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Endocrinology Vol. 140, No. 3 1294-1300
Copyright © 1999 by The Endocrine Society

ARTICLES

Role of Signal Transduction in Internalization of the G Protein-Coupled Receptor for Parathyroid Hormone (PTH) and PTH-Related Protein1

Zhengmin Huang2, Tom Bambino, Ying Chen3, Jelveh Lameh4 and Robert A. Nissenson5

Endocrine Research Unit (Z.H., T.B., Y.C., R.A.N.), Veterans’ Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, California 94121; and Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry (J.L.), School of Pharmacy, University of California, San Francisco, California 94143

Address all correspondence and requests for reprints to: Dr. Robert A. Nissenson, Virginia Medical Center (111N), 4150 Clement Street, San Francisco, California 94121. E-mail: chicago{at}itsa.ucsf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
For G protein-coupled receptors, limited information is available on the role of agonist binding or of the second-messenger products of receptor signaling on receptor endocytosis. We explored this problem using the opossum PTH/PTH-related protein (PTHrP) receptor, a prototypical Class II G protein-coupled receptor, as a model. In one approach, we evaluated the endocytic properties of mutated forms of the opossum PTH/PTHrP receptor that we had previously shown to be impaired in their ability to initiate agonist-induced signaling when expressed in COS-7 cells. A point mutation in the third cytoplasmic loop (K382A) that severely impairs PTH/PTHrP receptor signaling significantly reduced internalization, whereas two mutant receptors that displayed only partial defects in signaling were internalized normally. To explore more directly the role of second-messenger pathways, we used a cleavable biotinylation method to assess endocytosis of the wild-type receptor stably expressed in human embryonic kidney (HEK) 293 cells. A low rate of constitutive internalization was detected (<5% over a 30-min incubation at 37 C); the rate of receptor internalization was enhanced about 10-fold by the receptor agonists PTH(1–34) or PTHrP(1–34), whereas the receptor antagonist PTH(7–34) had no effect. Forskolin treatment produced a minimal increase in constitutive receptor endocytosis, and the protein kinase (PK)-A inhibitor H-89 failed to block agonist-stimulated endocytosis. Similarly, activation of PK-C, by treatment with phorbol 12-myristate 13-acetate, elicited only a minimal increase in constitutive receptor endocytosis; and blockade of the PK-C pathway, by treatment with a bisindolylmaleimide, failed to inhibit agonist-induced receptor endocytosis. Immunofluorescence confocal microscopic studies of PTH/PTHrP receptor internalization confirmed the results using receptor biotinylation. These findings suggest that: 1) agonist binding is required for the efficient endocytosis of the PTH/PTHrP receptor; 2) receptor activation (agonist-induced receptor conformational change) and/or coupling to G proteins plays a critical role in receptor internalization; and 3) activation of PK-A and PK-C is neither necessary nor sufficient for agonist-stimulated receptor internalization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RECEPTOR-MEDIATED endocytosis is an important cellular process that serves many biological functions, from uptake of nutrients to regulation of receptor signaling (down-regulation and desensitization/resensitization) (1, 2). There are two types of receptor-mediated endocytosis: constitutive endocytosis and ligand-induced endocytosis. Constitutive endocytosis is commonly used by transport receptors, such as transferrin receptors, which internalize and recycle to the plasma membrane independent of ligand binding. Many signaling receptors [e.g. G protein-coupled receptors (GPCRs)] are known to be rapidly endocytosed in the presence of agonist but to undergo little, if any, constitutive endocytosis (3, 4, 5, 6, 7, 8, 9). Thus, at least for these signaling receptors, activation of the receptor is associated with (and may be required for) efficient endocytosis. This is further suggested by the observation that targeted mutations that prevent or inhibit receptor signaling can reduce the efficiency of agonist-stimulated receptor endocytosis (10, 11, 12, 13, 14).

Endocytosis of GPCRs generally occurs via association with clathrin-coated pits (15, 16, 17, 18, 19, 20, 21, 22). A number of mechanisms have been suggested to contribute to agonist-stimulated association of these receptors with the coated pit and their consequent internalization, including the presence of cytoplasmic endocytic motifs (7, 17, 23), arrestin binding to phosphorylated receptors (24, 25), and ubiquitination of the receptor (26, 27). However, little direct attention has been paid to the possible role of the second-messenger products of receptor activation on the endocytosis of GPCRs. This is of particular interest, in light of recent observations that activation of second-messenger pathways, such as protein kinases (PK)-A and PK-C, can regulate the endocytosis of cell surface receptors (28, 29).

