown.

Many cellular effects of PTH and PTHrP are also partly dependent on the activation of the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (ERK1/2). Activation of ERK1/2 has been described in various target cells, including osteoblasts, bone marrow stromal cells, and distal/proximal convoluted tubule kidney cells. In these cells, ERK1/2 activation is associated with differentiation, proliferation, survival, and calcium transport (18–24). The molecular mechanisms mediating regulation of ERK1/2 by PTH and PTHrP have been examined by several investigators. Goltzman (21) and Partridge (22) and their respective co-workers independently found that PTH1R-mediated ERK1/2 activation is protein kinase C-dependent. In contrast, cAMP-mediated attenuation of ERK1/2 activation observed in osteoblasts, stromal cells, and osteogenic cells results in anti-proliferative actions (18, 25) and apoptosis (23, 26). In addition, transactivation of the ubiquitously expressed epidermal growth factor receptor (EGFR) has been shown to contribute to PTH1R-mediated ERK1/2 activation (24). Clearly, a balance exists between Gs- and Gq-mediated signaling pathways and EGFR transactivation in ERK1/2 activation. However, it still remains unclear whether activation of ERK1/2 occurs via trafficking-dependent or -independent mechanisms. Furthermore, the mechanisms that couple G protein-mediated signaling and receptor trafficking to the underlying activation of MAPKs are yet to be fully elucidated.

Here, we determine that PTH1R endocytosis occurs exclusively via both a {beta}-arrestinand a dynamin-dependent mechanism in HEK-293 cells. Although PTH1R trafficking is not required for the regulation of cAMP signaling, we demonstrate that arrestin- and dynamin-dependent PTH1R internalization and Gq signaling independently contribute to ERK1/2 activation, predominantly by transactivation of the EGFR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Anti-p44/p42 MAPK (ERK1/2) and phospho-p44/p42 MAPK (pERK1/2) (Thr202/Tyr204) rabbit polyclonal antibodies and a horseradish peroxidase-conjugated goat anti-rabbit antibody were purchased from Cell Signaling Technology (Beverly, MA). Alexa Fluor 546-tagged goat anti-rabbit antibody was obtained from Molecular Probes (Eugene, OR). Tyrphostin AG1478 and protease inhibitor mixture set I were obtained from Calbiochem. Dulbecco's modified Eagle's medium was from Cambrex BioScience (Walkersville, MD). Both fetal bovine serum and penicillin-streptomycin were from Invitrogen. Human epidermal growth factor (EGF) and all other reagents were from Sigma-Aldrich.

Cell Culture—Human embryonic kidney HEK-293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified 5% CO2, 95% air incubator at 37 °C. Cells were plated at 1.0 x 105 cells/well in 24-well plastic dishes (Corning Inc., Corning, NY) for adenylyl cyclase, radioligand binding, and radioligand internalization assays and transfected as described previously (15). Cells were plated at 1.0 x 105 cells/well in 6-well plastic dishes (Costar, Corning, NY) for MAPK assays, transfected, and then serum-starved overnight prior to performing the assay. All subsequent experiments were performed 24 h after transfection.

Peptides—The synthesis, purification, and characterization of [Nle8,18,Tyr34]bovine PTH-(1–34)-NH2 (abbreviated PTH-(1–34)), [p-benzoylphenylalanine1,Ile5,Arg11,13,Tyr36]PTHrP-(1–36)-NH2 (abbreviated Bpa1-PTHrP-(1–36)), PTH-(7–34)-NH2, and rhodamine (Rho)-labeled PTH-(1–34) and PTH-(7–34) were performed as described previously (12). The pure products were characterized by analytical high pressure liquid chromatography, electron spray mass spectrometry, and amino acid analysis. Radioiodination and high pressure liquid chromatography purification of PTH-(1–34) were carried out as described previously (12).

Radioligand Binding and Internalization—Radioligand receptor binding and internalization assays were performed as described previously (19, 31) using high pressure liquid chromatography-purified [125I-Nle8,18,Tyr34]PTH(1–34)-NH2. The binding affinity of PTH-(1–34) in the presence or absence of either wild-type or dominant negative dynamin was determined by Scatchard analysis of radioreceptor binding assays performed in triplicate as described previously (12, 15).

