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Originally published In Press as doi:10.1074/jbc.M306019200 on August 15, 2003 Originally published In Press as doi:10.1074/jbc.M306019200 on August 14, 2003

J. Biol. Chem., Vol. 278, Issue 44, 43787-43796, October 31, 2003
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Activation-independent Parathyroid Hormone Receptor Internalization Is Regulated by NHERF1 (EBP50)*

W. Bruce Sneddon{ddagger}, Colin A. Syme§, Alessandro Bisello§, Clara E. Magyar{ddagger}||||, Moulay Driss Rochdi¶||, Jean-Luc Parent¶, Edward J. Weinman**, Abdul B. Abou-Samra{ddagger}{ddagger}, and Peter A. Friedman{ddagger}§§¶¶

From the {ddagger}Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, the §Department of Medicine, Division of Endocrinology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, the Rheumatology Division, Faculty of Medicine and Clinical Research Centre, University of Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada, the **Departments of Medicine and Physiology, University of Maryland School of Medicine, and Department of Veterans Affairs Medical Center, Baltimore, Maryland 21201, the {ddagger}{ddagger}Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, and the §§Department of Medicine, Renal Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Received for publication, June 9, 2003 , and in revised form, August 13, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Parathyroid hormone (PTH) regulates extracellular calcium homeostasis through the type 1 PTH receptor (PTH1R) expressed in kidney and bone. The PTH1R undergoes {beta}-arrestin/dynamin-mediated endocytosis in response to the biologically active forms of PTH, PTH-(1–34), and PTH-(1–84). We now show that amino-truncated forms of PTH that do not activate the PTH1R nonetheless induce PTH1R internalization in a cell-specific pattern. Activation-independent PTH1R endocytosis proceeds through a distinct arrestin-independent mechanism that is operative in cells lacking the adaptor protein Na/H exchange regulatory factor 1 (NHERF1) (ezrin-binding protein 50). Using a combination of radioligand binding experiments and quantitative, live cell confocal microscopy of fluorescently tagged PTH1Rs, we show that in kidney distal tubule cells and rat osteosarcoma cells, which lack NHERF1, the synthetic antagonist PTH-(7–34) and naturally circulating PTH-(7–84) induce internalization of PTH1R in a {beta}-arrestin-independent but dynamin-dependent manner. Expression of NHERF1 in these cells inhibited antagonist-induced endocytosis. Conversely, expression of dominant-negative forms of NHERF1 conferred internalization sensitivity to PTH-(7–34) in cells expressing NHERF1. Mutation of the PTH1R PDZ-binding motif abrogated interaction of the receptor with NHERF1. These mutated receptors were fully functional but were now internalized in response to PTH-(7–34) even in NHERF1-expressing cells. Removing the NHERF1 ERM domain or inhibiting actin polymerization allowed otherwise inactive ligands to internalize the PTH1R. These results demonstrate that NHERF1 acts as a molecular switch that legislates the conditional efficacy of PTH fragments. Distinct endocytic pathways are determined by NHERF1 that are operative for the PTH1R in kidney and bone cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular calcium homeostasis in vertebrate animals is primarily under the endocrine control of the parathyroid hormone (PTH)1/type I PTH receptor (PTH1R). The PTH1R, predominantly expressed in kidney and bone cells, belongs to class B of the large superfamily of G protein-coupled receptors (GPCRs) that consists of receptors for peptide hormones and neuropeptides (1). Class B GPCRs are characterized by a common topology and by their ability to couple to multiple signaling pathways via distinct G proteins.

PTH is synthesized by the parathyroid glands as a mature peptide of 84 amino acids that is stored in secretory vesicles and dense core granules. Reductions of extracellular calcium levels are detected by the calcium-sensing receptor on parathyroid chief cells and promote the release of PTH, which acts on bone (to increase resorption) and kidney (to augment reabsorption), thereby restoring serum calcium levels. PTH-(1–84) is usually the major form of PTH secreted by the parathyroid glands. However, recent analyses reveal that PTH fragments that are likely to be PTH-(7–84) are also secreted by the parathyroid glands and generated by peripheral metabolism (2, 3). These PTH fragments or their synthetic analogs are thought to be inactive on the PTH1R because, despite binding to the receptor, they fail to promote activation of the classical effectors adenylyl cyclase and phospholipase C (4–7). In fact, NH2-terminally truncated PTH fragments behave as competitive PTH antagonists (8).

As with most GPCRs, the responses of the PTH1R to agonists are regulated by multiple mechanisms, including a well characterized and highly conserved process involving receptor phosphorylation by G protein-coupled receptor kinase 2 (9, 10) and arrestin recruitment (11–13). These processes contribute directly to PTH1R desensitization by facilitating the uncoupling of the receptor from its cognate G proteins, Gs and Gq. Following desensitization, the PTH1R is endocytosed into intracellular compartments, from which it can be either recycled to the membrane, leading to receptor resensitization (14), or targeted for degradation, leading to receptor down-regulation (15, 16).

Increasing evidence demonstrates that PTH1R activation and endocytosis can be dissociated, with each event requiring distinct and specific receptor conformational states. PTH peptide analogs that efficiently activate the PTH1R but fail to induce arrestin-mediated internalization have been described (17). Conversely, PTH1R mutants have been generated that exhibit impaired ability to transduce G protein-mediated signaling but are phosphorylated by G protein-coupled receptor kinase 2 and internalized in response to PTH-(1–34) (18). These observations raise the possibility that PTH analogs that are unable to activate the PTH1R may be capable of inducing receptor endocytosis. In the present work, we tested this hypothesis. We now show that amino-terminally truncated PTH peptides internalize the PTH1R without antecedent or concomitant activation. We uncovered the molecular mechanism underlying this novel phenomenon and found that it occurs in a cell-specific manner that depends on the expression of the scaffolding protein EBP50, also known as NHERF1.2

