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Endocrinology, doi:10.1210/en.2007-0343
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Endocrinology Vol. 148, No. 8 4073-4079
Copyright © 2007 by The Endocrine Society

ß-Arrestin-Dependent Parathyroid Hormone-Stimulated Extracellular Signal-Regulated Kinase Activation and Parathyroid Hormone Type 1 Receptor Internalization

W. Bruce Sneddon and Peter A. Friedman

Departments of Pharmacology (W.B.S., P.A.F.), and Medicine (P.A.F.), Renal Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: Peter A. Friedman, Ph.D., Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261. E-mail: paf10{at}pitt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH regulates renal calcium homeostasis by actions on the distal nephron. PTH-induced calcium transport in mouse distal convoluted tubule (DCT) cells requires activation of ERK1/2. ERK activation by ß-adrenergic receptors occurs in a biphasic manner and involves receptor internalization. An early rapid phase is ß-arrestin (ßAr) independent, whereas prolonged activation is ßAr dependent. We characterized PTH-stimulated ERK activation and the involvement of receptor internalization and ßAr dependence. In DCT cells, PTH transiently activated ERK maximally at 5 min and then returned to baseline. ßAr dependence of PTH receptor (PTH1R)-mediated ERK stimulation was assessed using mouse embryonic fibroblasts (MEFs) from ßAr1- and -2-null mice. In wild-type MEFs, PTH(1–34)-stimulated ERK activation peaked after 5 min, was 50% maximal after 15 min, and then recovered to 80% of maximal stimulation by 30 min. In MEFs null for ßAr1 and -2, PTH-stimulated ERK activation peaked by 5 min and returned to baseline. The effect was identical in ßAr2-null MEFs. In ßAr1-null MEFs, ERK exhibited delayed activation and remained elevated. PTH-stimulated ERK activation and receptor endocytosis were not inhibited by the clathrin-binding domain of ßAr1 [Ar(319–418)]. Coexpression of the sodium proton exchanger regulatory factor 1 (NHERF1) with Ar(319–418) blocked PTH1R internalization. We conclude that PTH-stimulated ERK activation in DCT cells proceeds with a rapid but transient phase that may involve ßAr1. Furthermore, the ßAr-dependent late phase of ERK activation by PTH requires the participation of ßAr2 and PTH1R internalization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH IS THE PRIMARY REGULATOR of calcium homeostasis and bone remodeling. Its effects are mediated by the PTH type 1 receptor (PTH1R). PTH stimulates calcium transport in the renal distal convoluted tubule (DCT) by a mechanism that requires activation of ERK1 and -2 (1). ERK activation by G protein-coupled receptors (GPCR) may be initiated by three distinct mechanisms that involve classical signaling pathways involving Gs and Gq, epidermal growth factor transactivation, or GPCR internalization. Receptor internalization and ß-arrestin (ßAr) recruitment play a role in ERK phosphorylation by ß-adrenergic receptors and the angiotensin II type IA receptor (2, 3, 4). PTH-stimulated ERK activation has been demonstrated recently to require ßAr recruitment (5, 6, 7). PTH-stimulated ERK activation proceeds with a biphasic time course in HEK-293 cells with an early transient phase that is G protein dependent and a sustained phase that requires ßAr (7). Inhibition of ßAr-mediated receptor endocytosis partially inhibits PTH-stimulated ERK activation in HEK-293 cells (5, 6). In contrast, in defined kidney DCT cells that are the physiological target of PTH action and where calcium absorption is regulated exhibit only a transient early phase of ERK activation by PTH. In this setting, PTH(1–34), which activates cAMP formation and protein kinase C (PKC), recruits ßAr to the plasma membrane and internalizes the PTH1R (8). The present studies were performed to characterize the role of arrestin abundance, recruitment, and receptor internalization in PTH-stimulated ERK activation. The present results show that PTH-stimulated ERK activation proceeds in a transient manner in DCT cells by a mechanism that requires G protein-mediated PKC activation and involves ßAr1 translocation but does not require PTH1R endocytosis. Furthermore, ßAr2 levels are insufficient to promote sustained ERK activation by PTH in DCT cells. ßAr2 overexpression introduced a sustained phase of ERK activation. The results show that the abundance of ßAr determines the pattern of arrestin-dependent ERK activation in native cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
The following materials were obtained from commercial sources: bisindolylmaleimide I (BisI) (Calbiochem, La Jolla, CA), H89 (Sigma Chemical Co., St. Louis, MO), and Nle8.18Tyr34-hPTH(1–34) (Bachem, Torrance, CA).