Adenylyl Cyclase Activity—Cyclic AMP accumulation was determined in subconfluent cell cultures in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) as described previously (15, 27). Briefly, cells were pretreated with either vehicle (0.1% BSA in PBS) or PTH-(1–34) for 30 min followed by a 2-h washout at 37 °C in cell culture medium containing [3H]adenine (2 µCi/ml). Subsequently, the acute response and resensitization of cAMP signaling was determined in the presence of IBMX (1 mM) by PTH-(1–34) stimulation for 30 min of unstimulated cells and PTH-(1–34)-pretreated cells, respectively. Residual cAMP accumulation was measured for 30 min in the presence of IBMX (1 mM) in PTH-(1–34)-pretreated cells. In all cases, reactions were stopped with 1.2 M trichloroacetic acid, and cAMP was isolated by the two-column chromatographic method. To prevent de novo synthesis of PTH1R, in some experiments cells were preincubated for 2 h with cycloheximide (25 µg/ml) at 37 °C and maintained in the presence of cycloheximide for the duration of the experiment.

ERK1/2 Phosphorylation Assays—Cells were incubated for 18–24 h in Dulbecco's modified Eagle's medium, 0.1% BSA. Peptide treatments were carried out for the times indicated (2–30 min), after which cells were washed once in ice-cold PBS and solubilized in 300 µl of Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 8) supplemented with protease inhibitor mixture set I and the phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM glycerophosphate). Cell lysates were incubated for 20 min and subsequently centrifuged at 12,000 rpm for 10 min at 4 °C. Cellular debris was then removed, cell lysates were resuspended in 2x Laemmli buffer, and resolved by electrophoresis on a 12% SDS-polyacrylamide gel. The proteins were transferred to nitrocellulose at 250 mA for 1.5 h at 4 °C. Blots were blocked for 1 h at room temperature using Tris-buffered saline blocking solution containing 5% (w/v) milk powder, 0.1% v/v Tween 20. Membranes were immunoblotted with 1:1000 phospho-p44/p42 MAPK (Thr202/Tyr204) rabbit polyclonal antibody for 1 h at room temperature, washed extensively, and incubated in secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG; 1:2000). After the detection of phosphorylated ERK1/2 bands by chemiluminescence and autoradiography, the membranes were stripped with 1 M glycine, pH 2.3–2.6 for 30 min at room temperature, rinsed with wash buffer, and immunoblotted as above with 1:1000 anti-p44/p42 MAPK rabbit polyclonal antibody. Autoradiographs were digitized, and subsequent analyses were performed with Image J software (National Institutes of Health).

Fluorescence Microscopy—For fluorescence microscopy studies with Rho-PTH-(1–34) and Rho-PTH-(7–34), HEK-293 cells were plated on 25-mm glass coverslips (1.5 x 105 cells/coverslip), transfected with the indicated GFP-tagged PTH1Rs as described above, and cultured for 24 h. Coverslips were then rinsed twice in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and incubated with the fluorescent peptides (100 nM each) for 15 min at 37 °C. The unbound ligands were removed by washing three times with cold PBS, and coverslips were fixed using 2% paraformaldehyde for 30 min on ice. For immunofluorescence studies of ERK1/2 activation, HEK-293 cells transiently expressing the GFP-tagged PTH1R (PTH1R-GFP) were grown on glass coverslips as described above. Cells were then treated as detailed in the figure legends (Figs. 1, 2, 3, 4, 5, 6 and 8) with 100 nM PTH-(1–34), transferred to ice, rinsed in PBS, and fixed and permeabilized using a 2% paraformaldehyde, 0.2% Triton X-100 solution for 30 min. Blocking was performed by incubating the cells for 10 min at room temperature in 4% fetal bovine serum. Primary antibody (anti-phospho-p44/p42 MAPK (Thr202/Tyr204) rabbit polyclonal antibody) diluted 1:250 in PBS plus 1% BSA was applied to the specimens for 1 h at room temperature followed by three washes with the same buffer. Alexa Fluor 546-tgged secondary antibody was diluted 1:200 and applied under the same conditions as the primary antibody. Coverslips were then rinsed in PBS and mounted for immunofluorescence microscopy. Coverslips were analyzed with a Zeiss LSM5 Pascal confocal microscope with a 63x oil immersion objective (Carl Zeiss, Thornwood, NY).