The receptor for PTH and PTH-related protein (PTHrP) is a class II GPCR that undergoes phosphorylation (30, 31) and rapid internalization (17, 32, 33, 34, 35, 36) upon agonist-binding. In addition, the agonist-occupied receptor couples to the G proteins Gs and Gq, leading to increased cellular levels of cAMP, inositol trisphosphate (IP3), and intracellular free calcium (Cai2+) (37, 38). In the present study, we used biochemical and morphological approaches, to evaluate the role of PTH/PTHrP receptor activation in promoting receptor endocytosis. The results suggest that maximal rates of receptor endocytosis require an active conformation of the receptor but do not require the participation of the second-messenger products of receptor signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Synthetic bovine PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and human PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) were purchased from Bachem California, Inc. (Torrance, CA). Restriction enzymes and Taq polymerase were purchased from Life Technologies (Gaithersburg, MD). Sequenase 2.0 sequencing kit was obtained from USB Corp. (Cleveland, OH). Cell culture media and reagents were obtained from the University of California, San Francisco (UCSF) cell culture facility. All oligonucleotides used were synthesized and purified at the UCSF Biomolecular Resource Facility. The human embryonic kidney (HEK) cell line 293-EBNA and expression vectors pcDNA1/AMP and pCEP4 were obtained from Invitrogen (San Diego, CA). Ionomycin, staurosporine, GF 109203X, and H89 were purchased from Calbiochem (La Jolla, CA). Forskolin, phorbol 12-myristate 13-acetate (TPA), diethylaminoethyl (DEAE)-Dextran, and chloroquine were purchased from Sigma Chemical Co. (St. Louis, MO). NHS-SS-biotin was obtained from Pierce Chemical Co. (Rockford, IL).

Cell culture and transfection
COS-7 cells were maintained in DMEM supplemented with 10% FBS. For transient transfection of COS-7 cells, we used a modification of the DEAE-Dextran/Chloroquine method (39). In brief, COS-7 cells, cultured in T75 flasks, were incubated for 3 h with a mixture of 5 µg plasmid DNA (opossum kidney PTH/PTHrP receptor complementary DNAs in the expression vector pcDNA1/AMP), 400 µg/ml DEAE-Dextran, and 0.1 mM chloroquine, followed by a 2-min 10% dimethyl sulfoxide shock. The following day, cells were subcultured into 6-well cluster plates for functional studies, which were carried out 72 h post transfection. For stable transfection, HEK-293 cells were used; cells were maintained in DMEM supplemented with 10% FBS. Stable transfection was carried out as described (30). Briefly, cells were grown in 10-cm dishes, to reach 10% confluence 1 day after subculturing. Ten micrograms of plasmid DNA (opossum kidney PTH/PTHrP receptor complementary DNAs in the expression vector pCEP4) was mixed with 0.5 ml 0.25 M CaCl2 and then mixed carefully with 0.5 ml sterile 280 mM NaCl/10 mM KCl/1.5 mM Na2HPO4/12 mM dextrose/50 mM HEPES (pH 7.05). The mixture was left at room temperature for 20 min, then was added dropwise to medium, bathing the cells in 10-cm dishes. After an overnight incubation at 37 C, the cells were rinsed twice with calcium-free PBS, grown for another 2 days in medium, then subcultured and subjected to selection with 200 µg/ml hygromycin for at least 3 weeks. Pooled hygromycin-resistant cells were used for subsequent functional characterization.

PTH/PTHrP receptor binding and internalization
PTH/PTHrP receptor binding and internalization studies were carried out essentially as described (17). In brief, COS-7 cells were incubated in 1 ml of media containing 20 mM HEPES, 0.1% BSA, 50,000 cpm 125I-hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)amide, plus varying concentrations of unlabeled bovine (b) PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Under these conditions, the concentration of hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) added as radioligand was approximately 0.1 nM. After a 1-h incubation at room temperature, the cells were rinsed with ice-cold PBS, collected in 1.5 ml of 0.8 N NaOH, and bound 125I-hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)amide was assessed. For internalization studies, after a 30-min incubation at room temperature with 125I-hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)amide, cells were rinsed twice with ice-cold PBS. Surface-bound ligand was then 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 radioligand was extracted by exposing cells to 0.8 N NaOH. Internalization studies in HEK-293 cells were carried out identically, except that the incubation time with 125I-hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)amide was reduced to 10 min. Receptor internalization is expressed as the percent of cell-associated radioligand remaining after acid rinsing.

Confocal laser microscopic studies of internalization of the PTH/PTHrP receptor stably expressed in HEK-293 cells
Cells were prepared for confocal microscopy, as described previously (15). Briefly, for time-course studies, HEK-293 cells stably expressing the PTH/PTHrP receptor were grown overnight at low density in complete medium on chamber slides (Nunc Inc., Naperville, IL). Cells were exposed to PTH-containing or serum-free medium at 37 C for specified times. After aspirating the medium and fixing in 50% methanol and acetone for 5 min at room temperature, cells were made permeable in PBS containing 0.25% cold-water fish gelatin, 0.04% saponin, and 0.05% NaN3 (IMF/saponin buffer). Cells were incubated at room temperature for 1 h with 1:1000 rabbit polyclonal antibody against an amino acid sequence in the N-terminal region of the opossum kidney PTH/PTHrP receptor (17), rinsed four times with PBS, then incubated with 1:500 Cy3-conjugated antirabbit antibody for 30 min at room temperature, rinsed three times with PBS and once with water, and mounted. The specificity of immunofluorescence staining was confirmed by the absence of staining in untransfected HEK-293 cells and in cells stained with secondary antibody alone (data not shown). Cells were visualized using a krypton-argon laser coupled with an MRC-600 confocal head (Bio-Rad Laboratories, Inc., Hercules, CA), as described previously (15). Optical sections along the z-axis of isolated cells were stored on 940 MB optical disc (Panasonic Inc., Secaucus, NJ). Images from a central section of the cell were compared with assess receptor internalization.