NHERF1 is a cytoplasmic adaptor protein that contains tandem PDZ domains that have been implicated in protein targeting and in the assembly of protein complexes (19, 20). NHERF1 also possesses an ERM domain, which binds to the actin-associated proteins, ezrin, radixin, moesin, and merlin (21). NHERF1 recruits various cellular receptors, ion transporters, and other proteins to the plasma membrane of epithelia and other cells (22). Recently, Mahon and co-workers (23) reported that NHERF1 and -2 bind to the PTH1R through a COOH-terminal PDZ recognition motif and determined a role for NHERF2 in PTH signaling. Our findings demonstrate a novel action for NHERF1 as a molecular switch that determines the conditional efficacy of PTH fragments in bone and kidney cells. Additionally, the results establish the existence of distinct endocytic pathways for the PTH1R in response to either agonists or nonactivating PTH fragments. As such, they provide a new cellular mechanism for the regulation of GPCR function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—The preparation, subcloning, characterization, and culture conditions for kidney and bone cells were as described (24, 25). Kidney cells were grown in a 50:50 mix of Dulbecco's modified Eagle's medium/Hamm's F-12 (10-092-CV; Mediatech, Inc., Herndon, VA), supplemented with 5% heat-inactivated fetal calf serum (Invitrogen) and 1% PSN (5 mg of penicillin, 5 mg of streptomycin, and 10 mg of neomycin/ml; Invitrogen). SaOS2 cells were grown in RPMI supplemented with 10% fetal calf serum. HEK-293 and ROS cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transient transfections of HEK-293 cells grown to 75–90% confluence were performed using FuGENE 6TM (Roche Applied Science) according to the manufacturer's instructions. Empty pcDNA3 vector was added to keep constant the total DNA amount added per plate. Cells grown on 100-mm (DCT) and 60-mm (ROS) plates were transfected using 6 µg of total DNA. DCT and ROS cells stably expressing NHERF1 were generated by transiently transfecting NHERF1 cDNA in pcDNA 3.1 Hygro using FuGENE 6TM. After 48 h, transiently transfected cells were trypsinized and plated on 150-mm dishes containing culture medium supplemented with 300 µg/ml hygromycin (Invitrogen) to select stable transfectants.

Complementary DNA Constructs—pEGFP-N2 plasmid encoding a full-length human PTH1R carboxyl-terminal EGFP fusion protein (PTH1R/C-EGFP) was kindly provided by C. Silve (INSERM, Paris, France). The PTH1R with EGFP introduced in the E2 extracellular domain (PTH1R/N-EGFP) has been previously described (26).

Mutation of the terminal amino acid of PTH1R, in PTH1R/C-EGFP, from methionine to alanine (M593A) was performed by PCR using the QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA). The fidelity of the construct was confirmed by sequencing (ABI PRISM 377; Applied Biosystems, Foster City, CA) and subsequent sequence alignment (NCBI BLAST) with human PTH1R (GenBankTM accession number XM 002895).

Cyclic AMP and Inositol Phosphate Assays—Ligand-stimulated accumulation of cAMP was determined in the presence of 1 µM 3-isobutyl-1-methylxanthine. Phosphatidylinositol hydrolysis was determined in the presence of 10 mM LiCl. cAMP and total inositol phosphates were measured chromatographically as previously described (17).

Receptor Internalization—PTH1R internalization was measured by quantitative, live cell confocal fluorescence microscopy in cells stably transfected with the hPTH1R-EGFP. Cells were plated on poly-D-lysine-coated 25-mm glass coverslips and analyzed at room temperature by confocal microscopy (Amersham Biosciences) equipped with a 488-nm argon/krypton laser. Emitted fluorescence was detected with a 515–540-nm band pass filter. PTH1R/EGFP internalization was analyzed by selecting the entire plasma membrane through a plane normal to and ~2–3 µm above the basal membrane surface (ImageScan; Amersham Biosciences). 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 15–30 min to ensure that internalization was complete with any given maneuver. Internalization of PTH1R/EGFP was reflected by a loss of plasma membrane fluorescence, quantified as changes in pixel intensity. Fluorescence intensity was digitized at 16-bit resolution and converted to 256 grayscale levels for each image. The product of the number of pixels within the defined membrane area and the average pixel intensity was calculated for each time point. Kinetic parameters were determined by fitting the data to a sigmoidal nonlinear equation, where PTH1R internalization = bottom + (top - bottom)/(1 + 10(log EC50 - log[PTH])) and plotted using Prism (GraphPad Software, Inc.). Results are presented as the mean ± S.E. for the indicated number of independent observations.

Radioligand Binding and Internalization—Cells (100,000–200,000) prepared as described above were incubated on ice for 2 h with ~100,000 cpm of high pressure liquid chromatography-purified [125I][Nle8,18,Tyr34]PTH-(1–34)NH2 in 250 µl of Dulbecco's modified Eagle's medium/F-12 medium containing 5% fetal bovine serum, essentially as described (27, 28). In brief, cells grown to confluence in 24-well plates were incubated for 2 h at room temperature (to achieve equilibrium binding). Under these conditions, the concentration of radioligand was ~0.1 nM. Following incubation, the cells were washed twice with ice-cold phosphate-buffered saline and collected in 0.5 ml of 0.1 N NaOH, and bound [125I]PTH was assessed by {gamma} spectrometry. Ligand internalization was measured as follows. Cells (100,000–200,000) were washed twice with ice-cold phosphate-buffered saline and incubated in 0.5 ml of Dulbecco's modified Eagle's medium/F-12 medium containing 5% fetal bovine serum at room temperature. At the indicated time points, surface-bound ligand was extracted by two 5-min incubations on ice with 50 mM glycine buffer (pH 3.0) containing 0.1 M NaCl. After acid extraction, the remaining cell-associated (internalized) radioligand was collected in 0.5 ml of 0.1 N NaOH. The amount of radioligand in each fraction was assessed by {gamma} spectrometry. Radioligand internalization is expressed as the ratio (percentage) of internalized fraction over the total cell-associated ligand (surface plus internalized). Nonspecific binding and internalization were measured in parallel experiments carried out in the presence of 1 µM unlabeled PTH-(1–34). Curves were fit using a four-point logistic algorithm (Prism, GraphPad Software, San Diego, CA).