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  		FIG. 1.
Effect of {beta}-arrestin1-(319–418) on agonist-stimulated PTH1R internalization and cAMP accumulation. A, internalization of radiolabeled PTH-(1–34). HEK-293 cells were transiently cotransfected with PTH1R (0.2 µg DNA/well) and either pcDNA3 ({square}) or {beta}-arrestin1 (319–418) ({blacksquare})) (0.5 µg DNA/well). Each point represents the mean percentage ± S.E. of total cell-associated ligand from triplicate experiments. Average values of all experiments for internalization at 45 min were as follows: PTH1R plus pcDNA3, 82 ± 8% of the total cell-associated radioligand; PTH1R plus {beta}-arrestin1 (319–418), 34 ± 1% (*, p < 0.005 compared with PTH1R + pcDNA3). B, a dominant negative form of {beta}-arrestin had no effect on cAMP signaling. HEK-293 cells transiently expressing PTH1R (0.2 µg DNA/well) and either pcDNA3 ({square}) or {beta}-arrestin (319–418) ({blacksquare}) (0.5 µg DNA/well) were treated with either vehicle (0.1% BSA) or PTH-(1–34) (100 nM) for 30 min followed by a 2-h washout at 37 °C. Re-challenged and residual cAMP accumulation was then measured for 30 min in the presence of IBMX (1 mM) with or without re-exposure to PTH-(1–34), respectively. Results are presented as mean ± S.E. from three independent experiments (*, p < 0.005 compared with acute PTH-(1–34) response).

 



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  		FIG. 2.
Effect of K44A-dynamin on agonist-stimulated PTH1R internalization and cAMP accumulation. A, internalization of radiolabeled PTH-(1–34). HEK-293 cells were transiently cotransfected with PTH1R (0.2 µg DNA/well) and either pcDNA3 ({square}) or K44A-dynamin ({blacksquare}) (0.5 µg DNA/well). Average values of all experiments for internalization at 45 min were as follows: PTH1R plus pcDNA3, 78 ± 4% of the total cell-associated radioligand; PTH1R plus K44A, 30 ± 1% (*, p < 0.005 compared with PTH1R plus pcDNA3). B, a dominant negative form of dynamin had no effect on cAMP signaling. HEK-293 cells transiently expressing PTH1R (0.2 µg DNA/well) and either pcDNA3 ({square}) or K44A-dynamin ({blacksquare}) (0.5 µg DNA/well) were treated with either vehicle (0.1% BSA) or PTH-(1–34) (100 nM) for 30 min, followed by a 2-h washout at 37 °C. Re-challenged and residual cAMP accumulation was then measured for 30 min in the presence of IBMX (1 mM) with or without re-exposure to PTH-(1–34), respectively. Cyclic AMP accumulation was also measured in the presence of cycloheximide (25 µg/ml) ({graysqu}) to prevent de novo protein synthesis. Results are presented as mean ± S.E. from three independent experiments. *, p < 0.005 compared with acute PTH-(1–34) response.

 



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  		FIG. 3.
Time course of agonist-stimulated ERK1/2 phosphorylation. HEK-293 cells expressing PTH1R were serum-starved for 18 h and stimulated with either vehicle (control), fetal bovine serum (FBS) (10%), or PTH-(1–34) (100 nM) and incubated at 37 °C for the time shown. A, phosphorylated (top) and total (bottom) ERK1/2 in whole-cell lysates were determined by immunoblotting. A representative time course experiment is shown. B, data from three independent experiments are summarized as mean ± S.E. *, p < 0.05 compared with basal conditions.

 



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  		FIG. 4.
Selective activation of Gs-mediated signaling fails to induce ERK1/2 phosphorylation. HEK-293 cells were transfected with PTH1R (0.8 µg DNA/well). Serum-starved cells were stimulated with 10% fetal bovine serum (FBS), vehicle (Basal), PTH-(1–34) (PTH), or Bpa1-PTHrP-(1–36) (Bpa1-PTHrP) and incubated at 37 °C for the time indicated. Both peptides were used at a concentration of 100 nM. A, phosphorylated (top) and total (bottom) ERK1/2 in whole cell lysates are shown following immunoblotting. B, data are summarized as mean ± S.E. (n = 3). *, p < 0.05 versus 5 min of PTH-(1–34) stimulation.

 



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  		FIG. 5.
Effect of dominant negative dynamin and EGF receptor inhibition on PTH-stimulated ERK1/2 phosphorylation. HEK-293 cells were transfected with plasmids encoding PTH1R (0.4 µg DNA/well) and either empty vector (pcDNA3) or K44A-dynamin (K44A-dyn) (1.0 µg of DNA per well) as indicated. Serum-starved cells were preincubated for 15 min in the absence or presence of tyrphostin AG1478 (AG1478) as indicated, before stimulation for 5 min with vehicle (Basal) or PTH-(1–34) (100 nM). A, phosphorylated (top) and total (bottom) ERK1/2 in whole cell lysates were determined by immunoblotting. B, data are summarized as mean ± S.E. *, p < 0.05 versus pcDNA3.