PTH/PTHrP receptor internalization, assessed by surface biotinylation
HEK-293 cells expressing PTH/PTHrP receptors were subcultured into six-well clusters and allowed to grow to confluence. Cells were rinsed four times with cold PBS, and then were subjected to two 15-min incubations with 500 µg/ml NHS-SS-Biotin (a thio-cleavable biotinylation reagent) in PBS. After two rinses with cold DMEM containing 20 mM HEPES (pH 7.4) and 0.2% BSA (incubation buffer), cells were incubated at 37 C, under various conditions, to allow PTH/PTHrP receptor endocytosis. Endocytosis was stopped by placing the cluster dishes on ice, aspirating media, and adding cold incubation buffer. After two rinses with cold PBS containing 10% FBS, surface biotin moieties were released by two 20-min incubations with 1 ml of the reducing solution (310 mg glutathione-free acid was dissolved in 18 ml of 83 mM NaCl, and 0.12 ml 50% NaOH and 2 ml FBS were added just before use). After two rinses with cold incubation buffer, free SH groups on the cell surface were quenched with a 15-min incubation with 5 mg/ml iodoacetamide in iced PBS containing 1% BSA. The media was then aspirated, and cells were incubated in 167:l buffer A (150 mM NaCl, 50 mM Tris-HCl (pH 7.5, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS) containing 1 mg/ml iodoacetamide at 4 C for 1 h, with intermittent rocking. After clearing the lysate by microcentrifugation for 15 min at 4 C, the supernatant was added to antibodies immobilized on Sepharose beads, which had been prepared as follows: 75µl protein-A Sepharose (50% vol/vol, Repligen Corp., Needham, MA) were rinsed three times with 1 ml buffer (150 mM NaCl, 20 mM Tris-HCl, pH 8.0), then incubated with 170:l anti-OK-1–13-H4 monoclonal antibody (30 µg/ml; against an epitope in the N-terminal extracellular domain of the OK PTH/PTHrP receptor) for 30 min at room temperature, and then rinsed again three times with 1 ml buffer. The lysate supernatant was incubated with Sepharose-antibody complex for 2 h in a rotator at 4 C, then rinsed six times with 0.5 ml cold buffer A. After the last aspiration, the procedure was terminated by adding 100:l SDS-PAGE sample buffer, and an aliquot of 20:l was then resolved by 8% SDS-PAGE. The gels were run at 200 V for 40 min; soaked in 20 mM Tris, 192 mM glycine, 10% methanol for 30 min; and electroblotted onto a nitrocellulose filter for 1 h at 100 V. The blots were then blocked for 1 h at room temperature in calcium-magnesium-free PBS containing 0.1% Tween and 5% nonfat dry milk. After 3 rinses (calcium-magnesium-free PBS and 0.1% Tween), the blots were incubated with streptavidin conjugated with horseradish peroxidase in rinse buffer at room temperature for 1 h. After three rinses with rinse buffer, the blots were then incubated with the Amersham ECL detection agents and developed for appropriate lengths of time.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We have recently introduced targeted mutations in the third intracellular loop of the PTH/PTHrP receptor that variably disrupt signal transduction without affecting high-affinity agonist binding (40). One of these (L379A) resembled the wild-type (wt) receptor, with respect to agonist-stimulated cAMP production, but displayed reduced levels of agonist-stimulated inositol phosphates (30% of wt). A second mutant (T381A) showed modest decreases in signaling through both pathways (60–80% of wt). A third mutant (K382A) was markedly defective, with respect to both pathways (<=10% of wt). The impact of these mutations on agonist-dependent receptor internalization was assessed in transiently-transfected COS-7 cells (Fig. 1Go). After a 30-min incubation at room temperature with 125I-labeled PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), 50% of the bound radioligand was internalized by the wt receptor. No significant differences in radioligand internalization, compared with wt, were obtained with the first two mutant receptors. However, the mutant most severely uncoupled from signal transduction (K382A) displayed a significant (30%) decrease in radioligand internalization. A similar reduction in receptor internalization was evident in studies of the K382A PTH/PTHrP receptor expressed in HEK-293 cells (data not shown).



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  		Figure 1. Effects of site-directed mutations, in the third intracellular loop, on internalization of the PTH/PTHrP receptor transiently expressed in COS-7 cells. Internalization was measured as the uptake of 125I-PTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ), as described in Materials and Methods. Data are normalized to the internalization displayed by the wt receptor, defined as 100%. The asterisk denotes that the percent of internalization differed significantly from that displayed by the wt receptor (P < 0.05).

