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Endocrinology Vol. 143, No. 10 3854-3865
Copyright © 2002 by The Endocrine Society

ARTICLE

Dual Regulation of the Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Signaling by Protein Kinase C and ß-Arrestins

Marián Castro1, Frank Dicker1,2, Jean-Pierre Vilardaga, Cornelius Krasel, Manfred Bernhardt and Martin J. Lohse

Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078, Germany

Address all correspondence and requests for reprints to: Martin J. Lohse, Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078, Germany. E-mail: lohse{at}toxi.uni-wuerzburg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined here the role of second messenger-dependent kinases and ß-arrestins in short-term regulation of the PTH receptor (PTHR) signaling. The inhibition of protein kinase C (PKC) in COS-7 cells transiently expressing PTHR, led to an approximately 2-fold increase in PTH-stimulated inositol phosphate (IP) and cAMP production. The inhibition of protein kinase A increased cAMP production 1.5-fold without affecting IP signaling. The effects of PKC inhibition on PTHR-mediated Gq signaling were strongly decreased for a carboxy-terminally truncated PTHR (T480) that is phosphorylation deficient. PKC inhibition was associated with a decrease in agonist-stimulated PTHR phosphorylation and internalization without blocking PTH-dependent mobilization of ß-arrestin2 to the plasma membrane. Overexpression of ß-arrestins strongly decreased the PTHR-mediated IP signal, whereas cAMP production was impaired to a much lower extent. The regulation of PTH-stimulated signals by ß-arrestins was impaired for the truncated T480 receptor.

Our data reveal mechanisms at, and distal to, the receptor regulating PTHR-mediated signaling pathways by second messenger-dependent kinases. We conclude that regulation of PTHR-mediated signaling by PKC and ß-arrestins are separable phenomena that both involve the carboxy terminus of the receptor. A major role for PKC and ß-arrestins in preferential regulation of PTHR-mediated Gq signaling by independent mechanisms at the receptor level was established.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
G PROTEIN-COUPLED RECEPTORS (GPCRs) are the largest family of signal transducers for extracellular stimuli. Protein kinases, including second messenger-dependent kinases (PKA, PKC), G protein-coupled receptor kinases (GRKs), tyrosine kinases, and MAPKs are downstream effectors of GPCRs. Conversely, upstream regulation of GPCR signaling involves the interaction of the receptors with these families of proteins, as well as arrestins (1, 2, 3, 4).

Desensitization of GPCRs, that is, the loss of their ability to respond to an agonist, is a crucial physiological mechanism of adaptation to continuous or repeated presence of stimuli. Rapid (within minutes) events involved in this adaptive response include phosphorylation of GPCRs or other components within the G protein signaling pathway, after homologous or heterologous activation of second messenger-dependent kinases (1, 2). In addition, GRKs and arrestins are involved in the homologous desensitization of GPCR signaling (3, 5): phosphorylation of agonist-activated GPCRs by GRKs leads to recruitment of ß-arrestins, which bind to the phosphorylated receptors and uncouple the receptor from its cognate G proteins. In addition, ß-arrestins promote receptor internalization via clathrin-coated pits (3).

The type I PTH receptor (PTHR) belongs to the class II of GPCRs (6) and plays a fundamental role in the regulation of calcium homeostasis in bone and kidney, as well as in bone formation and resorption (7). Upon activation by agonist (PTH or PTHrP), PTHR triggers at least two signaling pathways: Gq/11-mediated PLCß stimulation, leading to inositol-1,4,5-trisphosphate (IP3) production, calcium mobilization, and PKC activation, and Gs-mediated activation of adenylyl cyclase, resulting in cAMP production and PKA activation (8, 9, 10). The observation of certain cell/tissue specificity in the PTHR responses adds an additional level of diversity to the PTHR physiology: whereas PTHR elicits both Gs- and Gq-mediated signals in bone and kidney, the PTHR responses are mediated through a Gq pathway in pancreatic insulinoma cells (11), and predominantly, if not exclusively, through a cAMP-dependent pathway in smooth muscle cells (12).

Second messenger-dependent kinases (13, 14, 15, 16, 17, 18), GRKs (19) and arrestins (20) have been implicated in the regulation of PTHR signaling. The mechanisms of PKC or PKA involved in desensitization of PTHR remain poorly understood. In diverse cell types, long-term (within hours) exposure to PTH or to stimulators of second messenger-dependent kinases [i.e. phorbol 12-myristate 13-acetate (PMA), cAMP-analogs or forskolin] resulted in the desensitization of PTH-stimulated receptor signaling (13, 14, 15, 16). However, a direct effect of receptor phosphorylation on desensitization cannot be established from these studies, as a decrease in receptor number or down-regulation of other downstream elements of the signaling cascade, such as the kinases, can take place under these conditions. Other studies have shown desensitization of the PTH-stimulated Ca2+-response after short-term (within minutes) pretreatment with PTH or PMA (17, 18). In these cases, no correlation was established between receptor phosphorylation and the functional effects observed.

GRKs, and particularly GRK2, phosphorylate PTHR upon agonist stimulation (19, 21), and they are involved in the regulation of PTHR signaling (19). In addition, agonist- stimulation of PTHR promotes translocation of both ß-arrestin1 and ß-arrestin2 to the plasma membrane, their physical association with the receptor and internalization of the receptor/ß-arrestin2 complexes (20, 22, 23, 24). However, the role of ß-arrestins in regulation of the PTHR signaling remains to be fully established.