 



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  		FIG. 6.
Autocrine and paracrine activation of ERK1/2 by PTH. HEK-293 cells were transfected with GFP-tagged PTH1R (0.8 µg of DNA per well). Serum-starved cells were stimulated with vehicle (basal) or PTH-(1–34) (100 nM) and incubated at 37 °C for 5 min. Cells were fixed, permeabilized, and sequentially incubated with the primary (rabbit polyclonal anti-pERK1/2) and secondary (goat anti-rabbit Alexa Fluor 546) antibodies to label phosphorylated ERK1/2. Confocal images are shown for pERK1/2 (left column, red), PTH1R-GFP (middle column, green), and an overlay image (right column) for both basal conditions and 5 min of stimulation with 100 nM PTH-(1–34). Phosphorylated ERK1/2 was detected in cells expressing PTH1R-GFP (arrow a) and in cells devoid of PTH1R-GFP (arrow p).

 



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  		FIG. 8.
Trafficking-stimulated and activation-independent ERK1/2 phosphorylation by PTH1R. A, HEK-293 cells were transfected with either GFP-tagged wild-type or mutant M593A-PTH1R stimulated with Rho-PTH-(1–34) or Rho-PTH-(7–34) (100 nM) for 15 min and analyzed by confocal microscopy. B, HEK-293 cells were transfected with plasmids encoding either PTH1R or M593A-PTH1R (0.8 µg). Serum-starved cells were stimulated for 5 min with vehicle (0), PTH-(1–34) (1–34), or PTH-(7–34) (7–34) at a final concentration of 100 nM. Phosphorylation of ERK1/2 in whole cell lysates was determined using phospho-ERK1/2-specific IgG as described (top). Total ERK1/2 are shown following detection with a specific p44/42 MAPK antibody (bottom). C, data are summarized as mean ± S.E. for pERK/ERK as a percentage of the PTH-(1–34) response. PTH-(1–34)-induced ERK1/2 activation for both the wild-type and the M593A-PTH1R was 4.8 ± 1.4 and 2.3 ± 0.5-fold over basal, respectively.

 
Statistics—Data are presented as means ± S.E. (n), where n indicates the number of independent experiments. Statistical analyses were performed using either a two-tailed Student's t test or one-way analysis of variance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Dominant Negative Forms of {beta}-Arrestin and Dynamin on PTH1R Internalization—Previously described dominant negative forms of {beta}-arrestin1 (28) and dynamin (29) were tested for their effects on PTH-induced PTH1R internalization. PTH1R was cotransfected with either {beta}-arrestin1-(319–418) or K44A-dynamin in HEK-293 cells. Radioligand internalization assays were performed (Figs. 1A and 2A). In control cells (cotransfected with empty vector pcDNA3), PTH1R was rapidly internalized (82 ± 8% after 45 min). In the presence of {beta}-arrestin1-(319–418), receptor endocytosis was significantly inhibited compared with endocytosis under control conditions, such that 45 min after agonist stimulation only 34 ± 1% was internalized (p < 0.005). Similarly, in the presence of K44A-dynamin, receptor endocytosis was significantly inhibited compared with endocytosis under control conditions, such that 45 min after agonist stimulation only 30 ± 1% was internalized (p < 0.005). Furthermore, membrane expression and the affinity of [125I-Nle8,18,Tyr34]PTH(1–34)-NH2 to the PTH1R under control conditions or in the presence of either wild-type or K44A-dynamin were equivalent irrespective of the plasmid combination used (Kd was 13 ± 1, 18 ± 2, and 22 ± 4 nM, respectively).