 
These results raised the possibility that the second-messenger products of receptor signaling contribute to the full endocytic activity of the wt receptor. To investigate this, we developed a procedure for directly assessing endocytosis of the PTH/PTHrP receptor in HEK-293 cells stably expressing the wt PTH/PTHrP receptor (Fig. 2Go). A thio-cleavable cross-linking reagent, NHS-SS-Biotin, was used to label cell surface proteins, and receptor internalization was then allowed to occur under various conditions. Surface biotinylation had no effect on either the affinity of the wt PTH/PTHrP receptor for bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) or on the receptor-mediated internalization of 125I-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (data not shown). After treating cells with cell-impermeant glutathione, to release biotin from noninternalized proteins, total cell lysates were subjected to immunoprecipitation with a monoclonal antibody against a peptide epitope in the N-terminal extracellular domain of the PTH/PTHrP receptor (30). Immunoprecipitates were subjected to SDS-PAGE, blotted, and probed with streptavidin-peroxidase. A single biotin-labeled band of Mr 80 kDa was identified (Fig. 3AGo), and this conforms to the reported Mr of the wt PTH/PTHrP receptor (30). As expected, all of the biotin-labeled receptor remained at the cell surface when cells were maintained at 4 C, as evidenced by the susceptibility of the receptor to glutathione treatment. When biotin-labeled cells were incubated for 30 min at 37 C, a low level of constitutive internalization of the receptor was detected; receptor internalization was enhanced severalfold by the presence of bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (1 µM).



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  		Figure 2. A schematic diagram of the receptor internalization assay, using a thio-cleavable biotin. A, Structure of the thio-cleavable biotin derivative, N-hydroxysuccinimidyl-SS-biotin; B, schematic representation of the receptor internalization assay using the thio-cleavable biotin derivative (see text for details).

 


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  		Figure 3. Characteristics of constitutive and agonist-stimulated PTH/PTHrP receptor endocytosis in HEK-293 cells. Total cellular biotinylated PTH/PTHrP receptors are evaluated in cells not exposed to glutathione (- GSH); internalized biotinylated PTH/PTHrP receptors are evaluated in cells exposed to glutathione (+ GSH). A, Temperature-dependence of PTH/PTHrP receptor endocytosis in cells treated without or with 0.24 µM bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) for 30 min. Streptavidin blots of duplicate samples electrophoresed in adjacent lanes are shown. B, Time-course for internalization of PTH/PTHrP receptors, at 37 C, after the addition of 0.24 µM bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ). Receptor internalization in cells expressing the wt PTH/PTHrP receptor (left) is compared with that in cells expressing a C-terminally truncated form of the PTH/PTHrP receptor (T474) previously shown to display a 50% reduction in endocytosis, evaluated by a radioligand uptake assay (15 ). C, Dose-dependence for bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 )-induced internalization of the wt PTH/PTHrP receptor at 37 C.

 
Using a radioligand internalization assay, we reported that a C-terminally truncated form of the PTH/PTHrP receptor, containing only a 16-amino acid cytoplasmic tail (T474) displayed a reduced rate of agonist-stimulated endocytosis (17). The time-courses for PTH-stimulated endocytosis of the wt and T474 PTH/PTHrP receptors, using the biotin labeling method, are compared in Fig. 3BGo. Consistent with our previous results, 50% of the wt receptor was internalized in less than 5 min, whereas the rate of internalization of T474 was reduced severalfold.

The effect of bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) on endocytosis of the wt receptor was dose-dependent, displaying an ED50 of approximately 2.4 nM (Fig. 3CGo). This is virtually identical to the equilibrium dissociation constant for bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) binding to the wt PTH/PTHrP receptor in these cells, and is an order of magnitude greater than the ED50 for bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)-stimulated cAMP production (30).

In contrast to the ability of the agonists bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) to drive endocytosis of the wt PTH/PTHrP receptor, treatment with the receptor antagonist PTH(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) was ineffective (Fig. 4AGo). These results, together with those in Fig. 1Go, suggest that receptor activation is a prerequisite for efficient endocytosis. Because receptor activation leads to signaling through both the cAMP and PL-C pathways, the second-messenger products of either or both of these pathways could contribute to agonist-stimulated receptor endocytosis. However, forskolin treatment of 293 cells expressing the wt PTH/PTHrP receptor minimally enhanced receptor endocytosis, and phorbol ester treatment also had only a modest effect (Fig. 4BGo). Thus, neither increased levels of cAMP nor activation of PK-C was sufficient to replicate the high rate of agonist-stimulated PTH/PTHrP receptor endocytosis. Interestingly, both forskolin and TPA produced a small (15%) increase in the receptor-mediated internalization of 125I-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)amide in 293 cells expressing the wt PTH/PTHrP receptor (data not shown). However, blockade of the PK-A and PK-C pathways by the addition of H-89 and the bisindolylmaleimide GF 109203X, respectively, had only a slight inhibitory effect on agonist-stimulated endocytosis of the wt PTH/PTHrP receptor (Fig. 4CGo). Small (10%) inhibitory effects of H-89 and GF 109203X were also seen in studies of the receptor-mediated internalization of 125I-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)amide in HEK-293 cells expressing the wt PTH/PTHrP receptor (data not shown).



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  		Figure 4. Relationship between PTH/PTHrP receptor signaling and endocytosis in HEK-293 cells stably expressing the wt PTH/PTHrP receptor. A, Comparison of the effects of PTH/PTHrP receptor agonists (bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) and PTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 )) vs. an antagonist [PTH(7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 )]; B, effects of forskolin (FSK, 100 µM) and/or TPA (400 nM), relative to that of bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (0.24 µM) on PTH/PTHrP receptor endocytosis; C, lack of effect of inhibitors of PK-A (H89, 10 µM) and PK-C [the bisindolylmaleimide (Bis) GF 109203X, 2 µM] on bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 )-stimulated PTH/PTHrP receptor internalization.