Arrestin Translocation—DCT cells grown on 100-mm dishes were transiently transfected with 1 µg of {beta}-arrestin-2/GFP and 5 µg of hPTH1R-pcDNA3 (courtesy of Dr. Marc Caron, Duke University). After 48 h, the cells were split onto collagen-coated 25-mm coverslips. Arrestin translocation in response to PTH ligands was assessed at room temperature using real time live cell confocal microscopy as reported (29).

Dynamin Dependence—DCT cells were split onto 25-mm coverslips and transiently transfected with 1 µg of PTH1R/EGFP in the presence of 1 µg of K44A-dynamin-pcDNA3.1 (Dr. Orson Moe, University of Texas, Dallas) or empty pcDNA3.1 vector. PTH1R internalization in response to PTH ligands was then measured and quantified as outlined previously.

Coimmunoprecipitation—Six-well plates of HEK-293 cells were transfected with the different combinations of DNA constructs as indicated. Forty-eight h after transfection, the cells were rinsed with ice-cold phosphate-buffered saline and harvested in 800 µl of lysis buffer (150 mM NaCl, 50 mM Tris, pH 8, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 10 mM Na2P2O7, 5 mM EDTA) supplemented with protease inhibitors (9 nM pepstatin, 9 nM antipain, 10 nM leupeptin, and 10 nM chymostatin) (Sigma). After lysis for 60 min at 4 °C, the lysates were clarified by centrifugation for 20 min at 14,000 rpm at 4 °C. Four µg of specific anti-GFP polyclonal antibody (Molecular Probes, Inc., Eugene, OR) were added to the supernatant. After a 60-min incubation at 4 °C, 50 µl of 50% protein A-agarose pre-equilibrated in lysis buffer was added, followed by an overnight incubation at 4 °C. Samples were then centrifuged for 1 min in a microcentrifuge and washed three times with 1 ml of lysis buffer. Immunoprecipitated proteins were eluted by the addition of 50 µl of SDS sample buffer followed by a 30-min incubation at room temperature. Initial lysates and immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotting using specific antibodies.

Immunoblot Analysis—Cells were grown to confluence in T-25 flasks, trypsinized, and collected by centrifugation. The resultant cell pellet was resuspended in 500 µl of Nonidet P-40 lysis buffer (50 mM Tris (pH 8), 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40). Total protein concentrations were measured (Bio-Rad Dc Protein Assay). 30 µg of lysate (solubilized in Laemmli sample buffer) were resolved on 10% polyacrylamide gels by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Immobilon-P; Millipore Corp.) according to standard methods. Membranes were blocked overnight at 4 °C with 5% nonfat dried milk in Tris-buffered saline plus Tween-20 (TBST), incubated with polyclonal anti-EBP50 antibody (Affinity Bioreagents) at 1:1000 dilution for 4 h at room temperature, washed, and incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (Pierce) at 1:5000 dilution for 1 h at room temperature. Protein bands were visualized with a luminol-based enhanced chemiluminescence substrate (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Statistics—Data are presented as means ± S.E., where n indicates the number of independent experiments. Effects of experimental treatments were assessed by paired comparisons within experiments and reported as the mean ± S.E. of n independent experiments. Paired results were compared by analysis of variance with post-test repeated measures analyzed by the Bonferroni procedure. Single comparisons to control were analyzed by Dunnett's test (Prism; GraphPad). Differences greater than p <= 0.05 were assumed to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PTH1R and PTH Endocytosis—The PTH1R and PTH-(1–34) were simultaneously localized in DCT cells stably expressing a human PTH1R COOH-terminally tagged with the enhanced green fluorescent protein (PTH1R/C-EGFP) as previously described (17, 28, 30). Cells were exposed to rhodamine-tagged PTH-(1–34) (rhoPTH(1–34) in Fig. 1). Initially, the PTH1R was largely limited to the plasma membrane, although some cytoplasmic and perinuclear fluorescence is evident (Fig. 1). The ligand was restricted to the plasma membrane. When the fluorescence images for receptor (green) and ligand (red) were merged, only PTH1R at the plasma membrane was associated with ligand (yellow). After 15 min, little PTH1R or PTH-(1–34) remained at the plasma membrane. The decreases of PTH1R and PTH fluorescence at the plasma membrane were accompanied by concomitant increases of cytoplasmic PTH1R/C-EGFP and rhodamine-labeled PTH-(1–34) fluorescence. These results are consistent with the view that PTH and the PTH1R colocalize and internalize together in response to receptor occupancy.



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  		FIG. 1.
Colocalization of hPTH1R/EGFP and PTH-(1–34). A single DCT cell stably expressing hPTH1R/EGFP is shown immediately upon (0 min) and 15 min after the addition of 10-7 M Rho-PTH-(1–34). At time 0, hPTH1R/EGFP (green) was largely membrane-delimited, although some trans-Golgi and perinuclear localization is visible. Rho-PTH-(1–34) (red) bound to PTH1R/EGFP on the cell membrane. Merge of hPTH1R/EGFP and Rho-PTH-(1–34) depicts regions where the receptor and ligand colocalize (yellow). After 15 min of treatment with Rho-PTH-(1–34), plasma membrane fluorescence was reduced, whereas fluorescence of both the PTH1R/EGFP and Rho-PTH-(1–34) increased and colocalized within the cytoplasm.

 

Cell- and Ligand-specific Internalization of the PTH1R—We determined the kinetics of PTH1R internalization by real time, quantitative confocal fluorescence microscopy monitoring of membrane-delimited fluorescence intensity of the PTH1R/C-EGFP fusion protein in live cells. Upon the addition of 10-7 M PTH-(1–34), PTH1R internalization began after a latency of 6–7 min and reached 50% at 15 min (Fig. 2A). The results obtained on single cells by fluorescent imaging were independently corroborated by measuring internalization of radiolabeled PTH-(1–34) in large (>100,000) populations of cells. Ligand internalization paralleled that of the receptor. During the first 5 min, less than 5% of [125I]PTH-(1–34) was sequestered and 50% was endocytosed by 15 min (Fig. 2B). These findings demonstrate that the PTH1R and PTH-(1–34) traffic together and are internalized in a spatially and temporally congruent manner. The results qualitatively and quantitatively validate the optical determination of PTH1R endocytosis.