Effect of Dominant Negative Forms of {beta}-Arrestin and Dynamin on cAMP Signaling—To address whether PTH1R trafficking affected receptor signaling, we investigated the effect of either {beta}-arrestin1-(319–418) or K44A-dynamin on cAMP accumulation in HEK-293 cells (Figs. 1B and 2B). Exposure of HEK-293 transiently expressing PTH1R to PTH-(1–34) (100 nM) for 30 min resulted in a 10-fold increase in acute levels of cAMP over basal levels. Identical cAMP levels were measured in the presence of {beta}-arrestin1-(319–418). In addition, following the 2-h washout period the residual cAMP accumulation was 18 ± 2% and 24 ± 5% of the acute response in the absence or presence of {beta}-arrestin1 (319–418), respectively, indicating that receptor desensitization had occurred (p < 0.005). Similarly, the receptor resensitized both in the absence (83 ± 5%) or presence (93 ± 3%) of {beta}-arrestin1-(319–418) compared with the acute response. Similar results were obtained in the presence of cycloheximide (25 µg/ml) (data not shown). Exposure of HEK-293 transiently coexpressing K44A-dynamin and PTH1R to PTH-(1–34) (100 nM) for 30 min resulted in a 9-fold increase in acute cAMP levels over basal. Following the 2-h washout period, the residual cAMP accumulation was approximately half-maximal that of the acute response in the absence (50 ± 5%) or presence (41 ± 8%) of K44A-dynamin, indicating that receptor desensitization had occurred (p < 0.005). Receptor resensitization was also similar in the absence (117 ± 4%) or presence (120 ± 12%) of K44A-dynamin. In the presence of cyclohexamide (25 µg/ml), K44A-dynamin had no effect on cAMP accumulation; the receptor desensitized (60 ± 8%) and resensitized (117 ± 12%) similarly as under control conditions. Collectively, these data suggest that although {beta}-arrestin and dynamin are required for internalization of PTH1R, PTH1R trafficking is not necessary for regulation of cAMP signaling.

PTH1R-mediated ERK1/2 Activation—PTH-(1–34) stimulated ERK1/2 activation in a time-dependent manner (Fig. 3). Fig. 3A shows an illustrative example of a MAPK assay over a 30-min time period; the data from several experiments are summarized in Fig. 3B. These data demonstrate the rapid activation of ERK1/2 in response to PTH-(1–34). After 2–5 min, ERK1/2 activation reached a peak (p < 0.05 versus basal) and then declined gradually over a 30-min time period.

To begin to elucidate the mechanisms involved in PTH1R-mediated activation of ERK1/2, we utilized a signaling-selective PTHrP-based analog, Bpa1-PTHrP-(1–36), that acts solely via Gs-mediated signaling (30). In addition, it does not internalize PTH1R, because it fails to induce {beta}-arrestin2 mobilization and thereby acts via a mechanism independent of receptor trafficking. We compared the activation of ERK1/2 in response to both PTH-(1–34) and Bpa1-PTHrP-(1–36) (Fig. 4). As described previously, PTH-(1–34) caused a 4.5 ± 0.8-fold activation of ERK1/2 after 5 min that decreased within 30 min (2.6 ± 1.2-fold over basal). In contrast, stimulation with the Gs-selective analog Bpa1-PTHrP-(1–36) for 5 or 30 min failed to induce ERK1/2 phosphorylation (resulting in only a 0.9 ± 0.1-fold and 0.9 ± 0.5-fold increase in ERK1/2 activation over basal, respectively; p < 0.005 versus PTH-(1–34)).

Collectively, these data demonstrate that Gs-mediated signaling is insufficient for ERK1/2 activation. Furthermore, a role for Gq-mediated signaling and PTH1R trafficking in ERK1/2 phosphorylation is implicated by these results.

PTH1R Trafficking and Gq Signaling Contribute to ERK1/2 Phosphorylation via Transactivation of EGFR—Previous reports have implicated the complex involvement of receptor phosphorylation, {beta}-arrestin recruitment, and clathrin-mediated endocytosis in MAPK activation by GPCRs (31–37). Therefore, we investigated the role of PTH1R trafficking in the activation of ERK1/2. In HEK-293 cells transiently expressing PTH1R, PTH-(1–34) (100 nM) stimulated an average 3.9 ± 0.6-fold increase in ERK1/2 phosphorylation over basal (n = 9). Overexpression of K44A-dynamin at levels that abrogated PTH1R endocytosis without affecting signaling (Fig. 2) reduced the PTH-stimulated increase in phosphorylated ERK1/2 to an average of 1.9 ± 0.3 of basal (n = 6; p < 0.05) (Fig. 5), indicating that PTH1R trafficking contributes to ERK1/2 activation. Furthermore, to test whether ERK1/2 activation resulted from transactivation of EGFR (24), we tested the effect of tyrphostin AG1478, a selective EGFR inhibitor, on ERK1/2 activation in response to PTH-(1–34). Tyrphostin AG1478 (100 nM) significantly reduced PTH-induced ERK1/2 activation to an average 1.1 ± 0.2-fold (n = 3). Tyrphostin AG1478 further reduced PTH-induced ERK1/2 phosphorylation in the presence of K44A-dynamin to 0.9 ± 0.2-fold (n = 3).