 
Confocal immunofluorescence imaging studies were carried out to confirm these biochemical findings. At steady state, wt PTH/PTHrP receptors were located predominantly on the cell surface, and agonist stimulation caused a time-dependent increase in receptor internalization, as evidenced by the appearance of small fluorescent intracellular vesicles (Fig. 5AGo). No consistent difference in the pattern of intracellular fluorescence was seen in cells treated with bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) vs. PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). In contrast to the actions of PTH/PTHrP receptor agonists, agents that activate PK-A (forskolin) or PK-C (TPA) failed to induce the appearance of the wt PTH/PTHrP receptor in endocytic vesicles (Fig. 5BGo).



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  		Figure 5. Endocytosis of the PTH/PTHrP receptor assessed by confocal immunofluorescence microscopy. A, Time course for receptor internalization after the addition of 0.24 µM bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ); B, PTH/PTHrP receptor localization in untreated HEK-293 cells (A) and in cells treated for 10 min at 37 C with 0.24 µM bPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (B), 0.24 µM PTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (C), 100 µM forskolin (D), or 400 nM TPA (E).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In recent years, much has been discovered concerning the mechanisms that underlie the endocytosis of cell surface receptors via clathrin-coated pits (1, 2). Constitutively internalizing receptors (e.g. transferrin receptors) contain cytoplasmic sequences, the prototype of which is the sequence Tyr-X-X-hydrophobic, which directly mediate the interaction of the receptor with subunits of the AP-2 complex of the coated pit. Many signaling receptors, including the PTH/PTHrP receptor, also contain such endocytic motifs (17); but in general, the endocytic function of these sequences is manifest only upon agonist binding to the receptor. This suggests that activation of the receptor is crucial to endocytosis of signaling receptors. It is therefore conceivable that activation of downstream second-messenger pathways could contribute to agonist-induced receptor internalization.

In previous alanine-scanning mutagenesis studies of the GPCR for PTH/PTHrP, we have demonstrated that a stretch of sequence in the cytoplasmic tail that includes the sequence Tyr-Gly-Pro-Met is required for optimal receptor-mediated endocytosis (17). The present study was designed to determine whether PTH/PTHrP receptor endocytosis is agonist-dependent and to evaluate the role of receptor activation and the products of receptor signaling in this process. From our results, we conclude that: 1) efficient endocytosis of the wt PTH/PTHrP receptor is dependent upon agonist binding; 2) the ability to effectively signal is a determinant of receptor endocytosis; and 3) the basis for the latter is largely independent of any direct role of receptor-mediated activation of PK-C or PK-A on receptor endocytosis.

A novel feature of these studies was the use of biotin labeling with a cleavable reagent to investigate the endocytosis of a GPCR. This strategy allowed us to demonstrate that, when expressed in HEK-293 cells, the wt PTH/PTHrP receptor undergoes a low (but detectable) rate of constitutive internalization, which is enhanced severalfold by the addition of the agonists PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) but not by the competitive antagonist PTH (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). A number of observations support the validity of this mode of assessing PTH/PTHrP receptor endocytosis. Internalization had a rapid onset, with 50% internalization in less than 5 min after the addition of agonist at 37 C; and no internalization occurred at 4 C. In addition, endocytosis was reduced (but not abolished) in HEK-293 cells expressing a mutated form of the PTH/PTHrP receptor (T474) lacking all but 16 residues in the cytoplasmic tail. These findings are in agreement with our previous results using receptor-mediated uptake of radiolabeled agonist as an index of receptor internalization (17).

The previous identification, by us, of mutated forms of the PTH/PTHrP receptor that displayed deficits in signaling via the Gq/phospholipase C and/or Gs/adenylyl cyclase pathway (40) allowed us to assess whether such deficits would be associated with diminished agonist-stimulated endocytosis. Indeed, a receptor mutation (K382A) that severely impaired both phospholipase C and adenylyl cyclase signaling also produced a significant (30%) reduction in receptor-mediated ligand internalization. Two additional mutations that produced lesser defects in receptor signaling resulted in little or no reduction in receptor internalization. These results, while correlative, suggest that G protein activation by the receptor may be requisite for optimal agonist-driven receptor endocytosis. Similar findings have been reported for other GPCRs (10, 11, 12, 13, 14).

One way in which G protein activation might contribute to receptor endocytosis is via the second-messenger products generated by effector targets of G proteins. Because the PTH/PTHrP receptor is known to couple to the activation of both adenylyl cyclase and phospholipase C, these second-messenger pathways were distinct candidates to mediate receptor endocytosis in response to agonist binding. However, when endocytosis was evaluated, using the surface biotinylation method, treatment with either forskolin (to raise cAMP levels) or PMA (to stimulate PK-C) produced only a minimal increase in PTH/PTHrP receptor endocytosis, in the absence of agonist. Furthermore, inhibitors of PK-A and PK-C (H-89 and the bisindolylmaleimide GF 109203X, respectively), under conditions where they effectively disrupt kinase activity in HEK-293 cells (28), only weakly inhibited agonist-stimulated receptor endocytosis. Similar conclusions were derived from confocal immunofluorescence studies of PTH/PTHrP receptors in these cells. Agonist binding produced a translocation of the receptor from the plasma membrane to a presumed vesicular submembranous compartment. This effect was not mimicked by the addition of forskolin or PMA. Our results are similar to those obtained for some other GPCRs where second-messenger pathways seem to play a minimal role in receptor internalization (41, 42, 43). However, the role of intracellular calcium was not investigated in the present study, and we therefore cannot rule out the possibility that agonist-induced Cai2+ mobilization contributes to PTH/PTHrP receptor endocytosis.