Cell culture
DCT cells stably expressing 105 PTH1R tagged at the C terminus with enhanced green fluorescent protein (EGFP) were used for these studies. The procedures used to create, characterize, and maintain this cell line have been described (8). Where indicated, DCT cells were transiently transfected using FuGENE6 (Roche Applied Science, Indianapolis, IN). Mouse embryonic fibroblasts (MEFs) from ßAr-null and wild-type mice were obtained from Dr. R. Lefkowitz (Duke University) (9). MEF cells were maintained in DMEM (15-092; Mediatech Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

Characterization of ERK1/2 phosphorylation
DCT cells expressing the PTH1R/EGFP were plated on six-well plates for measurement of PTH-stimulated ERK activation. MEF cells were passaged onto six-well plates, and 24 h later, they were infected with 50 multiplicity of infection (MOI) of an adenovirus containing the cDNA for the rat PTH1R. AdrPTH1R was obtained from Dr. M. Ungerer (Munich, Germany) (10). Forty-eight hours after plating or infection, cells were serum starved overnight in DMEM (15-017; Mediatech). Cells were treated with PTH peptides at 37 C for the indicated times. The plate was placed on ice, and the media were removed. Cells were lysed in 250 µl/well of 0.5% Nonidet P-40 lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40) (Sigma) supplemented with a protease inhibitor cocktail consisting of 0.5 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 150 nM aprotinin, 1 µM E-64, 0.5 mM EDTA, and 1 µM leupeptin (Calbiochem) and incubated for 15 min on ice. Cells were scraped and transferred to a 1.5-ml microcentrifuge tube on ice. The cell lysate was drawn four times through a 21-gauge needle attached to a 1-cc syringe and then placed on ice for an additional 15 min. Lysates were microcentrifuged at 14,000 rpm at 4 C, and the supernatants were transferred to a fresh 1.5-ml microcentrifuge tube on ice. Fifty microliters of lysate were added to 50 µl of 2x Laemmli SDS-PAGE loading buffer (Bio-Rad, Hercules, CA) containing 5% 2-mercaptoethanol and incubated at 95 C for 2 min. Forty microliters of the lysate were run on duplicate 10% SDS-PAGE gels and then transferred to Immobilon P membranes (Millipore, Bedford, MA) using the semi-dry method (Bio-Rad). Total ERK1/ERK2 and phospho-ERK1/ERK2 levels were assessed by immunoblotting using polyclonal antibodies (Cell Signaling Technologies, Beverly, MA).

ERK1/2 was measured by phosphospecific immunoblotting of phosphorylated ERK1/2 (pERK1/2) with a polyclonal antibody (9101) targeted to residues surrounding Thr202/Tyr204 of human p44 MAPK. Total endogenous nonphosphorylated ERK1/2 was measured in parallel. Results were normalized to total ERK2 and expressed as the fold change of pERK2 under unstimulated control conditions.