The aim of this study is to analyze the short-term effects of PTH-dependent activation of second-messenger dependent kinases and of ß-arrestins in PTHR signaling. Using the wild-type PTHR and a carboxy-terminally truncated receptor, transiently expressed in COS-7 cells, we report that PKC activation regulates PTHR-mediated Gq and Gs signaling pathways, whereas PKA activation preferentially affects PTHR-mediated Gs vs. Gq signaling. PKC regulation of PTH-stimulated inositol phosphate (IP) production involves mechanisms that require the integrity of the carboxy-terminal tail of the receptor. Regulation of PTHR signaling by PKC correlated with effects at the receptor level including agonist-mediated receptor phosphorylation and internalization, without compromising ß-arrestin2 mobilization to the plasma membrane. In addition, both ß-arrestin1 and ß-arrestin2 are also able to regulate PTHR signaling upon overexpression in COS-7 cells, preferentially impairing IP vs. cAMP signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Taq-DNA-polymerase, Fura-2/AM, and 12CA5 monoclonal antibody were obtained from Roche Diagnostics (Mannheim, Germany). The preparation of ß-arrestin1 antibody has been previously described (25). Bisindolylmaleimide I (GF109203X) and staurosporine were obtained from Alexis. Pluronic F-127 Protein Grade Detergent and the PKA-inhibitor H89 were from Calbiochem. Human (Nle8,18,Tyr34)-PTH (1–34) (here always referred to as PTH) was purchased from Bachem. Diethylaminoethyl-dextran, protein A-Sepharose CL-4B and human [125I]-(Nle8,18, Tyr34)-PTH-(1–34) used in binding experiments, were from Amersham Pharmacia Biotech. [125I]-Labeled human PTHrP-(1–86), used in internalization assays, was kindly provided by Dr. E. Blind (Department of Endocrinology, University of Würzburg, Vershacher Strasse, Germany). The other radiochemicals were obtained from NEN Life Science Products (Boston, MA). Cell culture reagents and media were from PAN-Systems, except for Roswell Park Memorial Institute 1640 medium without L-glutamine and I-inositol, which was obtained from ICN Biomedicals (Eschwege, Germany). Restriction and DNA-modifying enzymes were from New England Biolabs, Inc. (Beverly, MA). The other reagents and chemicals were of analytical grade and obtained either from Sigma (Taufkirchen, Germany) or from Merck (Darmstadt, Germany).

Plasmid constructs
Hemagglutinin (HA)-tagged receptors were generated by converting the sequence EDKEAPTGS (residues 93–101) in the N-terminal region of the human PTHR cDNA to YPYDVPDYA, as described for the rat PTHR (26), by PCR reaction. The cDNAs for HA-human PTHR and for a HA-truncated human PTHR lacking the carboxy-terminal tail and hence phosphorylation sites (T480 receptor) were subcloned into pCMV plasmid as previously described (19). ß-Arrestin1 (bovine) cDNA (27) subcloned into pcDNA3 and pRK5-ß-arrestin2 (bovine) (a generous gift from Dr. S. Cotecchia, Institut de Pharmacologie et de Toxicologie, Université de Lausanne) were used in the second messengers experiments. GFP-tagged ß-arrestin constructs used in translocation experiments were previously described (24, 28).

Cell culture and transfection
COS-7 cells were cultured in DMEM 4.5 g/liter glucose, supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37 C in a humidified atmosphere containing 7% CO2. Cells were transiently transfected by the diethylaminoethyl-dextran method as described previously (19). For IP and cAMP measurements, phosphorylation, and internalization assays with kinase inhibitors, cells grown on 100-mm dishes were transfected with 6 µg of receptor DNA, completed with empty vector to a total of 8 µg of DNA. The control (empty vector) DNA was replaced by ß-arrestin2-GFP DNA in experiments studying the effects of staurosporine on ß-arrestin2-GFP translocation by confocal microscopy. In transient transfections for second messenger experiments with overexpressed ß-arrestins, as well as for studies of translocation of GFP-tagged ß-arrestins, a ß-arrestin:receptor ratio of 3:1 was used, unless mentioned otherwise. HEK293 cells employed for quantification of receptor/ß-arrestin1-GFP interaction were cultured in DMEM 1.0 g/liter glucose, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37 C in a humidified atmosphere containing 7% CO2. Cells were transfected using a protocol based on the calcium-phosphate method as described previously (29). In all cases, assays were carried out 48–72 h after transfection.

Binding assays
The binding characteristics of the wild-type PTHR and the truncated T480 transiently transfected in COS-7 cells were determined in intact cells. Cells were washed with ice-cold DMEM/F12 supplemented with 1% BSA and incubated in the same medium for 90 min on ice. After this preequilibration time, medium was substituted for DMEM/F12 containing 1% BSA and [125I]PTH (50,000 cpm/well, 2200 Ci/mmol), and cells were incubated on ice for additional 90 min. Incubations were stopped by placing the cells on ice and rapidly washing with 1 ml ice-cold PBS before solubilization with 1 ml 0.8 N NaOH. Apparent Ki and Bmax values were obtained from inhibition curves carried out in the presence of increasing concentrations of unlabeled PTH (10 pM-1 µM), following the equations for homologous competition binding: KI = IC50-L, where L is the concentration of free radioligand, Bmax = B0*(IC50/L), where B0 is the specific binding. Nonspecific binding was defined by using 1 µM unlabeled ligand.

Total IP production
The assays were carried out following previously published procedures (19). To study the effect of protein kinase inhibitors, myo-[2-3H-(N)]-inositol-loaded cells were preincubated for 1 h at room temperature with the different inhibitors or vehicle, at the indicated concentrations, in HEPES buffer A (137 mM NaCl; 5 mM KCl; 1 mM CaCl2; 1 mM MgCl2; and 20 mM HEPES, pH 7.3). Subsequently, stimulation was initiated by placing the cells in a water bath at 37 C and by adding 10 mM LiCl together with the appropriate concentration of agonist or vehicle. Cells were stimulated for 1 h in these conditions, in the absence or continuous presence of the inhibitors. Incubations were stopped by adding ice-cold HClO4 to the cells and placing the plates on ice.

cAMP accumulation
The measurement of cAMP production was carried out by the RIA method (Immunotech, Krefeld, Germany) as described (19). To test the effects of protein kinase inhibitors on agonist-stimulated cAMP production, cells were washed in HEPES buffer A and preincubated in the same buffer with the different inhibitors at the indicated concentrations for 1 h at room temperature. Subsequently, cells were placed in a water bath at 37 C and 0.5 mM 3-isobutyl-1-methylxanthine was added. After 5 min, cells were stimulated with the appropriate concentration of agonist or vehicle for 15 min in these conditions, in the absence or continuous presence of the inhibitors. Incubations were stopped by adding ice-cold HClO4 to the cells and placing the plates on ice.