Further confirmation for the presence of an EGFR-mediated mechanism linking PTH1R stimulation to ERK1/2 phosphorylation was obtained by fluorescence microscopy studies. Fig. 6 shows confocal microscopy images of HEK-293 cells transfected with PTH1R-GFP either under basal conditions or following stimulation with 100 nM PTH-(1–34). Under basal conditions, low levels of phosphorylated ERK1/2 were detected (Fig. 6, top row, left). In addition, PTH1R-GFP was predominantly localized to the plasma membrane (Fig. 6, top row, middle). Following PTH-(1–34) stimulation (100 nM), ERK1/2 activation was observed (Fig. 6, bottom row, left) as an increase of pERK1/2 labeling in parallel with PTH1R internalization (bottom row, middle). Phosphorylated ERK1/2 was predominantly localized to the cytoplasm. Furthermore, whereas pERK1/2 increased in PTH1R-expressing cells (Fig. 6, bottom row, right, arrow a), pERK1/2 activation was also observed in cells devoid of PTH1R (bottom row, right, arrow p), suggesting that both autocrine and paracrine events occurring concomitantly with PTH1R endocytosis contribute to ERK1/2 activation. To specifically address whether transfection affected EGFR function, we examined the direct effect of EGF on ERK1/2 activation under our experimental conditions (Fig. 7). Following 5 min of stimulation with EGF (10 ng/ml), a robust activation of ERK1/2 was observed that could be inhibited by tyrphostin AG1478 (100 nM). However, neither K44A-dynamin nor {beta}-arrestin1-(319–418) affected ERK1/2 activation in response to EGF, indicating that the effects of dominant negative dynamin and arrestin on PTH-stimulated ERK1/2 activation are due to impaired trafficking of PTH1R.



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  		FIG. 7.
Effect of dominant negative forms of dynamin and arrestin on EGF-stimulated ERK1/2 phosphorylation. HEK-293 cells were transfected with 1 µg of DNA per well of empty vector (pcDNA3) or plasmids encoding either K44A-dynamin or {beta}-arrestin1 (318–419) as indicated. Serum-starved cells were preincubated for 15 min in the absence or presence of tyrphostin AG1478 as indicated before stimulation for 5 min with vehicle (basal) or EGF (10 ng/ml). Phosphorylated ERK1/2 (top) and total ERK1/2 (bottom) in whole cell lysates were determined by immunoblotting.

 
Activation-independent ERK1/2 Phosphorylation by PTH1R— We characterized previously a point mutation in the C terminus of the PTH1R where the terminal methionine 593 is replaced by alanine (M593A). This mutant receptor does not require activation for endocytosis to occur in distal convoluted tubule cells (38). To confirm activation-independent PTH1R internalization in HEK-293 cells, we examined the internalization of GFP epitope-tagged wild-type or mutant (M593A) PTH1R in response to 15 min of stimulation with Rho-labeled agonist PTH-(1–34) or antagonist PTH-(7–34) (Fig. 8A). Both wild-type and M593A-PTH1R were rapidly internalized in response to Rho-PTH-(1–34) (100 nM), and both ligand and receptor co-localized intracellularly (Fig. 8A, left column). In contrast, Rho-PTH-(7–34) (100 nM) bound to the wild-type PTH1R-GFP on the cell membrane but did not induce internalization (Fig. 8A, bottom row, right). However, similar to the agonist PTH-(1–34), Rho-PTH-(7–34) (100 nM) stimulated M593A-PTH1R-GFP internalization (Fig. 8A, top row, right). We then examined whether stimulation of M593A-PTH1R by either the PTH agonist or PTH antagonist was able to induce ERK1/2 phosphorylation (Fig. 8, B and C). PTH-(1–34) (100 nM) induced ERK1/2 activation for both the wild-type and the M593A-PTH1R (4.8 ± 1.4 and 2.3 ± 0.5-fold over basal, respectively). PTH-(7–34) (100 nM) had little effect on ERK1/2 phosphorylation in cells expressing PTH1R (24 ± 7% of PTH-(1–34)-induced ERK1/2 activation). In contrast, PTH-(7–34) stimulation of the M593A-PTH1R resulted in robust ERK1/2 activation (77 ± 11% of PTH-(1–34)-induced ERK1/2 activation). These data indicate that induction of PTH1R endocytosis in the absence of detectable G protein-mediated signaling is sufficient to induce ERK1/2 phosphorylation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Several studies from different groups document the observation that upon agonist occupancy, PTH1Rs are internalized and recycle to the cell membrane (12, 14, 15, 39). However, the functional significance of PTH1R trafficking for the overall signaling activity of and cellular responses to PTH and PTHrP remains unclear. The goal of this study was to determine the contribution of PTH1R trafficking to signal transduction in response to PTH. First, we examined the effect of inhibition of PTH1R endocytosis on cAMP activity. In HEK-293 cells, PTH1R endocytosis occurs almost exclusively through an arrestin- and dynamin-dependent pathway, because overexpression of dominant negative forms of either molecule largely inhibits PTH1R internalization. Under these conditions, expression and affinity of the PTH1R for PTH were unchanged, as was the acute cAMP production in response to PTH. Interestingly, desensitization of cAMP signaling was also unaffected by the inhibition of PTH1R internalization, indicating that receptor sequestration does not contribute to desensitization. Rather, these data demonstrate that desensitization of cAMP signaling is exclusively dependent on the interaction between receptor and arrestin at the cell membrane. For many GPCRs, internalization and recycling are fundamental steps for recovering responsiveness to further stimulation (receptor resensitization) (40). Surprisingly, we found this not to be the case for the PTH1R, because inhibition of receptor endocytosis did not affect the ability of desensitized PTH1R to fully resensitize. Therefore, cellular trafficking of PTH1R does not contribute either to desensitization or resensitization of Gs-mediated cAMP activity.