Quantitative, real-time fluorescence measurement of PTH1R internalization
DCT cells stably transfected with 105 PTH1R tagged at the C terminus by the EGFP were used for one set of studies. MEF cells from ßAr-null mice transiently transfected with PTH1R/EGFP using Effectene (QIAGEN, Valencia, CA) were used for another set of experiments. For studies involving the ß2-adrenergic receptor, DCT cells were transiently transfected using a ß2-adrenergic receptor tagged with EGFP (courtesy of Dr. R. Lefkowitz, Duke University). Receptor internalization was measured as detailed previously (8). Briefly, cells were plated on poly-D-lysine-coated 25-mm glass coverslips, and 24 h later, the cells were analyzed at room temperature by confocal microscopy equipped with a 488-nm Ar/Kr laser (Leica Microsystems, Bannockburn, IL). Emitted fluorescence was detected with a 515- to 540-nm bandpass filter. Sequential images were acquired at 1-min intervals. After obtaining three control images, the indicated ligand was introduced, and images were obtained for an additional 30–60 min to ensure that internalization was complete with any given maneuver. Internalization of PTH1R was reflected by the reduction of plasma membrane fluorescence, quantified as changes in pixel intensity. Receptor internalization was analyzed by selecting the entire plasma membrane through a plane normal to and approximately 2–3 µm above the basal membrane surface. Fluorescence intensity was digitized at 16-bit resolution and converted to 256 grayscale levels for each image using Metamorph Offline version 6.2 (Meta Imaging Series; Molecular Devices, Sunnyvale, CA). The product of the number of pixels within the defined membrane area and the average pixel intensity was calculated for each time point. Membrane fluorescence was normalized to percentage of control. Percent receptor internalization was defined as (100 – membrane fluorescence percent) and is reported as the change from control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTH(1–34) stimulated ERK1/2 phosphorylation in DCT cells stably expressing 105 PTH1R per cell. The time course resembled the G protein-dependent and arrestin-independent transient activation of ERK1/2 by PTH in HEK-293 cells observed by Gesty-Palmer et al. (7). ERK1/2 phosphorylation peaked at 5–10 min and returned to baseline by 60 min (Fig. 1Go). To characterize more completely the role of ßAr in PTH1R-mediated activation of ERK, we employed MEFs isolated from mice that have targeted disruption of ßAr1 and/or -2. The PTH1R was introduced into these cells using an adenoviral (Ad) vector. MEF cells infected with Ad-PTH1R displayed an 8-fold increase in cAMP formation in response to PTH(1–34) as compared with cells infected with a virus driving the expression of EGFP alone and lacking the PTH1R cDNA. In wild-type MEF cells that are replete with both ßAr1 and ßAr2, PTH-stimulated ERK activation had a biphasic time course. There was an early transient phase that peaked at 5 min and a late sustained activation that was elevated at 30–60 min (Fig. 2Go). In MEF cells that were null for ßAr1 and ßAr2, the early phase of PTH-activated ERK was still present, but the sustained activation was absent. This implies that one or both of the ßAr is required for sustained ERK phosphorylation. In MEF cells that were null for ßAr1, the time course was qualitatively different. The transient early phase of activation by PTH was absent. ERK stimulation was maximal at 10 min and remained elevated at 30 min. This is consistent with a role for ßAr1 in the early phase of activation of ERK by PTH. In MEF cells null for ßAr2, ERK activation by PTH was transient with peak levels achieved by 5 min and remained elevated at 50% maximum after 30 min. These data demonstrate that ßAr2 plays a pivotal role in the sustained activation of ERK by PTH.


Figure 1
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  		FIG. 1. PTH(1–34) transiently stimulates ERK phosphorylation in DCT cells. A, Representative immunoblot is shown. DCT cells expressing 105 PTH1R/EGFP per cell were cultured as described in Materials and Methods. After overnight incubation in serum-free media, the cells were treated for the indicated times with 100 nM PTH(1–34). Cell lysates were prepared, and ERK phosphorylation was assessed and quantified by immunoblotting and densitometry using NIH Image. ERK is total ERK. B, Data from three independent experiments are shown. Results are normalized to percent maximal PTH(1–34)-stimulated ERK activation, which was 8.6-fold.

 

Figure 2
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  		FIG. 2. PTH(1–34)-stimulated ERK activation in wild-type MEF cells exhibits transient and sustained phases. A, Representative experiment illustrating sample results for each cell line. 1/2KO, MEF cells null for ßAr1 and ßAr2; 1KO, MEF cells null for ßAr1; 2KO, MEF cells null for ßAr2; WT, wild-type MEF cells. ERK is total ERK. MEF cells from mice containing targeted disruptions for ßAr1 and ßAr2, as indicated, were infected with Ad-PTH1R/EGFP as outlined, and 48 h later, cells were serum starved overnight and the cells treated for the indicated times with 100 nM PTH(1–34). Cell lysates were prepared, and ERK phosphorylation was assessed and quantified as above. B, Results from three independent experiments. Data are normalized to percent maximal PTH(1–34)-stimulated ERK activation.