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  		FIG. 2.
Ligand-induced PTH1R endocytosis. A, effect of 10-7 M PTH-(1–34) on PTH1R internalization in PCT and DCT convoluted tubule cells. Receptor endocytosis was measured by real time quantitative confocal microscopy. Confocal images were quantified at 60-s intervals as described under "Experimental Procedures." n = 3 for PCT; n = 4 for DCT. B, radioligand internalization of PTH-(1–34) in DCT cells. Results are the means ± S.D. of triplicate determinations in two independent experiments. C, cell-specific PTH1R internalization by PTH-(7–34) in DCT but not PCT cells. 10-7 M PTH-(7–34) was added at 0 min. n = 3 for DCT; n = 3 for PCT. D, effect of full-length PTH peptides (10-7 M) on PTH1R endocytosis in DCT cells. E, ligand-induced PTH1R endocytosis in bone cells. PTH-(7–34) evokes PTH1R endocytosis in ROS cells but not in SaOS2 cells. Receptor endocytosis was measured after the addition of 10-7 M PTH-(1–34) or PTH-(7–34) as described. The extent of receptor endocytosis after 15 min is depicted. n = 3 for ROS; n = 3 for SaOS2.

 

PTH-(1–34) also internalized the PTH1R in kidney proximal tubule (PCT) cells (Fig. 2A). Internalization began promptly without delay in PCT cells but by 15 min reached levels equivalent (50%) to that in DCT cells.

We next examined the effects of the PTH antagonist PTH-(7–34) on PTH1R internalization. As expected, PTH-(7–34) (10-7 M) did not promote PTH1R internalization in PCT cells (Fig. 2C). However, PTH-(7–34) promptly and efficiently induced receptor sequestration in kidney DCT cells. PTH1R endocytosis evoked by PTH-(7–34) was greater (82 ± 4.2 versus 49 ± 3.7%, p < 0.01) and more rapid (t = 2.5 versus >8 min) than that elicited by PTH-(1–34). From concentration dependence curves, half-maximal internalization (EC50) of the PTH1R occurred at 0.90 x 10-9 M PTH-(1–34) and 10-8 M for PTH-(7–34). We confirmed that PTH-(7–34) lacks agonist activity (31, 32). In DCT cells, 10-7 M PTH-(7–34) had no effect on either adenylyl cyclase or phospholipase C, whereas PTH-(1–34) activated both adenylyl cyclase and phospholipase C (21.4 ± 0.1- and 3.2 ± 0.5-fold, respectively). These results additionally substantiate that EGFP ligation to the intracellular tail of the PTH1R does not interfere with its signaling in DCT cells, as in HEK-293 cells or COS cells (17).

To verify that the observed ligand-specific internalization effects of PTH-(7–34) were not due to the presence of EGFP on the intracellular tail of the PTH1R/C-EGFP, identical experiments were performed with a PTH1R, where EGFP is located in the extracellular domain (PTH1R/N-EGFP) (26). We previously established that this receptor construct signals and traffics normally in response to PTH-(1–34) in LLC-PK1 kidney cells (26). PTH1R/N-EGFP internalization induced by PTH-(7–34) was 76 ± 8.7% at 15 min and did not differ from that of the PTH1R/C-EGFP, 81 ± 4.2%. PTH-(1–34) also efficiently internalized the PTH1R/N-EGFP. Thus, the functional properties of the PTH1R/C-EGFP and PTH1R/N-EGFP are not different from the native receptor.

The actions of PTH-(1–84) and PTH-(7–84) were assessed to determine if the full-length circulating forms of PTH exerted similar effects on PTH1R internalization. At comparable peptide concentrations (10-7 M), these naturally occurring forms of PTH evoked PTH1R endocytosis in DCT cells that was indistinguishable from that displayed by their respective shorter synthetic analogs (Fig. 2D).

Similar cell-specific effects of PTH-(1–34) and PTH-(7–34) on PTH1R internalization were observed in two bone-derived cell lines, human SaOS2 and rat ROS 17/2.8 cells (Fig. 2E). In SaOS2 cells, as in PCT kidney cells, only PTH-(1–34) induced PTH1R internalization. In contrast, in ROS cells both PTH-(1–34) and PTH-(7–34) induced PTH1R endocytosis, similar to kidney DCT cells.

NHERF1 Expression Determines PTH-(7–34) Effects on PTH1R Endocytosis—NHERF1 is expressed by PCT and SaOS2 cells (Fig. 3), where PTH-(7–34) has no effect on PTH1R internalization, but not by DCT or ROS cells (Fig. 3), where PTH-(7–34) induces endocytosis (33, 34). Therefore, we theorized that the presence or absence of NHERF1 determines the cell-specific pattern of internalization of PTH1R in response to inactive PTH peptides.



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  		FIG. 3.
NHERF1 expression. Immunoblot of mouse kidney, PCT, DCT, human osteosarcoma SaOS2, and rat osteosarcoma ROS 17/2.8 cells. Lanes were loaded with 5 µg (kidney) or 20 µg (cell lines) of protein per lane.

 

To test this idea, we introduced NHERF1 in cells normally lacking it and determined the effect of PTH-(7–34) on PTH1R internalization. DCT and ROS cells were stably transfected with NHERF1. DCT/NHERF1 cells exhibited PTH1R internalization in response to PTH-(1–34) similar to that seen in vector-transfected or non-transfected control cells (Fig. 4A). Now, however, PTH1R internalization in response to PTH-(7–34) was significantly attenuated (Fig. 4A). PTH-(7–34)-induced PTH1R internalization was also largely inhibited in ROS cells stably expressing NHERF1 (Fig. 4B). PTH-(1–34) and PTH-(7–34) promoted the internalization of a PTH1R tagged with EGFP in the extracellular domain (PTH1R/N-EGFP) (Fig. 4C). NHERF1 similarly inhibited PTH-(7–34)-induced internalization of the PTH1R/N-EGFP as it did the PTH1R/C-EGFP (Fig. 4C). PTH-(1–34)-stimulated PTH1R/N-EGFP endocytosis was not affected. Thus, the actions of NHERF1 are independent of the location of EGFP within the PTH1R.