PKC in vitro phosphorylation assays on cell membranes
Cell membranes were prepared from transiently transfected COS-7 cells as described (19). Phosphorylation of the HA-tagged PTHR and T480 was carried out with recombinant purified PKC{delta}, kindly provided by Dr. H. Mischak (Institut für Klinische Molekularbiologie, München, Germany). Membranes from cells expressing the nontagged PTHR were used as a control. Control of receptor levels in the membrane preparations were performed by determining [125I]PTH specific binding following the protocol described above, where serum was substituted by 0.1% 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The specific activity of the PKC{delta} preparation was 2.0 nmol/min·mg using histone as substrate. Receptor-containing cell membranes (2 pmol receptor/reaction) were incubated with 1 µg total protein of the PKC{delta} preparation in buffer containing 20 mM Tris/HCl, pH 7.4; 2 mM EDTA; 6.5 mM MgCl2; 0.65 mM CaCl2; 1 mM dithiothreitol; 8 µM PMA; and 0.1 mM [{gamma}-32P]-ATP (5 cpm/fmol) in a final volume of 50 µl. The phosphorylation was carried out for 30 min at 30 C and was stopped by addition of 500 µl ice-cold 20 mM Tris/HCl, pH 7.4, 2 mM EDTA, and subsequent centrifugation at 21,000 x g, at 4 C for 30 min. The membrane pellet was solubilized in 50 µl of RIPA buffer (1% Nonidet P-40; 0.5% Na-deoxycholate; 0.1% sodium dodecyl sulfate (SDS); 50 mM Tris, pH 7.4; 100 mM NaCl; 2 mM EDTA; 50 mM NaF) on a shaking incubator for 30 min on ice. The HA-tagged PTHRs were immunoprecipitated with 12CA5 (2 µg) monoclonal antibody using protein A-Sepharose. Immunoprecipitated proteins were analyzed on 8.5% SDS-polyacrylamide gels and visualized by autoradiography. Quantification of phosphorylation was carried out by PhosphorImager analysis (Molecular Dynamics, Inc.).

Receptor phosphorylation in intact cells
Transiently transfected COS-7 cells were washed with 3 x 1 ml phosphate-free carbonate buffer (137 mM NaCl; 5 mM KCl; 1 mM CaCl2; 1 mM MgCl2; 4.5 g/liter glucose; and 2.5 g/liter NaHCO3) and incubated for 2 h at 37 C in the same buffer supplemented with 100 µCi/well [32P]orthophosphate. During this period and before stimulation of the cells, inhibitors or vehicle were added for 30 min. Labeled cells were either stimulated with 100 nM PTH, 1 µM PMA, 50 µM forskolin or vehicle for 5 min and solubilized in 0.8 ml RIPA buffer as above. The HA-tagged PTHRs were immunoprecipitated with 12CA5 (10 µg) monoclonal antibody. Immunoprecipitated proteins were resolved and analyzed as described above.

Determination of receptor internalization kinetics
Transiently transfected COS-7 cells were preincubated for 30 min at 37 C with 1 ml DHB buffer (DMEM containing 20 mM HEPES and 0.1% BSA), and incubations were continued for another 30 min in DHB in the absence (vehicle) or presence of kinase inhibitors at the indicated concentrations. Internalization kinetics were assayed at room temperature by replacing the preincubation buffer with 1 ml of DHB buffer containing [125I]-PTHrP-(1–86) (50,000 cpm/well) in the continuous absence or presence of the inhibitors. Incubations were stopped at the indicated times by placing the cells on ice and rapidly washing with 1 ml ice-cold PBS. The cells were incubated for 2 x 5 min in 0.5 ml acid wash solution (150 mM glycine/50 mM acetic acid, pH 3) to remove the surface-bound radioligand. The supernatants containing the acid-released radioactivity were collected and 1 ml 0.8 N NaOH was added to the cells to solubilize the acid-resistant radioactivity. The nonspecific binding was measured in parallel samples using 10-6 M PTH. The radioactivity was quantified in an automatic {gamma}-counter (1480 Wizard 3", Wallac, Inc.). The percent internalization was calculated after deduction of the respective nonspecific value: . The curves represent the single-phase exponential fit of the data. The endocytic rate constant, ke (min-1), was determined from the slope of the line obtained by plotting the amount of internalized radioligand against the integral of the surface-bound radioligand (30).

Visualization of agonist-stimulated mobilization of GFP-tagged ß-arrestins by confocal microscopy
COS-7 cells transiently expressing PTHR or T480 together with ß-arrestin1-GFP or ß-arrestin2-GFP were incubated with 100 nM PTH for different times at 37 C. The incubation was stopped by placing the plates on ice and washing the cells with ice-cold PBS before fixation with 4% paraformaldehyde for 10 min at room temperature. The effects of kinase inhibition on PTH-stimulated ß-arrestin2-GFP translocation were assayed by preincubation of the cells with 3 µM staurosporine, for 30 min at 37 C, before stimulation with 100 nM PTH for 5 min at 37 C. Fixed cells were observed by confocal scanning in a Leica Corp. True Confocal Scanner (TCS4D) confocal laser microscope using fluorescein filters. Representative sections corresponding to a middle plane of the cells are presented.

Quantification of agonist-stimulated ß-arrestin1-GFP mobilization by Western blot
HEK293 cells transiently transfected with ß-arrestin1-GFP and PTHR or T480 were stimulated with 100 nM PTH at 37 C for different times. Cells were place on ice, washed with ice-cold PBS, and detached with a rubber policeman. Cells pelleted by low speed centrifugation were lysed in PBS containing a protease inhibitor mixture (Calbiochem) by sonication. The lysate was centrifuged at 1,000 x g for 10 min to remove unbroken cells and the supernatant was further centrifuged at 20,000 x g for 30 min. The resulting supernatant represents the cytosolic fraction and the pellet the membrane fraction. Membrane proteins were solubilized in RIPA buffer (1% Nonidet P-40; 0.5% Na-deoxycholate; 0.1% SDS; 50 mM Tris, pH 7.4; and 150 mM NaCl) supplemented with protease inhibitors. Equal amounts of cytosolic and soluble membrane proteins were resolved on 7.5% SDS-polyacrylamide gels. Proteins were transferred onto an Immobilon P transfer membrane (Millipore Corp., Bedford, MA). Membranes were reacted with anti-ß-arrestin1 antibody (1:3,000), and antirabbit IgG (1:20,000) coupled to peroxidase and immunoreactive bands were visualized using chemiluminiscence (Pierce Chemical Co., Rockford, IL). Immunoreactive bands were quantified by densitometry and percent translocation was calculated as: .