 
It is clear that PTH is able to activate ERK both transiently and in a sustained manner. We hypothesized that the complement and relative levels of the two ßAr determine transient and sustained ERK stimulation. Figure 1Go illustrates that PTH(1–34) transiently activated ERK in DCT cells. These cells present a physiologically relevant phenotype for PTH in that they display PTH-stimulated calcium transport that is dependent on activation of protein kinase A (PKA) and PKC (11). We predicted that prolonged activation of ERK by PTH requires increased ßAr2 expression. We overexpressed ßAr2 in these cells and examined its effect on the time course of PTH-stimulated ERK phosphorylation (Fig. 3Go). PTH-stimulated ERK activation was elevated at all time points from 10–30 min and was sustained at maximal levels at 30 min in DCT cells transfected with ßAr2. Overexpression of ßAr1 had a more modest effect. PTH-stimulated ERK phosphorylation peaked at 10 min and remained elevated at 75% of maximal levels at 30 min. Constitutive ßAr expression levels in the DCT cells was assessed by immunoblot. Both ßAr1 and ßAr2 were detectable (Fig. 4Go). Taken together, these data suggest that the endogenous level of ßAr2 expression is insufficient to promote sustained ERK phosphorylation by PTH(1–34).


Figure 3
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  		FIG. 3. Overexpression of ßAr2 promotes sustained ERK activation in DCT cells. A, Representative experiment is shown. EV means empty vector transfected, ßAr1 means ßAr1 transfected, and ßAr2 means ßAr2 transfected. ERK is total ERK, and FLAG is FLAGßAr. Cells were transfected with ßAr1, ßAr2, or empty vector, as indicated, and cultured as described, and 48 h after transfection, cells were incubated overnight in serum-free media. The cells were treated for the indicated times with 100 nM PTH(1–34). Cell lysates were prepared, and ERK phosphorylation was assessed as before. ßAr expression was confirmed by immunoblotting with a polyclonal anti-FLAG antibody (Sigma). B, Data summarizing mean of three experiments. Data are normalized to percent maximal PTH(1–34)-stimulated ERK activation, which was 6-fold.

 

Figure 4
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  		FIG. 4. ßAr1 and ßAr2 expression in DCT cells. Cell lysates were prepared from DCT cells, and 20 µg of total cell lysates was analyzed by immunoblot for ßAr1 and ßAr2 expression. For ßAr1, a goat polyclonal anti-ßAr1 antibody (Santa Cruz) was used at 1:100 dilution. For ßAr2, a rabbit polyclonal antibody AR2CT (courtesy of R. Lefkowitz) was used at a 1:2000 dilution. Positions of molecular weight markers are presented.

 
We previously demonstrated that PTH-stimulated ERK activation was PKC dependent. In light of the present findings and the emerging view that the time course of ERK activation is regulated by both G proteins and ßAr, we examined the relative importance of signaling by PKA and PKC on transient and sustained ERK activation by PTH(1–34). Inhibition of PKC with BisI abolished PTH-stimulated ERK phosphorylation at 5 min (Fig. 5Go). At 30 min, PTH-stimulated pERK was 20% maximal in the absence of inhibitors. In the presence of BisI, this was further reduced almost to zero. These data confirm earlier observations and demonstrate that PKC is required for transient ERK activation by PTH (1). PKA inhibition by H89 had no effect on pERK after a 5- or 30-min challenge with PTH (Fig. 5Go).


Figure 5
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  		FIG. 5. PKC inhibition blocks PTH-stimulated ERK activation. DCT cells were cultured in six-well plates and transfected with the clathrin-binding domain of ßAr1, Ar(319–418), as indicated, and 48 h later, cells were serum starved overnight and treated with 100 nM PTH(1–34) for 5 or 30 min in the absence or presence of the indicated agent, 10 µM BisI to block PKC or 10 µM H89 to inhibit PKA. Cell lysates were prepared, and ERK phosphorylation was assessed. Data represent mean ± SEM of three experiments. Data are normalized to percent maximal PTH(1–34)-stimulated ERK activation, which was 7.6-fold.