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  		FIG. 4.
NHERF expression inhibits PTH1R endocytosis in DCT and ROS cells. A, effect of 10-7 M PTH-(1–34) or PTH-(7–34) on PTH1R internalization in DCT cells after 15 min in the absence (filled bars) or presence (stippled bars) of NHERF1 (n = 3). **, p < 0.01 versus DCT. B, effect of 10-7 M PTH-(7–34) on PTH1R internalization in ROS cells in the absence (filled bars) or presence (stippled bars) of NHERF1. Receptor endocytosis was measured and quantified as outlined in Fig. 1A. The results show the average ± S.E. of three independent observations for each condition. **, p < 0.01 versus ROS. C, PTH-(7–34)-induced internalization of the PTH1R with an extracellular EGFP tag (PTH1R/N-EGFP). n = 3. **, p < 0.01 versus PTH-(1–34).

 

The role of NHERF1 in determining sensitivity to PTH-(7–34) was further and independently established by expressing a dominant negative form of NHERF1 (NHERH-(1–326), NHERF1{Delta}ERM) (35, 36) in PCT cells that endogenously express NHERF1. Cells transfected with NHERF1{Delta}ERM now displayed PTH1R internalization in response to PTH-(7–34) (Fig. 5), whereas they are normally refractory to PTH-(7–34), as shown by the cells transfected with empty vector.



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  		FIG. 5.
ERM-deficient dominant negative NHERF (NHERF-(1–326)) permits PTH-(7–34) to internalize the PTH1R in PCT cells. The effect of 10-7 M PTH-(7–34) on PTH1R internalization in PCT cells in the presence or absence of NHERF-(1–326). Receptor endocytosis was measured and quantified as outlined in Fig. 1A. n = 4 for each condition.

 

Direct Interaction between NHERF1 and PTH1R Determines PTH-(7–34) Effects on PTH1R Endocytosis—The interaction between PTH1R and NHERF1 was directly demonstrated in HEK-293 cells co-expressing EGFP-tagged wild-type PTH1R with HA-tagged NHERF1. HEK-293 cells were used because they are normally devoid of PTH1R and have been previously employed for GPCR coimmunoprecipitation with NHERF1 (37, 38). Cell lysates were immunoprecipitated with a GFP-specific polyclonal antibody, and blotting was performed with an HA-specific monoclonal antibody. As shown in Fig. 6 (top), NHERF1 efficiently coimmunoprecipitated with the PTH1R. This finding also establishes that such interaction occurs constitutively (i.e. without ligand occupancy).



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  		FIG. 6.
Coimmunoprecipitation of EBP50 with PTH1R(ETVM)-EGFP and PTH1R(ETVA)-EGFP. HEK-293 cells transfected with the indicated constructs were harvested, lysed, and immunoprecipitated as described under "Experimental Procedures." Immunoprecipitation experiments were performed by incubating the cell lysates with an EGFP-specific polyclonal antibody followed by incubation with protein A-agarose. Immunoprecipitated proteins were eluted from protein A-agarose with SDS sample buffer. Eluates and cell extracts were subjected to SDS-PAGE. Immunoblotting was performed with a HA-specific monoclonal antibody (upper panel). The amount of immunoprecipitated EGFP-tagged receptor in each sample was verified by immunoblotting with the EGFP-specific polyclonal antibody (middle panel). The quantity of HA-EBP50 present in each cell extract was evaluated by immunoblotting with the HA-specific monoclonal antibody (lower panel). A representative Western blot is shown.

 

We then generated a full-length PTH1R/EGFP construct wherein the terminal methionine of the PTH1R was changed to alanine (M593A) (ETVA-PTH1R/C-EGFP), a modification that disrupts the interaction of the PTH1R with the PDZ domain of NHERF1 (23). The ETVA-PTH1R/C-EGFP was expressed in a functional form and stimulated cAMP production in response to 10-7 M PTH-(1–34) similarly to wild type PTH1R (7.5 ± 0.5- and 8.5 ± 0.7-fold above basal level for ETVA-PTH1R and wild-type PTH1R, respectively).

In contrast to the wild-type ETVM-PTH1R, however, ETVA-PTH1R coimmunoprecipitation with NHERF1 was negligible (Fig. 6, top). Immunoblotting of the cell lysates with an anti-GFP antibody showed that both wild-type and ETVA-PTH1R were expressed at similar levels (Fig. 6, middle). Likewise, immunoblotting of the cell lysates with the HA-specific antibody showed that HA-NHERF1 expression levels were similar in all conditions (Fig. 6, bottom). Thus, mutation of a single residue of the PDZ recognition domain was sufficient to disrupt the association of the PTH1R with NHERF1. The results further demonstrate that the presence of EGFP at the carboxyl terminus of the PTH1R does not occlude or interfere with the PDZ-recognition domain, binding to NHERF1, or PTH1R signaling.

The ETVA-PTH1R was used to test the hypothesis that disrupting the PDZ recognition domain of the PTH1R, which prevents binding to NHERF1, would permit internalization in response to PTH-(7–34). This was accomplished by introducing the ETVA-PTH1R/C-EGFP in PCT cells that constitutively express NHERF1. Fig. 7A shows that 10-7 M PTH-(7–34), which normally has a negligible effect on PTH1R internalization, now effectively internalized the ETVA-PTH1R. PTH-(1–34) stimulated internalization of the ETVA-PTH1R as efficiently as that of the wild-type PTH1R. Comparable results were obtained with ROS/NHERF cells (Fig. 7B). These experiments provide strong evidence that the PDZ-binding domain of the PTH1R is necessary and sufficient to mediate the association with NHERF1. Moreover, they establish that disrupting this interaction confers sensitivity to PTH-(7–34).