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of the PTHR and T480 receptor transiently expressed in COS-7 cells
Wild-type PTHR and the carboxy-terminally truncated T480 receptor transiently expressed in COS-7 cells were tested for their ability to bind [125I]-PTH (Fig. 1AGo). The receptors displayed an affinity for PTH of Ki (mean ± SEM, n = 3) = 16.02 ± 2.65 nM for PTHR and 7.83 ± 0.06 nM for T480. Thus, T480 receptor showed an affinity for PTH twice as high as PTHR wild type, whereas the maximal levels of expression achieved for T480 were 24% lower than those for PTHR (Table 1Go). This was in agreement with previous works reporting higher affinity for PTH of this truncated receptor in comparison with wild type (19, 31, 32).



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  		Figure 1. Characteristics of PTHR and T480 receptor transiently expressed in COS-7 cells. A, Competition by PTH for the binding of [125I]PTH to intact COS-7 cells transiently expressing PTHR or T480. Data are the mean ± SEM of three independent experiments performed in triplicate. B, Concentration-response effects of PTH on IP and cAMP signaling mediated by PTHR and T480. COS-7 cells transiently expressing PTHR or T480 were stimulated with increasing concentrations of PTH for 1 h (IP production) or 15 min (cAMP accumulation), at 37 C. Dotted lines show the percent of maximal response achieved at PTH concentrations of 100 nM (IP) and 10 nM (cAMP) chosen in single-concentration experiments. Basal values were subtracted from agonist-stimulated values and expressed as % of control for each receptor. Data are the mean ± SEM of three (IP measurements) or five (cAMP measurements) independent experiments performed in duplicate. C, Phosphorylation of PTHR in intact cells upon heterologous activation of PKC and PKA. COS-7 cells transiently expressing HA-tagged PTHR were treated with 1 µM PMA or 50 µM forskolin for 5 min. Phosphorylation of anti-HA-immunoprecipitated receptors was quantified by PhosphorImager analysis. Basal phosphorylation was set to 100%. Bars represent the mean ± SEM of three independent experiments. D, Cell membranes from COS-7 cells transiently expressing HA-tagged PTHR or T480, or nontagged PTHR as a control, were prepared for in vitro PKC{delta} phosphorylation assays as described in Materials and Methods. Receptors were immunoprecipitated, analyzed on 8.5% SDS-polyacrylamide gels, and visualized by autoradiography. The picture shows a representative gel from one of three independent experiments.

 

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  		Table 1. Ligand binding and signaling characteristics of the wild-type PTHR and truncated T480 receptor transiently expressed in COS-7 cells

 
In COS-7 cells transiently expressing PTHR or T480 receptors, PTH stimulated IP and cAMP responses in a concentration-dependent manner (Fig. 1BGo). EC50 values are reported in Table 1Go. The EC50 values obtained for PTHR-mediated signaling in our system are comparable to previous data obtained for transiently transfected PTHRs in COS-7 cells (25 ± 3 nM and 0.32 ± 0.04 nM for IP and cAMP, respectively, for rat PTHR) (33).

Despite a lower expression level, the truncated receptor T480 displayed an increased PTH-stimulated IP and cAMP signaling in comparison to the wild-type receptor ({approx}2.1-fold and {approx}1.2-fold increase, respectively) (Table 1Go).

The characteristics of PTHR- and T480-phosphorylation by second messenger-dependent kinases were studied. In intact cells, heterologous activation of PKA by treatment with 50 µM forskolin slightly stimulated PTHR phosphorylation (1.1 ± 0.02-fold of basal, n = 3). However, treatment of the cells with 1 µM PMA promoted PTHR phosphorylation to a much higher extent (2.3 ± 0.4-fold of basal, n = 3) (Fig. 1CGo), reaching values similar to that obtained after 5 min stimulation with 100 nM PTH ({approx}2.3 ± 0.2-fold of basal) (see Fig. 3AGo). None of these conditions resulted in phosphorylation of the T480 receptor (not shown). In addition, PTHR but not T480 was a substrate for in vitro PKC phosphorylation in membranes prepared from COS-7 cells transiently transfected with the receptors (Fig. 1DGo). Thus, PTHR but not T480 was a substrate for PKC phosphorylation in vitro as well as in intact cells.



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  		Figure 3. A, Effects of kinase inhibitors on PTH-mediated phosphorylation of PTHR in intact cells. [32P]Orthophosphate loaded COS-7 cells transiently transfected with PTHR were preincubated with vehicle (control), GF109203X (30 µM), or H89 (10 µM) for 30 min at 37 C. Agonist-stimulation was started by adding PTH (100 nM) for 5 min at 37 C. Phosphorylation of anti-HA-immunoprecipitated receptors was quantified by PhosphorImager analysis and basal (nonstimulated) phosphorylation was set to 100% (lower panel). Bars represent the mean ± SEM of three to five independent experiments (* means significantly different with respect to basal phosphorylation P < 0.05, ** P < 0.01, # means significantly different with respect to PTH-stimulated phosphorylation in control conditions P < 0.05, Mann Whitney test). B, Visualization of a gel from a representative experiment.

 
Activation of second-messenger kinases upon PTH-stimulation regulates PTHR IP and cAMP signaling
The effects of activation of second-messenger kinases upon agonist stimulation on PTHR signaling were studied by using three different kinase inhibitors: H89 and bisindolylmaleimide I (GF109203X), which are selective for PKA and PKC, respectively, and staurosporine, which inhibits a broad spectrum of second messenger-dependent kinases.