We did observe a weak effect of both forskolin and TPA, to increase both constitutive and agonist-dependent internalization of the PTH/PTHrP receptor, suggesting a minor role for PK-A and PK-C in regulating receptor endocytosis. Surprisingly, H-89 and the bisindolylmaleimide GF 109203X also produced a small increase in constitutive internalization of the PTH/PTHrP receptor. The mechanisms underlying these effects are unclear. Importantly, inhibition of PK-A and PK-C produced minimal inhibition of agonist-dependent PTH/PTHrP receptor internalization, further emphasizing the primary role of mechanisms independent of activation of PK-A and PK-C. Although there are examples of membrane receptor systems in which cAMP (28) and/or PK-C-activation (29) seem to play a prominent role in receptor endocytosis, the present results demonstrate that this is not the case with the PTH/PTHrP receptor.

There are a number of possible explanations for the need for the PTH/PTHrP receptor to be competent to signal in order to be endocytosed with optimal efficiency. G protein activation per se could contribute to receptor internalization independent of effector activation. Alternatively, the PTH/PTHrP receptor is known to be phosphorylated in an agonist-dependent manner by what seems to be a GRK-like mechanism (30), and this modification could enhance receptor endocytosis. Indeed, it seems that phosphorylation of the ß-adrenergic receptor by GRK-2, followed by the binding of ß-arrestin to the receptor, contributes to receptor endocytosis, perhaps via the direct association of ß-arrestin with clathrin (24, 25). Moreover, Fukayama et al. (44) recently reported that a dominant inhibitory form of GRK-2 reduces the efficiency of PTH/PTHrP receptor internalization in human osteosarcoma cells. However, we have found that targeted mutations that eliminate phosphorylation of the PTH/PTHrP receptor do not affect its ability to be endocytosed in HEK-293 cells (45). Finally, it is possible that endocytosis is not mechanistically related to the ability of the receptor to activate G proteins, but that both G protein activation and optimal endocytosis require a common agonist-induced conformational state of the receptor.

Gene knockout studies of the PTH/PTHrP receptor have indicated that the function of this gene is required for normal embryonic skeletal development and for postnatal survival (46). The receptor seems to provide morphogenetic cues in response to locally produced PTHrP in a number of organs, as well as specific endocrine homeostatic signals in response to circulating PTH in bone and kidney. Previous studies of the PTH/PTHrP receptor have suggested that homologous desensitization and agonist-induced receptor down-regulation are important mechanisms for the regulation of target cell responsiveness to PTH and PTHrP, and work on other GPCRs indicates that agonist-driven receptor endocytosis is an important component of receptor desensitization/resensitization and down-regulation. Elucidation of the molecular events by which PTH and PTHrP promote receptor internalization therefore promises to shed light on the mechanisms involved in homologous regulation of this important GPCR.


   Acknowledgments
 
The authors wish to express gratitude to Dr. Keith Mostov and his colleagues for their assistance in the use of cell surface biotinylation for the assessment of receptor endocytosis.


   Footnotes
 
1 This work was supported by a Merit Review grant from the Department of Veterans’ Affairs (to R.A.N.); by NIH Grant DK-35323 (to R.A.N.); and by NIH NRSA award DK-09187 (to Z.H.). Back

2 Current address: Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110. Back

3 Current address: Department of Pathology, University of Arkansas for Medical Science, Little Rock, Arkansas 72205. Back