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  		FIG. 7.
Mutation of the carboxyl-terminal PDZ interaction domain in the PTH1R abolishes NHERF-mediated inhibition of PTH-(7–34)-induced PTH1R endocytosis. A, PCT cells expressing either the wild-type (WT) PTH1R/EGFP or mutated (ETVA) PTH1R/EGFP were challenged with 10-7 M PTH-(1–34) or PTH-(7–34) for 15 min as indicated. n = 3–4 for each condition. **, p < 0.01 versus wild-type PTH-(1–34). B, ROS/NHERF cells expressing either the wild-type PTH1R/EGFP (n = 4) or mutated ETVA-PTH1R/EGFP (n = 3) were challenged with 10-7 M PTH-(7–34), and endocytosis was measured as described. **, p < 0.01 versus ETVA-PTH1R.

 

Role of ERM Domain on PTH1R Endocytosis—NHERF1 possesses a carboxyl-terminal ERM domain. To determine whether the ERM domain of NHERF1 is required for ligand-induced PTH1R internalization, we expressed ERM-deficient NHERF1 (NHERF{Delta}ERM) in DCT cells. In contrast to full-length NHERF1, which substantially inhibited PTH1R internalization initiated by PTH-(7–34), NHERF{Delta}ERM exerted no significant inhibitory action (Fig. 8). This finding is consistent with the idea that NHERF1 tethers the PTH1R to the actin cytoskeleton through the ERM domain of NHERF1 and, in the absence of the ERM domain, the PTH1R is unconstrained. In this setting, receptor occupancy by PTH is sufficient to induce endocytosis.



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  		FIG. 8.
ERM-deficient NHERF-(1–326) does not inhibit PTH-(7–34)-induced PTH1R endocytosis in DCT cells. DCT cells were transiently transfected with PTH1R/EGFP in the presence of pcDNA 3.1 (empty vector), NHERF1, or ERM-deficient NHERF-(1–326), and PTH1R internalization was assessed after challenge with 10-7 M PTH-(7–34) for 15 min. n = 3 for each condition. **, p < 0.01 versus empty vector.

 

The ERM domain of NHERF1 binds actin-associated proteins (39). We reasoned that if the PTH1R is tethered through NHERF to ezrin and the actin cytoskeleton, disrupting the actin cytoskeleton should unleash the PTH1R from NHERF1 and permit PTH-(7–34) to internalize the receptor. We tested this theory in PCT cells because they express NHERF1 and because PTH-(7–34) normally has a minimal effect on PTH1R endocytosis. Treatment with 1 µM cytochalasin D, a membrane-permeant inhibitor of actin polymerization, fully allowed PTH-(7–34) to promote receptor internalization (Fig. 9). Since the ETVA-PTH1R does not interact with NHERF1, cytochalasin D should not interfere with PTH-(7–34)-initiated PTH1R sequestration. As predicted, actin depolymerization did not alter PTH-(7–34)-stimulated internalization of the ETVA-PTH1R (Fig. 9). In contrast to the inhibitory action of cytochalasin D on PTH (7–34)-induced PTH1R internalization, microtubule disruption with colchicine (1 µM) had no effect on PTH1R internalization (data not shown). These results support the view that the presence of an intact actin network and NHERF1 determine the fate of PTH1R trafficking in response to activating or inactivating PTH peptides.



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  		FIG. 9.
Disruption of the actin cytoskeleton permits PTH-(7–34)-induced PTH1R internalization in PCT cells. PCT cells were transiently transfected with PTH1R/EGFP or ETVA-PTH1R/EGFP. Where indicated, cells were treated with 1 µM cytochalasin D for 15 min before the addition of 10-7 M PTH-(7–34). Receptor endocytosis was then measured after 15 min and quantified as described. Results show internalization at t = 15 min.

 

PTH-(7–34)-induced PTH1R Internalization Is Independent of {beta}-Arrestin but Requires Dynamin—To define further the molecular mechanisms underlying antagonist-induced PTH1R endocytosis in DCT cells, we sought to determine whether PTH1R sequestration involves {beta}-arrestin and dynamin. DCT cells were transiently transfected with {beta}-arrestin-2-GFP and monitored by fluorescence microscopy. Under resting conditions, {beta}-arrestin-2 was uniformly distributed throughout the cytoplasm but excluded from the nucleus (Fig. 10, top). Within 5 min of the addition of 10-7 M PTH-(1–34), {beta}-arrestin-2 moved from the cytoplasm to the plasma membrane, exhibiting a characteristic punctate distribution. By 25 min, {beta}-arrestin had translocated to the cytoplasm. This observation is consistent with arrestin-mediated trafficking of the PTH1R to clathrin-coated pits for internalization. In contrast, PTH-(7–34) (10-7 M) exerted no detectable effect on {beta}-arrestin-2 movement (Fig. 10, bottom).



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  		FIG. 10.
PTH-(1–34) translocates {beta}-arrestin-2 in DCT cells. DCT cells were transiently transfected with {beta}-arrestin-2-GFP and hPTH1R as outlined under "Experimental Procedures." Real time, live cell confocal images were taken after 0, 5, and 25 min of treatment with 10-7 M PTH-(1–34) or -(7–34), as indicated.

 

DCT cells expressing PTH1R-EGFP were transfected with a dominant-negative form of dynamin, [K44A]dynamin (40). In these cells, PTH1R internalization was significantly inhibited, both in response to the agonist PTH-(1–34) and to the antagonist PTH-(7–34) (Fig. 11). Hence, whereas agonist-occupied PTH1R internalizes in a classical {beta}-arrestin- and dynamin-dependent fashion, receptor occupancy by the nonactivating analog PTH-(7–34) induces PTH1R endocytosis independently of {beta}-arrestin. Dynamin function, however, is required.