Treatment of cells expressing PTHR with kinase inhibitors significantly increased PTH-mediated IP and cAMP responses (Fig. 2Go, A and B). We used concentrations of 100 nM and 10 nM PTH to promote IP and cAMP production, respectively, to achieve levels of receptor occupancy that lead to a similar extent of maximal response for each pathway ({approx}85% of the maximum) (Fig. 1BGo). PTHR responses under control conditions were 4-fold increase of basal IP production and 23.9 ± 2.7 pmol cAMP/well (Fig. 2Go, A and B). Exposure of the cells to 3 µM staurosporine resulted in a similar effect on both PTHR-mediated IP and cAMP responses: a 2.5-fold increase in PTH (100 nM)-induced IP signal and a 2.3-fold increase in PTH (10 nM)-induced cAMP accumulation were observed (Fig. 2Go, A and B). Selective inhibition of PKC by 30 µM GF109203X resulted in a 1.8-fold increase in PTHR-mediated IP signal and a 1.7-fold increase in the cAMP signal. A differential regulation of both pathways was observed after selective inhibition of PKA by 10 µM H89, which affected the cAMP signal (1.5-fold increase in PTH (10 nM)-induced cAMP accumulation) but not the IP signal (Fig. 2Go, A and B). None of these treatments had effects on basal IP and cAMP levels (data not shown).



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  		Figure 2. Regulation of PTH-induced IP and cAMP signaling by PKA and PKC. COS-7 cells transiently expressing PTHR or T480 were preincubated for 1 h at room temperature with vehicle (control), H89 (10 µM), GF109203X (30 µM) or staurosporine (3 µM), and PTH (100 nM, 1 h)-stimulated IP production (A) or PTH (10 nM, 15 min)-stimulated cAMP accumulation (B) was determined as described in Materials and Methods. Basal values were subtracted from agonist-stimulated values and expressed as % of control for each receptor. Bars represent the mean ± SEM of three to five independent experiments performed in duplicate (* means significantly different with respect to control, P < 0.05, ** P < 0.01, Mann Whitney test). C, Concentration-response curves for PTH-stimulated IP production in cells expressing PTHR or T480, in control conditions or upon treatment with staurosporine (3 µM). PTHR-mediated maximal effect (1 µM PTH) under control conditions was set to 100%. D, Idem for PTH-stimulated cAMP production. Data represent means ± SEM of two independent experiments performed in duplicate.

 
The carboxy-terminal tail of PTHR is an important determinant for the PKC-mediated regulation of the IP signaling
As mentioned above, the phosphorylation-deficient T480 receptor showed an increased agonist-stimulated IP response in comparison to the wild-type receptor (7.2-fold increase of basal IP production and 25.3 ± 5.5 pmol cAMP/well under control conditions). Inhibition of PKC by GF109203X or staurosporine potentiated the IP signaling of the T480 receptor to a lower extent than that of the PTHR (1.3 and 1.4-fold increase for T480 vs. 1.8- and 2.5-fold increase for PTHR, respectively) (Fig. 2AGo). Truncation of the carboxy-terminal tail, however, did not impair the observed potentiation of the cAMP response by the different kinase inhibitors: H89 (1.3-fold increase for T480 vs. 1.5-fold increase for PTHR), GF109203X (2.0-fold increase for T480 vs. 1.7-fold increase for PTHR) or staurosporine (2.5-fold increase for T480 vs. 2.3-fold increase for PTHR) (Fig. 2BGo).

Concentration-response curves for PTH-stimulated IP and cAMP signals in control cells or cells treated with 3 µM staurosporine confirmed the higher potentiation of IP signals mediated by PTHR vs. T480 receptor (Fig. 2CGo), as well as the similar behavior of both receptors in terms of staurosporine effects on cAMP signals (Fig. 2DGo).

Mechanisms contributing to the regulation of PTHR signaling at the receptor level by PKC
The effects of kinase inhibitors on PTH-stimulated PTHR phosphorylation were tested in intact cells. Exposure of the cells to the PKC inhibitor GF109203X (30 µM) lowered the basal levels of PTHR phosphorylation by 25%, and, significantly, the PTH (100 nM)-stimulated phosphorylation of PTHR (by 50%) (Fig. 3Go). In contrast, the PKA inhibitor H89 (10 µM) did not influence basal or PTH-mediated phosphorylation of the PTHR. These data show a correlation between PKC-dependent phosphorylation of PTHR upon agonist stimulation and PKC-mediated regulation of the PTHR signaling at the receptor level.

Given that internalization of GPCRs is one of the possible mechanisms that regulate receptor signal responsiveness, we then examined whether PKC inhibition altered PTHR internalization. As shown in Fig. 4AGo, GF109203X and staurosporine significantly decreased the extent of PTHR internalization in transiently transfected COS-7 cells (72.0 ± 1.4% and 66.1 ± 1.6% of control, respectively), whereas PKC inhibition did not modify the endocytic rate constant of PTHR internalization: ke(control) = 0.09 ± 0.02 min-1; ke(GF109203X) = 0.09 ± 0.03 min-1 (Fig. 4BGo). PKC-dependent internalization of the PTHR requires the integrity of the carboxy-terminal tail of the receptor, as the T480 receptor showed a deficient internalization, which was PKC-insensitive (Fig. 4BGo). Neither the extent nor the rate constant of internalization of T480 were affected by GF109203X (ke (control) = 0.14 ± 0.01 min-1; ke(GF109203X) = 0.12 ± 0.01 min-1) (Fig. 4BGo). Thus, selective inhibition of PTHR phosphorylation and internalization by PKC inhibitors was concomitant to the observed regulation of PTHR-mediated IP signals by PKC at the receptor level.