 
Recruitment of the ßAr to a GPCR can initiate endocytosis of the receptor in clathrin-coated pits. PTH(1–34) recruits ßAr1 and ßAr2 to the PTH1R in DCT cells (8, 12). Furthermore, the PTH1R is internalized maximally after a 15-min exposure to PTH(1–34). We inquired whether PTH1R internalization was required for hormone-stimulated ERK phosphorylation in DCT cells. To answer this question, we used the clathrin-binding domain of ßAr1 [Ar(319–418)]. This construct blocks arrestin-associated receptors from being internalized. Ar(319–418) failed to modulate pERK after 5 and 30 min exposure to PTH(1–34) (Fig. 5Go). Likewise, Ar(319–418) failed to inhibit PTH(1–34)-stimulated PTH1R internalization in DCT cells (Fig. 6Go). However, Ar(319–418) blocked PTH1R internalization in HEK-293 cells (Fig. 6Go), clearly demonstrating that this construct functions as expected in this cell line. To exclude the possibility that transfection efficiency of Ar(319–418) in DCT cells might be insufficient and thereby accounting for the failure of Ar(319–418) to suppress PTH1R endocytosis, we transfected EGFP-tagged ß2-adrenergic receptor into the parental DCT cell line. Ar(319–418) blocked isoproterenol-induced ß2-adrenergic receptor internalization (Fig. 6Go). The results confirm that Ar(319–418) functions as expected at least with respect to the ß2-adrenergic receptor. Thus, arrestin recruitment is opportunistic but not required for PTH1R internalization in DCT cells. Previous work established that PTH(1–31), which stimulates PKC but does not internalize the PTH1R (13), activates ERK in DCT cells (12). Taken together, these data indicate that that arrestin-dependent PTH1R internalization is not required for transient PTH-activated ERK phosphorylation.


Figure 6
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  		FIG. 6. The clathrin-binding domain of ßAr1, Ar(319–418), inhibits PTH(1–34)-stimulated PTH1R internalization in HEK-293 cells but not DCT cells. DCT or HEK-293 cells were transiently transfected with PTH1R/EGFP or the EGFP-tagged ß2-adrenergic receptor (ß2AR) in the presence or absence of Ar(319–418), and 48 h later, agonist-induced receptor internalization was measured after a 15-min treatment with ligand. For the PTH1R, 100 nM PTH(1–34) was used. For the ß2AR, 1 µM isoproterenol was used. Data represent mean ± SEM of three experiments.

 
The sodium/proton exchanger regulatory factor (NHERF1) is expressed at high levels in HEK-293 cells (14) and not expressed in DCT cells (8). NHERF1 is a critical regulator of PTH1R internalization (8). NHERF1 also blocks ßAr-independent PTH1R internalization and impairs ßAr recruitment to the PTH1R (15) but fails to block agonist-induced PTH1R internalization in DCT cells. Based on these considerations, we hypothesized that the PTH1R is internalized by two distinct mechanisms, one ßAr dependent and the other ßAr independent. We further predicted that blockade of one pathway is insufficient to inhibit PTH(1–34)-stimulated PTH1R internalization. We tested the effect of blocking both putative PTH1R endocytosis pathways using Ar(319–418) to impair ßAr-dependent internalization and NHERF1 to block ßAr-independent internalization (Fig. 6Go). Expression of Ar(319–418) together with NHERF1 abolished PTH(1–34)-stimulated PTH1R endocytosis in DCT cells. These data confirm the existence of two separate mechanisms of PTH1R internalization.

In MEF cells expressing ßAr1 and ßAr2, PTH-stimulated ERK activation is sustained at maximal levels at 30 min. We inquired whether PTH1R internalization plays a role in this prolonged action. To address this question, we examined PTH1R endocytosis in wild-type, ßAr1-, ßAr2-, and ßAr1/2-null MEF cells. The PTH1R/EGFP was transiently transfected into MEF cells, and PTH1R internalization was measured using real-time confocal fluorescence microscopy (Fig. 7Go). PTH(1–34)-induced PTH1R endocytosis 50–60% in wild-type and ßAr1-null MEF cells. In contrast, the PTH1R did not internalize in ßAr2-null or ßAr1/2-null MEF cells. ßAr2, therefore, plays a critical role in agonist-induced PTH1R sequestration in MEF cells and, by extension, for sustained ERK activation. These data show that PTH(1–34) recruits ßAr2, targets the PTH1R for internalization, and leads to prolonged ERK activity.