PTH has been shown to regulate activity of MAPKs, specifically the extracellular signal-regulated ERK1/2, in a variety of endocrine target cells, resulting in effects on proliferation, differentiation, or apoptosis (18–26, 41). ERK1/2 activation via GPCRs involves a variety of independent but not necessarily exclusive mechanisms, including the following: (i) receptor endocytosis (31–37); (ii) G protein-mediated signaling (18, 22, 25, 35, 36); and (iii) transactivation of tyrosine kinase receptors (24, 29, 32, 42–44). In particular, for class II GPCRs (including the PTH1R) that are able to couple to multiple G proteins, the contribution of different pathways to ERK1/2 activation is likely to depend on the particular receptor, the cell type, the relative contribution of opposing Gs- and Gq-mediated signaling, and the ability to undergo endocytosis.

To address these questions, we investigated the role of PTH1R signaling and trafficking in the activation of ERK1/2. We have shown previously that modifications of residue 1 in the biologically active N-terminal fragment of PTHrP result in ligands (e.g. PTHrP-(2–36) and Bpa1-PTHrP-(1–36)) that are unable to stimulate Gq/protein lipase C/protein kinase C-mediated signaling but still cause cAMP accumulation as a result of Gs signaling (30). In addition, these analogs fail to stimulate translocation of {beta}-arrestin2 to the cell membrane with a resultant absence of PTH1R endocytosis. Therefore, we used Bpa1-PTHrP-(1–36) as a pharmacological tool to address the role of Gs-mediated signaling in the activation of MAPK. In our studies, selective Gs activation of PTH1R failed to activate ERK1/2. Indeed, several studies have suggested that Gs/cAMP/protein kinase A-mediated signaling inhibits ERK1/2 activity and results in reduced cell proliferation (18, 22, 25, 45). These observations indicate that Gq-mediated signaling and/or receptor trafficking is necessary for activating ERK1/2. To dissect the relative contribution of Gq-mediated signaling and PTH1R trafficking to ERK1/2 activation, we used two distinct approaches.

Inhibition of PTH1R endocytosis by K44A-dynamin, which leaves the ability to signal via G proteins unaffected, resulted in a significant reduction of ERK1/2 activation in response to PTH, demonstrating that receptor endocytosis directly contributes to PTH signaling. In agreement with our findings, several reports have demonstrated that the inhibition of dynamin partially inhibits MAPK activation by GPCRs, including {beta}-adrenergic (31), serotonin 1A (32), {delta}-opioid (43), gonadotropin-releasing hormone (34, 35), and thyrotropin-releasing hormone (36) receptors. On the other hand, to determine directly whether PTH1R endocytosis alone is sufficient to induce ERK1/2 activation, we used a mutant form of PTH1R (M593A-PTH1R) in which the PDZ-binding domain is disrupted by a point mutation of the N-terminal methionine. This mutation results in a receptor that is internalized in an activation-independent manner in response to PTH-(7–34) and naturally circulating PTH-(7–84) (38). In our studies, phosphorylation of ERK1/2 by M593A-PTH1R occurred in response to both PTH-(1–34) and PTH-(7–34). In contrast, ERK1/2 phosphorylation by wild-type PTH1R occurred in response to the agonist PTH-(1–34) only. Therefore, stimulation of PTH1R endocytosis in the absence of G protein-mediated signaling is sufficient to induce ERK1/2 activation. It is noteworthy that both approaches (the inhibition of PTH1R trafficking by K44A-dynamin and the use of the combination of M593A-PTH1R and PTH-(7–34)) resulted in quantitatively comparable effects; in both cases the contribution of PTH1R trafficking is 70–80% of the total ERK1/2 activity. Nonetheless, it is plausible that different cellular environments (such as receptor density, efficacy of Gs versus Gq coupling, and efficiency of the endocytic machinery) would result in differences in the relative importance of the signaling and trafficking components for ERK1/2 activation.