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  		Figure 4. A, Effects of PKC inhibition on internalization of PTHR. COS-7 cells transiently transfected with PTHR were preincubated with vehicle, GF109203X (30 µM) or staurosporine (3 µM) for 30 min at 37 C. After that, PTH-induced internalization of PTHR was measured at room temperature as described in Materials and Methods, in the continuous absence or presence of the kinase inhibitors. B, Effects of PKC inhibition on the kinetics of internalization of PTHR and T480. COS-7 cells transient transfected with PTHR or T480 were preincubated with vehicle or GF109203X (30 µM) for 30 min at 37 C. After that, PTH-induced internalization of the receptors was measured at different time points as described in Materials and Methods, in the continuous absence or presence of the kinase inhibitors. Internalized radioligand is expressed as percent of total specific radioligand bound. Inset, Kinetics of the binding of radiolabelled [125I]PTH to these cells. Data represent the mean ± SEM of three independent experiments performed in duplicate (* means significantly different with respect to control, P < 0.05, Mann Whitney test).

 
Given that ß-arrestin2 is involved in the internalization process of PTHR (22, 23), we investigated the effects of PKC inhibition on the recruitment of ß-arrestin2-GFP to the plasma membrane upon agonist stimulation of PTHR in COS-7 cells (Fig. 5Go). ß-Arrestin2-GFP showed a homogeneous cytosolic distribution in nonstimulated cells both in the absence (panel a) or presence (panel c) of staurosporine. Addition of 100 nM PTH to the cells expressing PTHR produced a rapid translocation of ß-arrestin2-GFP to the plasma membrane (panel b) that was not inhibited by treatment of the cells with staurosporine (panel d). Thus, the observed effects of PKC inhibition on PTHR internalization are not due to an impaired translocation of ß-arrestin2.



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  		Figure 5. Visualization of PTH-induced ß-arrestin2-GFP translocation to the plasma membrane. COS-7 cells transiently coexpressing PTHR and ß-arrestin2-GFP were pretreated with vehicle (a, b) or staurosporine 3 µM (c, d) for 30 min at 37 C. Unstimulated cells (basal) or cells stimulated by 100 nM PTH for 5 min in the continuous absence or presence of staurosporine were fixed and examined by confocal scanning fluorescence microscopy.

 
Altogether, our data relate the effects of PKC on PTHR-mediated IP pathway at the receptor level with receptor phosphorylation and internalization events independent of ß-arrestin2.

ß-Arrestin1 and ß-arrestin2 differentially desensitize PTH-mediated PTHR signaling
To further elucidate the mechanisms of regulation of both PTHR signaling pathways, we analyzed the contribution of ß-arrestin-dependent mechanisms to the short-term regulation of PTHR signaling. Figure 6AGo showed that the overexpression of ß-arrestin 1 reduced the PTHR-mediated IP response by 56% and led to a smaller, although significant, reduction of the cAMP response (by 19%). A similar pattern was observed after overexpression of ß-arrestin 2, which significantly reduced PTHR-mediated signaling responses by 86% for IPs and by 18% for cAMP (Fig. 6BGo). Concentration-response curves reflected the preferential inhibition of the PTHR-mediated IP signals vs. cAMP signals, in cells overexpressing ß-arrestin2 (Fig. 6Go, C and D).



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  		Figure 6. Effects of overexpression of ß-arrestins on PTH-induced signaling. A, COS-7 cells transiently expressing PTHR or T480 were cotransfected with empty vector (control) or ß-arrestin1. Cells were assessed for PTH (100 nM, 1 h)-stimulated IP and PTH (10 nM, 15 min)-stimulated cAMP responses as described in Materials and Methods. B, Idem for cells coexpressing ß- arrestin2. Basal values were subtracted from agonist-stimulated values and expressed as percent of control values for each receptor. Bars represent the mean ± SEM of three independent experiments performed in triplicate (IP measurements) or seven independent experiments performed in duplicate (cAMP measurements). C, Concentration- response curves for PTH-stimulated IP production mediated by PTHR and T480 in control cells or cells coexpressing ß-arrestin2. PTHR-mediated maximal effect (1 µM PTH) in control cells was set to 100%. D, Concentration-response curves for PTH-stimulated cAMP production mediated by PTHR and T480 in control cells or cells coexpressing ß-arrestin2. E, PTH (100 nM, 1 h)-stimulated IP production in cells expressing PTHR or T480 cotransfected with ß-arrestin2 at different ß-arrestin2:receptor DNA ratios. PTH-induced IP response in control cells (cotransfected with empty vector) was set to 100% in each case. Bars represent the mean ± SEM of three independent experiments performed in duplicate. (* means significantly different with respect to control, P < 0.05, ** P < 0.01, Mann Whitney test).

 
Overexpression of ß-arrestins resulted in a predominant inhibition of T480-mediated IP signals vs. cAMP signals as well (Fig. 6BGo). Although the overall pattern of effects of overexpression of ß-arrestins on PTHR or T480 signaling was similar, the magnitude of the impairment observed for T480 signaling was reduced in comparison to that observed for PTHR signaling (Fig. 6Go, A and B). This was highlighted in the case of ß-arrestin1 overexpression (Fig. 6AGo). In the case of ß-arrestin2, strong differences in the sensitivity of PTHR and T480 signaling to ß-arrestin2 were revealed by lowering ß-arrestin2 overexpression levels (Fig. 6EGo).

These data indicate that ß-arrestin1 or ß-arrestin2 were able to regulate PTHR signaling in COS-7 cells with a preferential impairment of IP vs. cAMP signaling, and that the truncation of the carboxy-terminal tail of the receptor compromised the ability of ß-arrestins to regulate PTHR signaling.

The interaction of ß-arrestins with both receptors was further analyzed by visualization of GFP-tagged ß-arrestin translocation to the plasma membrane. In the absence of agonist, both ß-arrestin1-GFP and ß-arrestin2-GFP showed a homogeneous distribution throughout the cytosol in COS-7 cells expressing PTHR or T480, as observed by confocal microscopy (Fig. 7AGo, panels a, e, i, m). After PTH stimulation for 5 min, both ß-arrestins translocated to the plasma membrane in cells expressing PTHR (Fig. 7AGo, panels b and j). Longer (60 min) exposition to PTH resulted in the localization of ß-arrestins in intracellular vesicles, presumably reflecting endocytosis of receptor-ß-arrestin complexes (Fig. 7AGo, panels d and l).