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  		FIG. 11.
PTH1R endocytosis is dynamin-dependent. DCT cells were transiently transfected with PTH1R/EGFP in the presence of dominant negative K44A-dynamin or empty vector. PTH1R internalization was analyzed after treatment for 15 min with 10-7 M PTH-(1–34) or PTH-(7–34) as indicated. n = 3 independent observations for each condition. *, p < 0.05; **, p < 0.01 versus vector plus PTH-(1–34).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A feature common to GPCRs is the cyclical process of activation, desensitization and internalization, resensitization, and recycling to the plasma membrane (41). These coordinated events protect against excessive receptor stimulation or periods of prolonged inactivity. In this manner, receptor activation, desensitization, and trafficking are thought to go hand-in-hand, thereby regulating the physiological balance of GPCR activity. In some instances, however, GPCR activation can be dissociated from subsequent desensitization and internalization. For the PTH1R, this has been shown with synthetic analogs of the PTH-related protein that stabilized an active, G protein-coupled PTH1R state. In this configuration, persistent signaling is maintained, and the receptor is not competent to interact with {beta}-arrestin-2 and is not desensitized or internalized (17). Conversely, PTH1R mutants have been generated that exhibit severely blunted signaling activities but are phosphorylated by G protein-coupled receptor kinase 2 and endocytosed in a {beta}-arrestin-2-dependent manner (18). Similar findings were reported following mutagenesis of {beta}2-adrenergic (42) and angiotensin type 1 receptors (43).

This raises the important question of whether ligands that bind but do not activate signaling through G proteins are capable of inducing receptor sequestration. In the present studies, this possibility was explored by using structural analogs of PTH on cells derived from kidney (proximal and distal tubule cells) and bone (SaOS2 and ROS cells), since these organs are major targets of PTH action. We found that PTH1R internalization occurs in response to NH2-terminally truncated fragments of PTH that do not activate signaling either through Gs or Gq proteins. Strikingly, this PTH1R internalization occurs in a markedly cell-specific fashion and proceeds through a mechanism that is distinct from that induced by PTH agonists. The agonist PTH-(1–34) activated the PTH1R, mobilized {beta}-arrestin, and internalized the receptor, as previously shown in HEK-293, COS-7, and LLC-PK1 cells (11, 44, 45). PTH-(7–34), a competitive inhibitor of the PTH1R, however, robustly promoted receptor internalization in DCT and ROS cells without accompanying activation or {beta}-arrestin-2 translocation. These results demonstrate that PTH1R activation and internalization can be dissociated in a ligand- and cell-selective fashion. Some precedent for this phenomenon may be found with the class A serotonin, endothelin, and cholecystokinin GPCRs, where both agonists and synthetic antagonists internalize these receptors (46–48). It has been suggested that the distinct biological effects of different ligands acting through a common GPCR partially depend upon their abilities to induce endocytosis (49). Thus, the dissociation between receptor activation and internalization as shown here may represent a more common biological phenomenon that contributes to ligand- and cell-specific hormone and drug action for multiple classes of GPCRs.

NHERF1 has been shown previously to affect the function of some GPCRs containing PDZ-binding motifs. NHERF1, for instance, enhances the rate of recycling of K-opioid (36) and {beta}2-adrenergic receptors (50). Disrupting the interaction of NHERF1 with the {beta}2-adrenergic receptor causes sorting of endocytosed receptor to the lysosomal pathway instead of the recycling pathway (50). Segre and co-workers (23) reported that NHERF1 and -2 bind to the PTH1R through a COOH-terminal PDZ-binding domain (ETVM) and determined a role for NHERF2 in PTH signaling. The present work illustrates a different role of NHERF1 that is distinct from its effect on receptor recycling, on the one hand, and apparently unrelated to the signaling switch, on the other.

The capacity of NHERF1 to establish cell-specific effects on PTH1R internalization was tested in three independent ways. In the first, we mutated the PDZ-binding domain of PTH1R by changing the terminal methionine to alanine (M593A). This mutation is sufficient to abolish the association of the full-length PTH1R with NHERF1 (Fig. 6). The ETVA-PTH1R was fully functional, and, as expected, in cells lacking NHERF1 was endocytosed equivalently to the wild-type PTH1R by both PTH-(1–34) and PTH-(7–34). The ETVA-PTH1R, however, was also efficiently internalized in response to PTH-(7–34) in cells expressing NHERF1, whereas the wild-type PTH1R was not. Thus, interfering with the ability of the PTH1R to associate with NHERF1 is sufficient to permit the nonsignaling PTH-(7–34) to internalize the receptor.

PTH1R fusion proteins containing EGFP within the extracellular domain or at the carboxyl-terminus of the receptor were used. These receptor constructs exhibit signaling behavior that is indistinguishable from the native receptor (26, 30). As shown here, the EGFP-tagged receptors were both internalized in response to PTH-(7–34), and this effect was absent in cells constitutively expressing or transfected with NHERF1. Further, the wild-type PTH1R sequence COOH-terminally ligated to EGFP efficiently coimmunoprecipitated with NHERF1. Single residue mutagenesis of the terminal Met of the PTH1R was sufficient to abrogate interaction of the PTH1R/C-EGFP with NHERF1. Therefore, the interaction of NHERF1 with the PTH1R/EGFP fusion protein involves the ETVM recognition motif within the PTH1R sequence. These findings further indicate that COOH-terminal ligation of EGFP does not interfere with the physical interaction or functional effects of NHERF1 with the PTH1R. In this regard, the EGFP-tagged PTH1R behaves like certain other proteins such as nNOS that recognize internal PDZ motif-mediated interactions (51). An 18-amino acid linker between the COOH terminus of the PTH1R sequence and the start of the EGFP sequence may facilitate interaction between the PDZ recognition motif and NHERF1. The two PDZ domains of NHERF1 can dimerize, preferentially through homologous binding interactions (52, 53). NHERF1 dimerization may permit interaction with non-canonical COOH-terminal PDZ recognition motifs as in the PTH1R/EGFP fusion protein.

The second strategy to examine the role of NHERF1 in regulating PTH1R endocytosis involved using a truncated form of NHERF1 that lacks the ERM domain (NHERF-(1–326)) but contains both PDZ domains. In cells expressing NHERF1, NHERF-(1–326) exerted a dominant-negative function and permitted PTH-(7–34) to internalize the PTH1R. Furthermore, whereas introduction of full-length NHERF1 in cells normally lacking it suppressed the effect of PTH-(7–34) (Fig. 4, A and B), expression of NHERF-(1–326) alone had no effect (Fig. 8). These results further establish a role for the NHERF1 ERM domain in tethering the PTH1R and indicate that interfering with the association between PTH1R, NHERF1, and the actin cytoskeleton allows the occupied, but not activated, receptor to be endocytosed.