Figure 7
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  		FIG. 7. PTH1R internalization is impaired in ßAr2-null and ßAr1/2-null MEF cells. MEF cells were transiently transfected with PTH1R/EGFP, and 48 h later, agonist-induced receptor internalization was measured after a 15-min treatment with 100 nM PTH(1–34). Data represent mean ± SEM of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PTH regulates renal calcium transport by a mechanism that requires ERK activation (1). In DCT cells, the site of this regulation, PTH-stimulated ERK phosphorylation proceeds in a PKC-dependent manner primarily through transactivation of the epidermal growth factor receptor (12). PTH also stimulates ßAr translocation and PTH1R internalization in these cells (8). PTH(1–34)-stimulated PTH1R endocytosis likewise proceeds in a PKC-dependent manner. While performing the present work, a previously unappreciated role for the ßAr was reported (5). In addition to promoting desensitization and internalization of GPCRs, ßAr also induce formation of signaling platforms that are required by some receptors to activate the RAS-MEK-ERK signaling cascade (for review, see Ref. 16) as has been demonstrated for the PTH1R in HEK-293 cells, where arrestin-dependent PTH1R internalization plays a role in PTH(1–34)-stimulated ERK phosphorylation (5).

The objective of the present investigation was to characterize the role of the ßAr and receptor endocytosis in PTH1R-mediated activation of ERK. In DCT cells, PTH(1–34) stimulated ERK phosphorylation with a transient time course. Stimulation was maximal at 5 min and slowly returned to baseline by 60 min. This contrasts with results from heterologously expressed PTH1R in HEK-293 cells, where PTH(1–34) activated ERK signaling with a biphasic time course (7). The transient phosphorylation of ERK by PTH in DCT cells is kinetically similar to the G protein-dependent, arrestin-independent pathway described for HEK-293 cells but with some important differences. We provide evidence that implicates ßAr1 in the early transient phase of ERK activation. In ßAr1-null MEF cells, PTH(1–34)-stimulated ERK activation was delayed, with peak activation not occurring until 10–15 min, whereas in cells replete for ßAr1 and ßAr2, peak activation occurred at 5 min. In ßAr2- and ßAr1/2-null cells, the transient phase of PTH-stimulated ERK activation was intact, but sustained activation was not observed. We hypothesized that ßAr2 levels in DCT cells were insufficient to promote prolonged ERK phosphorylation. This theory was borne out in experiments where overexpression of ßAr2 enhanced ERK activation by PTH(1–34) at times longer than 5 min, and stimulation of ERK was sustained at maximal levels at 30 min. The delay of PTH-stimulated ERK activation in ßAr1-null MEF cells was absent in MEF cells null for both ßAr1 and ßAr2 (Fig. 2Go). These data are not intuitively reconcilable and suggest a complex relationship, where elimination of ßAr1 delays the early phase of ERK activation by PTH and further removal of ßAr2 restores the original time course. This interpretation is consistent with ßAr2 driving the late phase of ERK activation and, perhaps, promoting a delayed early phase of ERK activation in the absence of ßAr1. Such an explanation is, however, speculative and will require further investigation. The requirement for both ßAr1 and ßAr2 at different phases of PTH-stimulated ERK activity also contrasts with the angiotensin II type IA receptor, where down-regulation of ßAr1 augments ERK stimulation and down-regulation of ßAr2 eliminates ERK activation (17).

The PTH1R, perhaps by virtue of its ability to couple to multiple signaling pathways, exhibits remarkable cell specificity regarding the mechanism of ERK stimulation. In DCT cells, PTH stimulates ERK phosphorylation by a mechanism involving PKC activation and transactivation of the epidermal growth factor receptor (12). The requirement for receptor endocytosis varies between DCT cells and HEK-293 cells. From the present results it is clear that arrestin-mediated PTH1R internalization is not required for ERK activation in DCT cells. Blockade of arrestin-mediated receptor endocytosis by dominant-negative Ar(319–418) failed to inhibit PTH(1–34)-stimulated ERK activation in DCT cells. In addition, Ar(319–418) also failed to inhibit PTH(1–34)-stimulated PTH1R internalization but was entirely effective at inhibiting ß2-adrenergic receptor endocytosis. PTH(1–31) activates ERK without internalizing the PTH1R in DCT cells (12). PTH1R internalization is, therefore, not required for transient ERK activation by PTH. The opposite is true in HEK-293 cells, where arrestin-mediated PTH1R internalization plays an important role for ERK activation (13). These data underscore the need for caution in extrapolating results from heterologous expression to native cells inasmuch as the cellular complement of G proteins, ßAr, other proteins, and effectors importantly determines the mechanism of ERK activation.