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  		Figure 7. Analysis of the interaction of ß-arrestins with PTHR or T480 upon agonist-stimulation. A, Time-course of PTH-stimulated translocation of ß-arrestin1-GFP and ß-arrestin2-GFP to the plasma membrane in COS-7 cells transiently coexpressing PTHR or T480. Unstimulated cells or cells stimulated with 100 nM PTH for the indicated times at 37 C were fixed and examined by confocal scanning fluorescence microscopy. B, Immunoblot analysis of ß-arrestin1-GFP translocation in response to 100 nM PTH in HEK293 cells transiently coexpressing ß-arrestin1-GFP and PTHR or T480. Equal amounts of cytosolic and solubilized membrane proteins were separated by SDS-PAGE and ß-arrestin1-GFP was visualized by immunoblot analysis with a specific ß-arrestin1 antibody. Data represent the mean ± SEM of seven (PTHR) or three (T480) independent experiments. Inner panel shows a representative Western blot.

 
Recruitment of GFP-tagged ß-arrestins to the plasma membrane was less efficient in cells expressing the T480 receptor (Fig. 7AGo, panels e–h and m–p). In addition, the interaction of ß-arrestins with T480 seemed to be transitory, as certain redistribution of ß-arrestins in the cytosol was observed at long time (60 min) exposition to the agonist (Fig. 7AGo, panels h and p). This was especially notorious in the case of ß-arrestin1-GFP.

These results were confirmed by quantification of receptor/ß-arrestin1-GFP interactions by Western blot of cytosol and membranes from unstimulated or PTH-stimulated cells (Fig. 7BGo). These experiments were carried out in HEK293 transiently expressing ß-arrestin1-GFP and PTHR or T480, as these cells were more suitable than COS-7 cells for subcellular fractionation and further detection of ß-arrestin1 using our anti-ß-arrestin1 antibody. No ß-arrestin1 immunoreactivity was detected in membranes from unstimulated cells expressing PTHR or T480. Upon PTH (100 nM)-stimulation for 5–10 min, a high ß-arrestin 1 immunoreactivity was associated with the membrane fraction in cells expressing PTHR (40% of total ß-arrestin1-GFP immunoreactivity at 10 min). PTH-mediated ß-arrestin1-GFP recruitment to the plasma membrane was less efficient in cells expressing T480 ({approx}14% of total ß-arrestin1-GFP immunoreactivity at 10 min). For both receptors, the fraction of ß-arrestin1-GFP associated with the plasma membrane diminished at later times (20–40 min) (Fig. 7BGo). These data show the contribution of the carboxy-terminal tail of the receptor to the interaction of the PTHR with ß-arrestins.

Altogether, the effects of ß-arrestins on the signaling of PTHR and T480 receptor resembled the different ability of the two receptors to interact with ß-arrestins.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we investigate the involvement of second messenger-dependent kinases and ß-arrestins in the short-term regulation of PTHR signaling. Because PTHR stimulates both Gs and Gq signaling pathways, a major aim of this study was to explore whether common mechanisms were involved in the regulation of these two pathways.

Cross-talk between Gs and Gq signaling pathways in the regulation of PTHR signaling
Our results show a cross-talk between the Gs and the Gq pathways at the level of regulation of PTHR signaling by PKC because PKC inhibition by GF109203X up-regulated both IP and cAMP signals to a similar extent. PKA, however, differentially regulated the Gs signaling pathway, because selective inhibition of PKA by H89 resulted in up-regulation of the cAMP production without changes of the IP signal. In agreement with our data, short-term regulation of PTHR-mediated calcium and cAMP signals by PKC has been reported (18), whereas significant regulation of the IP or calcium signals by cAMP analogs or forskolin could not be observed in different cell types (15, 17, 18). Considering this regulatory cross-talk, the selective activation of Gs or Gq pathways by PTHR in certain cell types (11, 12) could lead to particularities in the regulation of the PTHR signaling in different tissues. In fact, regarding vascular tissue, where PTHR selectively activates the Gs signaling pathway, a recent study reported the lack of homologous desensitization of the vasodilatory responses to PTHrP in patients with hyperparathyroidism (34).

Mechanisms at, and distal to, the receptor differentially contribute to the regulation of PTHR-mediated IP and cAMP signals
Desensitization mechanisms operating both at, and distal to, the receptor level are known to regulate the signaling of other Gs- and Gq-coupled receptors (35). The dissociation of kinase effects on PTHR signaling at, and distal to, the receptor level was achieved in this study by analyzing the signaling of the carboxy-terminally truncated receptor, T480 receptor. This receptor, which lacks all but the juxtamembrane 16 amino acids of the carboxy-terminal tail of the PTHR, was phosphorylation-deficient upon stimulation by PTH (19), in agreement with other studies pointing out the carboxy-terminal tail of PTHR as the domain containing the sites of PTH-stimulated receptor phosphorylation (21, 36, 37). Our approach allows us to quantify, for the first time to our knowledge, the contribution of mechanisms distal to the receptor to the short-term desensitization of PTHR signaling.

The results revealed differences in the mechanisms of regulation of PTHR-mediated signals by kinases operating on the Gs and Gq pathways. The regulation of IP signals mediated by PKC was mainly exerted by mechanisms operating at the receptor level, with certain contribution of effects distal to the receptor. This was reflected by the higher potentiation of the IP signal mediated by PTHR in comparison to T480 signals. In contrast, regulation of cAMP signals by PKA or PKC was exerted mainly by mechanisms operating at levels down-stream from the receptor, without involvement of receptor phosphorylation events. This was shown by the similar effects of kinase inhibitors on the cAMP signals mediated by PTHR or T480. Possible targets for regulation of PTHR signaling by kinases at levels downstream from the receptor include PKC effects on PLCß (38, 39) and PKA and PKC effects on adenylyl cyclase (40, 41, 42).

Regulation of PTHR-mediated IP signaling by PKC is concomitant to receptor phosphorylation and internalization events independent of ß-arrestin mobilization
PKC regulation of IP signals at the receptor level correlated with PKC-mediated phosphorylation and internalization of PTHR. In addition, the internalization of the phosphorylation-deficient T480 receptor occurred to a lower extent than that of the wild-type, and was no more sensitive to PKC inhibition. Thus, the observed PKC effects on the Gq pathway could be mainly attributed to the C-terminus and its phosphorylation regulating internalization, with small contribution of mechanisms downstream from the receptor. The fact that the inhibition of PTHR internalization has greater effects on the PTHR Gq pathway than on Gs-mediated signals was expected, considering the more efficient coupling of the receptor to the Gs pathway.