4 Current address: Molecular Research Institute, Palo Alto, California 94304. Back

5 A Research Career Scientist of the Department of Veterans’ Affairs. Back

Received June 29, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mukherjee S, Ghosh RN, Maxfield FR 1997 Endocytosis. Physiol Rev 77:759–803[Abstract/Free Full Text]
  2. Mellman I 1996 Endocytosis and molecular sorting. Ann Rev Cell Devel Biol 12:575–625[CrossRef][Medline]
  3. Le Gouill C, Parent J-L, Rola-Pleszczynski M, Sta[icicr]nková J 1997 Structural and functional requirements for agonist-induced internalization of the human platelet-activating factor receptor. J Biol Chem 272:21289–21295[Abstract/Free Full Text]
  4. Petrou C, Chen L, Tashjian Jr AH 1997 A receptor-G protein coupling-independent step in the internalization of the thyrotropin-releasing hormone receptor. J Biol Chem 272:2326–2333[Abstract/Free Full Text]
  5. Shapiro MJ, Trejo J, Zeng D, Coughlin SR 1996 Role of the thrombin receptor’s cytoplasmic tail in intracellular trafficking. J Biol Chem 271:32874–32880[Abstract/Free Full Text]
  6. Chu P, Murray S, Lissin D, von Zastrow M 1997 {delta} and {kappa} opioid receptors are differentially regulated by dynamin-dependent endocytosis when activated by the same alkaloid agonist. J Biol Chem 272:27124–27130[Abstract/Free Full Text]
  7. Böhm SK, Khitin LM, Smeekens SP, Grady EF, Payan DG, Bunnett NW 1997 Identification of potential tyrosine-containing endocytic motifs in the carboxyl-tail and seventh transmembrane domain of the neurokinin 1 receptor. J Biol Chem 272:2363–2372[Abstract/Free Full Text]
  8. Hein L, Meinel L, Pratt RE, Dzau VJ, Kobilka BK 1997 Intracellular trafficking of angiotensin II and its AT1 and AT2 receptors: evidence for selective sorting of receptor and ligand. Mol Endocrinol 11:1266–1277[Abstract/Free Full Text]
  9. Naik N, Giannini E, Brouchon L, Boulay F 1997 Internalization and recycling of the C5a anaphylatoxin receptor: evidence that the agonist-mediated internalization is modulated by phosphorylation of the C-terminal domain. J Cell Sci 110:2381–2390[Abstract]
  10. Dhanwada KR, Vijapurkar U, Ascoli M 1996 Two mutations of the lutropin/choriogonadotropin receptor that impair signal transduction also interfere with receptor-mediated endocytosis. Mol Endocrinol 10:544–554[Abstract]
  11. Barak LS, MJnard L, Ferguson SSG, Colapietro A-M, Caron MG 1995 The conserved seven-transmembrane sequence NP(X)2,3Y of the G-protein-coupled receptor superfamily regulates multiple properties of the ß2-adrenergic receptor. Biochemistry 34:15407–15414[CrossRef][Medline]
  12. Van Koppen CJ, Sell A, Lenz W, Jakobs KH 1994 Deletion analysis of the m4 muscarinic acetylcholine receptor. Eur J Biochem 222:525–531[Medline]
  13. Nussenzveig DR, Heinflink M, Gershengorn MC 1993 Decreased levels of internalized thyrotropin-releasing hormone receptors after uncoupling from guanine nucleotide-binding protein and phospholipase-C. Mol Endocrinol 7:1105–1111[Abstract]
  14. Benya RV, Akeson M, Mrozinski J, Jensen RT, Battey JF 1994 Internalization of the gastrin-releasing peptide receptor is mediated by both phospholipase C-dependent and independent processes. Mol Pharmacol 46:495–501[Abstract]
  15. Tolbert LM, Lameh J 1996 Human muscarinic cholinergic receptor Hm1 internalizes via clathrin-coated vesicles. J Biol Chem 271:17335–17342[Abstract/Free Full Text]
  16. Moore RH, Sadovnikoff N, Hoffenberg S, Liu S, Woodford P, Angelides K, Trial J, Carsrud NDV, Dickey BF, Knoll BJ 1995 Ligand-stimulated ß2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes. J Cell Sci 108:2983–2991[Abstract]
  17. Huang Z, Chen Y, Nissenson RA 1995 The cytoplasmic tail of the G-protein-coupled receptor for parathyroid hormone and parathyroid hormone-related protein contains positive and negative signals for endocytosis. J Biol Chem 270:151–156[Abstract/Free Full Text]
  18. Trapaidze N, Keith DE, Cvejic S, Evans CJ, Devi LA 1996 Sequestration of the {delta} opioid receptor. J Biol Chem 271:29279–29285[Abstract/Free Full Text]
  19. Ishii I, Saito E, Izumi T, Ui M, Shimizu T 1998 Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor receptor. J Biol Chem 273:9878–9885[Abstract/Free Full Text]
  20. Gagnon AW, Kallal L, Benovic JL 1998 Role of clathrin-mediated endocytosis in agonist-induced down-regulation of the ß2-adrenergic receptor. J Biol Chem 273:6976–6981[Abstract/Free Full Text]
  21. Zhang J, Barak LS, Winkler KE, Caron MG, Ferguson SSG 1997 A central role for ß-arrestins and clathrin-coated vesicle-mediated endocytosis in ß2-adrenergic receptor resensitization. J Biol Chem 272:27005–27014[Abstract/Free Full Text]
  22. Koenig JA, Edwardson JM 1997 Endocytosis and recycling of G protein-coupled receptors. Trends Pharmacol Sci 18:276–287[Medline]
  23. Gabilondo AM, Hegler J, Krasel C, Boivin-Jahns V, Hein L, Lohse MJ 1997 A dileucine motif in the C terminus of the ß2-adrenergic receptor is involved in receptor internalization. Proc Natl Acad Sci USA 94:12285–12290[Abstract/Free Full Text]