The role of arrestin recruitment in ERK activation appears to be cell specific. In HEK-293 cells, ßAr2 translocation promotes PTH1R internalization that then leads to PTH1R endocytosis. Importantly, it is PTH1R endocytosis that appears to be the central requirement, and arrestin facilitates this phenomenon in these cells (5, 6). This may also be the case in MEF cells because ßAr2-null cells exhibit impaired PTH1R internalization and only transient ERK activation by PTH. ßAr2 overexpression in DCT cells conferred sustained ERK phosphorylation in response to PTH(1–34). Therefore, ßAr2 recruitment to the PTH1R is critical for prolonged PTH(1–34)-stimulated ERK activity. In DCT cells, ßAr1 may act as a scaffold by bringing other members of the RAS-MEK-ERK signaling cascade into optimal configuration because ßAr-mediated receptor internalization is not required for transient ERK phosphorylation. This phenomenon has been observed for other GPCRs. Agonist stimulation of the protease-activated receptor 2, for example, results in the formation of a complex containing the activated receptor, ßAr1, Raf-1, and pERK (18). Similarly, for the angiotensin II type IA receptor, Luttrell et al. (19) described an agonist-induced ß-arrestin2, Raf-1, MEK1, and ERK1/2 signaling complex. GPCR-activated ERK is generally translocated to the nucleus, where it phosphorylates and regulates transcription factors (20). In contrast, ßAr-activated ERK typically remains in the cytoplasm, where it phosphorylates a distinct set of effectors (21). These two pathways are both involved in activation of ERK by PTH. ßAr-dependent ERK stimulation may be relevant for PTH-stimulated calcium transport because transcriptional activation is not involved (22). In contrast, G protein-dependent phosphorylation of ERK by the PTH1R may be important for activation of cell proliferation (23).

Agonist-stimulated PTH1R internalization is blocked by expression of Ar(319–418) in HEK-293 cells (Fig. 6Go) (5). In contrast, Ar(319–418) failed to inhibit PTH(1–34)-stimulated PTH1R endocytosis in DCT cells. NHERF1, a critical regulator of PTH1R trafficking, is constitutively expressed in HEK-293 but not DCT cells. Introduction of NHERF1, along with Ar(319–418), blocked PTH(1–34)-induced PTH1R internalization in DCT cells. We proposed previously that the PTH1R can internalize by two mechanisms, one ßAr dependent and the other ßAr independent (15). Ar(319–418) blocks the former, and NHERF1 blocks the latter. The data presented here confirm this model. Therefore, we speculate that a novel bridge between the PTH1R and clathrin promotes receptor endocytosis in the absence of ßAr.

In summary, PTH activates ERK transiently in DCT cells by a PKC-dependent mechanism. In MEF cells replete with ßAr1 and ßAr2, PTH activates ERK both transiently and in a sustained manner. In cells deficient in ßAr1, the transient phase of PTH-stimulated ERK phosphorylation is delayed, whereas the sustained phase is missing in cells lacking ßAr2. Agonist-stimulated PTH1R endocytosis is also impaired in cells poor in ßAr2. Consistent with this view, overexpression of ßAr2 promotes prolonged activation of ERK by PTH. Ar(319–418) fails to inhibit PTH-stimulated ERK and PTH1R internalization in DCT cells. We conclude that transient ERK activation by PTH in these cells involves PKC activation and ßAr1 recruitment but does not require PTH1R endocytosis. Sustained ERK activation requires ßAr2 recruitment to the PTH1R and receptor internalization. Modulation of ßAr levels affects both the amplitude and kinetics of PTH1R signaling. This is an important consideration that, in turn, will affect the biological activity of the PTH1R in many cellular contexts.


   Footnotes
 
This work was supported by Grants DK-54171 and DK-69998 from the National Institutes of Health.

Disclosure Summary: The authors have nothing to disclose.

First Published Online May 24, 2007

Abbreviations: ßAr, ß-Arrestin; DCT, distal convoluted tubule; EGFP, enhanced green fluorescent protein; GPCR, G protein-coupled receptor; MEF, mouse embryonic fibroblast; NHERF1, sodium/proton exchanger regulatory factor; pERK, phosphorylated ERK; PKA, protein kinase A; PKC, protein kinase C.

Received March 12, 2007.

Accepted for publication May 11, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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