The requirement of PKC activation for PTHR internalization is a controversial question, as opposite results have been reported in different cell lines (20, 22, 43). In agreement with a recent study performed in HEK293 cells (20), our data support the existence of a PKC-dependent PTHR internalization pathway in COS-7 cells. This pathway appears either independent of ß-arrestin2 or subsequent to ß-arrestin2 recruitment to the plasma membrane. In this way, recent studies in ß-arrestin knockout cells suggest a novel mechanism of GPCR internalization through a dynamin- and clathrin-dependent pathway that is independent of ß-arrestins (44).

Interaction of both ß-arrestin1 and ß-arrestin2 with PTHR involves the carboxy terminus of the receptor and leads to preferential desensitization of the Gq signaling pathway
Here we report for the first time the functional consequences of the interaction of PTHR with ß-arrestin1. Given that PTHR and ß-arrestin1 are endogenously expressed in osteoblastic cells (45), a physiological role for ß-arrestin1 as regulator of PTHR signaling appears likely.

Overexpression of ß-arrestins rescued the internalization deficiency of the T480 receptor in HEK293 cells, whereas it had smaller effects on the internalization of the wild-type receptor (23). These effects on the internalization of both receptors do not correlate with the effects on the signaling reported here. The smaller sensitivity of T480 signaling vs. PTHR signaling to overexpression of ß-arrestins correlates, however, with the reduced ability of T480 to mobilize ß-arrestins to the plasma membrane. Thus, whereas the effects of PKC on PTHR signaling correlated with internalization events, the regulation of PTHR signaling by ß-arrestins seems to be associated with recruitment of ß-arrestins to the receptor rather than receptor internalization.

The reduced affinity of T480 for ß-arrestins indicates that the interaction of ß-arrestins with PTHR involves the carboxy-terminal tail of the receptor. However, as shown for other GPCRs (46), additional receptor domains seem to contribute to the interaction with ß-arrestins, as the lower sensitivity of T480 receptor signaling to ß-arrestin2 in our study was overcome by increasing the levels of ß-arrestin2 overexpression.

Our data reveal that ß-arrestins preferentially desensitized the PTHR-stimulated Gq vs. Gs pathway. This does not seem to be due to the different time-frame of measurement of both signals. A strong translocation of ß-arrestins to the plasma membrane was already visible upon 5 min of agonist stimulation of the cells, and it remains stable at least for 1 h. The efficient coupling of PTHR to the Gs pathway suggests a higher affinity of the receptor for Gs than for Gq, which could facilitate the preferential disruption of the PTHR-Gq vs. -Gs coupling by ß-arrestins. Thus, a competition of ß-arrestins for domains on the PTHR also involved in interaction with G proteins could be postulated as the mechanism underlying the selective desensitization of PTHR-mediated IP signaling by ß-arrestins. Other authors have shown impairment of cAMP signaling in COS-7 cells upon overexpression of a ß-arrestin2-GFP construct (20). However, effects on IP signaling were not determined in parallel in this study, which makes a comparison to the effects observed in our system difficult. Our data highlight a differential desensitization of the two PTHR signaling pathways by ß-arrestins and suggest the possibility of selective desensitization of a certain pathway leaving intact other receptor functions.

In conclusion, we have demonstrated different mechanisms of regulation of the PTHR Gq and Gs signaling pathways upon agonist-stimulation of the receptor in COS-7 cells. Mechanisms operating at, and distal to, the receptor contribute to a different extent to the short-term feedback regulation of PTHR IP and cAMP signaling by agonist-activated second messenger-dependent kinases. PKC- and ß-arrestin-mediated effects involving the carboxy-terminal tail of the receptor preferentially regulate the Gq vs. Gs signaling pathway. Whereas both PKC and ß-arrestin2 are implicated in PTHR internalization, PKC inhibition does not impair agonist-stimulated ß-arrestin2 recruitment to the plasma membrane. We conclude that regulation of PTHR signaling by PKC and ß-arrestins are separable phenomena which both involve the carboxy-terminal tail of the receptor.


   Acknowledgments
 
We thank Dr. E. Blind, Dr. S. Cotecchia, and Dr. H. Mischak for kindly providing material required to perform this study, as well as Dr. K. Nip and Dr. U. Quitterer for critically reading the manuscript and helpful discussion.


   Footnotes
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft, and the Fonds der Chemischen Industrie (grants to M.J.L.) and fellowships from the Alexander von Humboldt Foundation and Ministerio de Ciencia y Tecnología, Spain (to M.C.).

1 M.C. and F.D. contributed equally to the work. Back

2 Present address: Department of Gene Therapy, Roche Pharma Research Oncology, Nonnenwald 2, D-82372 Penzberg, Germany. Back

Abbreviations: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinases; HA, hemagglutinin; IP, inositol phosphate; IP3, inositol-1,4,5-trisphosphate; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PTHR, PTH receptor; SDS, sodium dodecyl sulfate.

Received February 27, 2002.

Accepted for publication June 27, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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S. L. Ferrari, D. D. Pierroz, V. Glatt, D. S. Goddard, E. N. Bianchi, F. T. Lin, D. Manen, and M. L. Bouxsein
Bone Response to Intermittent Parathyroid Hormone Is Altered in Mice Null for {beta}-Arrestin2
Endocrinology, April 1, 2005; 146(4): 1854 - 1862.
[Abstract] [Full Text] [PDF]

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C. A. Syme, P. A. Friedman, and A. Bisello
Parathyroid Hormone Receptor Trafficking Contributes to the Activation of Extracellular Signal-regulated Kinases but Is Not Required for Regulation of cAMP Signaling
J. Biol. Chem., March 25, 2005; 280(12): 11281 - 11288.
[Abstract] [Full Text] [PDF]