ERM proteins such as NHERF1 contain an F-actin binding site in the COOH-terminal 28 residues (39). Ezrin, a member of the ERM family, cross-links the actin cytoskeleton to the plasma membrane. Ezrin is abundantly expressed at the apical brush-border membrane of proximal tubules, the site of NHERF1 localization (34, 54, 55). Ezrin is likewise expressed by osteosarcoma cells (56). Therefore, the third tactic that was applied to test the role of NHERF1 in determining the effects of inactive ligands on PTH1R internalization was to disrupt the actin cytoskeleton. Application of cytochalasin D to cells possessing NHERF1 allowed PTH-(7–34) to internalize the PTH1R. This effect was quite specific for the actin cytoskeleton, since microtubule disruption with colchicine had no effect on PTH1R internalization. These independent approaches provide strong evidence that NHERF1 dictates the response of the PTH1R to occupancy by nonactivating PTH peptides. In cells lacking NHERF1, PTH1R occupancy is sufficient to promote receptor internalization without prior or concurrent activation.

The mechanism of PTH1R internalization in response to PTH-(7–34) in cells lacking NHERF1 is, at least in part, different from that commonly employed by the same receptor in response to agonists. Agonist-induced endocytosis of the PTH1R occurred in an arrestin-dependent manner. In the case of nonactivating analogs, PTH1R sequestration proceeds in a {beta}-arrestin-independent manner. In both instances, however, internalization requires dynamin. This distinct internalization pathway is operative in cells lacking NHERF1 or in cells expressing NHERF1{Delta}ERM. Taken together, these findings suggest that the role of NHERF1 is not necessarily an "active" one but rather that the interactions between the PTH1R, NHERF1, and cytoskeleton (through the ERM domain) confer sufficient membrane stability on the PTH1R to require full agonist occupancy for internalization. This possibility is supported by the observation that the interaction between PTH1R and NHERF1 is constitutive, since both proteins coimmunoprecipitated from preparations of nontreated cells.

It is now apparent that PTH1R activation, desensitization, and internalization can be dissociated with each event requiring distinct receptor conformational states. These specific states can be selectively stabilized by appropriate modifications of the ligand. PTH analogs containing specific modifications at the NH2 terminus have been shown to efficiently activate the PTH1R, but they fail to induce arrestin-mediated internalization (17). The postactivation response of the PTH1R depends on specific interactions between the NH2-terminal activation domain of the ligand and the third extracellular loop of the receptor (17). These interactions are distinct from those necessary for activation of G protein-mediated signal transduction. The present work shows that the presence of the adaptor protein NHERF1 and its interaction with the PTH1R legislates the cell-specific pattern of PTH1R internalization in response to otherwise inactive PTH fragments. Taken together, these observations indicate that PTH1R activation and desensitization/endocytosis are mediated through distinct structural states that derive from specific interactions between ligand and receptor. Thus, agonist- or antagonist-occupied receptor states induce distinct conformations or accessibility to intracellular domains. The differential or inducible involvement of these domains in coupling to G proteins may represent a molecular basis for ligand-selective responses not only for the PTH1R but also for other GPCRs. In the case of the PTH1R, these conformational states also depend on interactions between the PTH1R and NHERF1 at the cytoplasmic surface.

In addition to its relevance to GPCR regulation and trafficking, the present studies may have implications for understanding extracellular calcium homeostasis. After synthesis of mature PTH-(1–84), the protein is concentrated in secretory vesicles and granules. Morphologically distinct granule subtypes contain both PTH and the proteases cathepsin B and cathepsin H (59). The co-localization of proteases and PTH in secretory granules may explain the observation that a portion of the PTH secreted from parathyroid glands consists of amino-terminally truncated PTH fragments (60). These fragments do not activate the PTH1R. Therefore, the intracellular fragmentation of PTH is thought to represent an inactivating pathway to dispose of "excess" peptide in situations such as hypercalcemia (61, 62). As shown here (Fig. 2D), PTH-(1–84) and PTH-(7–84) exerted actions on PTH1R internalization comparable with their shorter respective synthetic analogs. This novel finding suggests that PTH-(7–84) is not so much an inactive peptide as an inactivating protein.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health (NIH) Grants DK-54171 (to P. A. F.) and DK-62078 (to A. B.) and the Canadian Institutes of Health Research (CIHR) and the Kidney Foundation of Canada (J-L. P.). 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

|||| Supported in part by NIH Training Grant DK54171. Back

|| Recipient of a CIHR doctoral award. Back

¶¶ To whom correspondence should be addressed: University of Pittsburgh School of Medicine, Dept. of Pharmacology, E-1347 Biomedical Science Tower, Pittsburgh, PA 15261. Tel.: 412-383-7783; Fax: 412-648-1945; E-mail: paf10{at}pitt.edu.

1 The abbreviations used are: PTH, parathyroid hormone; PTH1R, type 1 PTH and PTH-related peptide receptor; hPTH1R, human PTH1R; GPCR, G protein-coupled receptor; EBP50, ezrin-binding protein 50; NHERF1, Na/H exchange regulatory factor 1; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; DCT, distal convoluted tubule; PCT, proximal convoluted tubule; SaOS, human osteosarcoma cells; ROS, rat osteosarcoma cells; HA, hemagglutinin. Back

2 In the present work, we use the terms NHERF1 (EBP50) and NHERF2 (E3KARP) to distinguish between the two forms of NHERF. Back


   ACKNOWLEDGMENTS
 
HA-NHERF1 was generously provided by Dr. Mark von Zastrow (University of California, San Francisco), {beta}-arrestin-2-GFP was kindly contributed by Dr. Marc Caron, and K44A dynamin was provided by Dr. Orson Moe. We are especially grateful to Dr. Simon Watkins (University of Pittsburgh School of Medicine, Center for Biologic Imaging) for assistance and advice.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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