  24. Ferguson SSG, Downey III WE, Colapietro A-M, Barak LS, MJnard L, Caron MG 1996 Role of ß-arrestin in mediating agonist-promoted G protein-coupled receptor internalization. Science 271:363–366[Abstract]
  25. Goodman Jr OB, Krupnick JG, Santini F, Gurevich VV, Penn RB, Gagnon AW, Keen JH, Benovic JL 1996 ß-Arrestin acts as a clathrin adaptor in endocytosis of the ß2-adrenergic receptor. Nature 383:447–450[CrossRef][Medline]
  26. Hicke L, Riezman H 1996 Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell 84:277–287[CrossRef][Medline]
  27. Roth AF, Davis NG 1996 Ubiquitination of the yeast a-factor receptor. J Cell Biol 134:661–674[Abstract/Free Full Text]
  28. Foti M, Carpentier JL, Aiken C, Trono D, Lew DP, Krause KH 1997 Second-messenger regulation of receptor association with clathrin-coated pits: a novel and selective mechanism in the control of CD4 endocytosis. Mol Biol Cell 8:1377–1389[Abstract]
  29. Signoret N, Oldridge J, Pelchen-Matthews A, Klasse PJ, Tran T, Brass LF, Rosenkilde MM, Schwartz TW, Holmes W, Dallas W, Luther MA, Wells TNC, Hoxie JA, Marsh M 1997 Phorbol esters and SDF-1 induce rapid endocytosis and down-modulation of the chemokine receptor CXCR4. J Cell Biol 139:651–664[Abstract/Free Full Text]
  30. Blind E, Bambino T, Nissenson RA 1995 Agonist-stimulated phosphorylation of the G protein-coupled receptor for parathyroid hormone and parathyroid hormone-related protein. Endocrinology 136:4271–4277[Abstract]
  31. Blind E, Bambino T, Huang Z, Bliziotes M, Nissenson RA 1996 Phosphorylation of the cytoplasmic tail of the PTH/PTHrP receptor. J Bone Miner Res 11:578–586[Medline]
  32. Silve CM, Hradek GT, Jones AL, Arnaud CD 1982 Parathyroid hormone receptor in intact embryonic chicken bone: characterization and cellular localization. J Cell Biol 94:379–386[Abstract/Free Full Text]
  33. Teitelbaum AP, Silve CM, Nyiredy KO, Arnaud CD 1986 Down-regulation of parathyroid hormone (PTH) receptors in cultured bone cells is associated with agonist-specific intracellular processing of PTH-receptor complexes. Endocrinology 118:595–602[Abstract]
  34. Mitchell J, Rouleau MF, Goltzman D 1990 Biochemical and morphological characterization of parathyroid hormone receptor binding to the rat osteosarcoma cell line UMR-106. Endocrinology 126:2327–2335[Abstract]
  35. Brown RC, Silver AC, Woodhead JS 1991 Binding and degradation of NH2-terminal parathyroid hormone by opossum kidney cells. Am J Physiol 260:E544–552
  36. Bergwitz C, Abou-Samra AB, Hesch RD, Jüppner H 1994 Rapid desensitization of parathyroid hormone dependent adenylate cyclase in perfused human osteosarcoma cells (SaOS-2). Biochim Biophys Acta 1222:447–456[Medline]
  37. Jüppner H, Abou-Samra A-B, Freeman M, Kong XF, Schipani E, Richards F, Kolakowski Jr LF, Hock J, Potts Jr JT 1991 A G protein-linked receptor for parathyroid hormone, and parathyroid hormone-related peptide. Science 254:1024–1026[Abstract/Free Full Text]
  38. Abou-Samra A-B, Jhppner H, Force T, Freeman MW, Kong XF, Schipani E, Urena P, Richards J, Bonventre JV, Potts Jr JT, Kronenberg HM, Segre GV 1992 Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol triphosphates and increases intracellular free calcium. Proc Natl Acad Sci USA 89:2732–2736[Abstract/Free Full Text]
  39. Chen C, Okayama H 1987 High efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752[Abstract/Free Full Text]
  40. Huang Z, Chen Y, Pratt S, Chen T-H, Bambino T, Nissenson R, Shoback DM 1996 The N-terminal region of the third intracellular loop of the parathyroid hormone (PTH)/PTH-related peptide receptor is critical for coupling to cAMP and inositol phosphate/Ca2+ signal transduction pathways. J Biol Chem 271:33382–33389[Abstract/Free Full Text]
  41. Shockley MS, Burford NT, Sadée W, Lameh J 1997 Residues specifically involved in down-regulation but not internalization of the muscarinic acetylcholine receptor. J Neurochem 68:601–609[Medline]
  42. Tolbert LM, Lameh J 1998 Antibody to the epitope tag induces internalization of human muscarinic cholinergic receptor (hm1). J Neurochem 70:113–119[Medline]
  43. Hoelscher SR, Sairam MR, Ascoli M 1991 The slow rate of internalization of deglycosylated human chorionic gonadotropin is not due to its inability to stimulate cyclic adenosine monophosphate accumulation. Endocrinology 128:2837–2843[Abstract]
  44. Fukayama S, Kong G, Benovic JL, Meurer E, Tashjian Jr AH 1997 Beta-adrenergic receptor kinase- 1 acutely regulates PTH/PTHrP receptor signalling in human osteoblastlike cells. Cell Signal 6:469–474
  45. Malecz N, Bambino T, Bencsik M, Nissenson RA 1998 Identification of phosphorylation sites in the G protein coupled receptor for parathyroid hormone. Receptor phosphorylation is not required for agonist-induced internalization. Mol Endocrinol 12:1846–1856[Abstract/Free Full Text]
  46. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M, Defize LHK, Ho C, Mulligan RC, Abou-Samra A-B, Jhppner H, Segre GV, Kronenberg HM 1996 PTH/PTHrP receptor in early development and Indian Hedgehog-regulated bone growth. Science 273:663–666[Abstract]



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