Collectively, these observations indicate that Gq-mediated signaling and receptor endocytosis independently contribute to ERK1/2 activation by the PTH1R. Mechanistically, both signals converge to induce EGF receptor transactivation, because the inhibitor tyrphostin AG1478 abolished ERK1/2 activation by PTH, either alone or in combination with K44A-dynamin. Moreover, and in agreement with the findings of Luttrell and co-workers in primary osteoblasts (24), our evidence indicates that both autocrine and paracrine events occur, presumably originating from the release of soluble factors (including heparin-bound EGF) from the cell surface (known as "ectodomain shedding") following proteolysis by membrane metalloproteases (46). Heparin-bound EGF undergoes shedding in response to the stimulation of Gq-coupled receptors (47–49). Although we demonstrate in HEK-293 cells that cross-talk between EGFR and PTH1R represents the predominant mechanism of PTH1R-mediated activation of ERK1/2, we also show that Gq and trafficking contribute independently to this mechanism. Our data suggest that parallel, yet independent, pathways exist with Gq-mediated and trafficking-dependent components.

In conclusion, we demonstrate that in HEK-293 cells PTH1R endocytosis occurs exclusively via {beta}-arrestin- and dynamin-dependent mechanisms. PTH1R trafficking affects neither acute cAMP signaling nor desensitization and resensitization of Gs signals. In contrast, we show that both Gq signaling and PTH1R trafficking contribute to the activation of ERK1 and ERK2 through the transactivation of EGFR. Given the complexity and variety of mechanisms involved in PTH1R-mediated ERK1/2 phosphorylation, it is not surprising that many effects, in particular the mitogenic activity of PTH and PTHrP, are remarkably cell-specific. Understanding these mechanisms will allow us to determine the overall contributions of each of these components to the physiological effects of PTH and PTHrP in tissues as diverse as bone, kidney, and vasculature.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK-62078 (to A. B.) and DK-54171 (to P. A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, E1140 Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15261. Tel.: 412-648-7347; Fax: 412-648-3290; E-mail: biselloa{at}msx.dept-med.pitt.edu.

1 The abbreviations used are: PTH, parathyroid hormone; Bpa1-PTHrP-(1–36), [p-benzoylphenylalanine1, Ile5, Arg11,13, Tyr36]PTHrP-(1–36)NH2; BSA, bovine serum albumin; EGF, epidermal growth factor; EGFR, EGF receptor; HEK, human embryonic kidney; ERK1/2, extracellular signal-regulated kinases 1 and 2; IBMX, 3-isobutyl-1-methylxanthine; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; pERK, phosphorylated ERK; PTH(1–34), [Nle8,18,-Tyr34]bovine PTH-(1–34)NH2; PTH(7–34), [Nle8,18,D-Trp12,Tyr34]bovine PTH-(7–34)NH2; PTHrP, PTH-related protein; PTH1R, PTH type 1 receptor; Rho, rhodamine; Rho-PTH-(1–34), [Nle8,18,Lys13(N5-carboxymethylrhodamine)-L-2-Nal23,Arg26,27,Tyr34]bovine PTH-(1–34)-NH2; Rho-PTH-(7–34), [Nle8,18-D-2-Nal12,Lys13(N-5-carboxymethylrhodamine)-L-2-Nal23,Arg26,27,Tyr34]bovine PTH-(7–34)NH2. Back


   ACKNOWLEDGMENTS
 
We thank the following colleagues for kindly providing reagents: Dr. Orson Moe (University of Texas, Dallas TX) for K44A-dynamin, Dr. Marc Caron (Duke University, Durham NC) for {beta}-arrestin2-GFP, and Dr. Jeffrey Benovic (Thomas Jefferson University, Philadelphia PA) for {beta}-arrestin1-(318–419).



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 DISCUSSION
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