Endocytosis of Ligand-Human Parathyroid Hormone Receptor 1 Complexes Is Protein Kinase C-dependent and Involves β-Arrestin2

Endocytosis and intracellular trafficking of the human parathyroid hormone receptor subtype 1 (hPTH1-Rc) and its ligands was monitored independently by real-time fluorescence microscopy in stably transfected HEK-293 cells. Complexes of fluorescence-labeled parathyroid hormone (PTH)-(1–34) agonist bound to the hPTH1-Rc internalized rapidly at 37 °C via clathrin-coated vesicles, whereas fluorescent PTH-(7–34) antagonist-hPTH1Rc complexes did not. A functional C terminus epitope-tagged receptor (C-Tag-hPTH1-Rc) was immunolocalized to the cell membrane and, to a lesser extent, the cytoplasm. PTH and PTH-related protein agonists stimulated C-Tag-hPTH1-Rc internalization. Relocalization to the cell membrane occurred 1 h after removal of the ligand. Endocytosis of fluorescent PTH agonist-hPTH1-Rc complexes was blocked by the protein kinase C (PKC) inhibitor staurosporine but not by the specific protein kinase A inhibitorN-(2-(methylamino)ethyl)-5-isoquinoline-sulfonamide. Fluorescent PTH antagonist-hPTH1-Rc complexes were rapidly internalized after PKC activation by phorbol 12-myristate 13-acetate or thrombin, but not after stimulation of the cAMP/protein kinase A pathway by forskolin. In cells co-expressing the hPTH1-Rc and a green fluorescent protein-β-arrestin2 fusion protein (β-Arr2-GFP), PTH agonists stimulated β-Arr2-GFP mobilization to the cell membrane. Subsequently, fluorescent PTH-(1–34)-hPTH1Rc complexes and β-Arr2-GFP co-localized intracellularly. In conclusion, agonist-activated hPTH1-Rc internalization involves β-arrestin mobilization and targeting to clathrin-coated vesicles. Our results also indicate that receptor occupancy, rather than receptor-mediated signaling, is necessary, although not sufficient, for endocytosis of the hPTH1-Rc. Activation of PKC, however, is absolutely required.

signaling, and endocytosis. These issues are particularly interesting in light of recent models that propose multiple conformational states for GPCRs (21)(22)(23), which may be stabilized by different classes of ligands (i.e. agonists, partial agonists, antagonists, and inverse agonists) and may mediate distinct molecular and cellular events.
PTH is the major regulator of calcium and phosphate homeostasis and plays a central role in bone metabolism (24). Activation of the parathyroid hormone receptor subtype 1 (PTH1-Rc) by PTH stimulates both adenylyl cyclase/cAMP/protein kinase A (PKA) and the phospholipase C/inositol 1,4,5trisphosphate/cytosolic Ca 2ϩ and diacylglycerol/PKC pathways (25)(26)(27). In addition, PTH activates G protein-coupled receptor kinases, such as GPCR kinase 2 (28 -30). Which, if either, of these second messenger pathways is linked to receptor endocytosis has not been determined definitely. Previous studies have provided indirect evidence suggesting that PTH1-Rc internalization occurs upon agonist stimulation, possibly via clathrin-coated vesicles (31,32). Recovery of receptor function following blunted response to PTH stimulation has been attributed to receptor recycling to the cell surface (33). More recently, both positive and negative endocytic signals have been identified as originating in the cytoplasmic tail of the opossum PTH1-Rc, transiently expressed in COS-7 cells (32). These studies suggest that structural changes in the receptor might contribute to the internalization process. However, neither receptor phosphorylation nor protein kinase activation seemed to play a role in agonist-induced internalization of the opossum PTH1-Rc when expressed in a human cell background (30,34). Therefore, the relationship among ligand-receptor interactions, receptor activation, signaling, and endocytosis in the PTH system remains to be elucidated.
Real-time monitoring of biomolecules by fluorescence microscopy is a powerful methodology for the study of internalization and trafficking of both hormones and receptors in living cells. In the present study, we designed and synthesized fluorescence-labeled PTH agonists and antagonists, as well as an epitope-tagged human PTH1-Rc, in order to examine independently the trafficking of both receptor and ligand and to determine directly the relationships between receptor occupancy, activation of specific intracellular signaling pathways, and endocytosis of ligand-hPTH1-Rc complexes. Furthermore, by using a GFP-␤-arrestin2 fusion protein (␤-Arr2-GFP) (7), we gained insights into the involvement of this adaptor molecule in both endocytosis and trafficking of the hPTH1-Rc.

Adenylyl Cyclase Assay
Stably transfected HEK-293 cells (clones C-21, C-20, and HPTD8) were subcultured in 24-well plates and grown to confluence. Adenylyl cyclase activation by PTH agonists and inhibition of PTH-(1-34)-stimulated adenylyl cyclase activation by PTH antagonists was determined in the presence of 1 mM isobutylmethylxanthine as described (35). Cyclic AMP was isolated by the two-column chromatographic method (38). In experiments evaluating the effects of forskolin (10 -100 M), PMA (1-10 M), or staurosporine (1-5 M) on adenylyl cyclase activity, cells were incubated with these agents for 30 min at 37°C in the presence of isobutylmethylxanthine prior to PTH stimulation.

Detection of Fluorescent PTH Agonists and Antagonists in Living Cells
In order to perform real-time microscopy with fluorescence-labeled PTH ligands, C-21 cells were plated on 25-mm glass coverslips (1.5 ϫ 10 5 cells/coverslip) and cultured for 48 h. Coverslips were then rinsed twice in cold PBS and mounted in an open-air chamber. After 10 min in cold PBS/1% bovine serum albumin (blocking buffer), coverslips were incubated with fluorescence-labeled PTH agonists or antagonists for 5 min on ice, unless otherwise indicated, and the unbound ligand was subsequently removed by washing three times with cold PBS. Coverslips were then covered with 1 ml of PBS/0.1% bovine serum albumin, and the cell chamber was placed at 37°C in a temperature-controlled block for the duration of the experiment. Competition of fluorescent ligands binding was performed by preincubating the coverslips 30 min at 37°C with unlabeled PTH ligands, using concentrations as indicated in the figure legends. When co-localization of fluorescent PTH ligands with fluorescein-conjugated transferrin was investigated, coverslips were incubated for 5 min on ice with both reagents simultaneously; subsequent treatment was carried out as described above.
In some experiments, cells were treated with sucrose or with activators or inhibitors of PKA (forskolin or H8, respectively) and of PKC (PMA or staurosporine, respectively). In this case, cells on coverslips were preincubated with the pharmacological agents for 30 min at 37°C, using concentrations as detailed in the figure legends. Subsequently, these reagents were washed and cells were incubated with the fluorescent ligands as described above. Coverslips were then covered with 0.1% PBS/bovine serum albumin containing the pharmacological agents and maintained at 37°C for the duration of the observation. When thrombin was used as an activator of PKC, there was no preincubation, but coverslips were first incubated with the fluorescent ligands as described above and then treated with thrombin (1 M) at 37°C for the duration of the observation.
Fluorescence was viewed on a Nikon epifluorescence microscope (Nikon Diaphot 300®) with filters selective for rhodamine and fluorescein labeling. Micrographs were taken using a Sensys CCD® digital camera (Photometrics, Tucson, AZ) and the Image-pro Plus® software (media Cybernetics, Silver Spring, MD). Z-mode (confocal-like) analysis, in which sequential images were taken at 0.1-m intervals throughout the cell body, was used for co-localization of two differentially fluorescencelabeled entities.

␤-Arrestin2-GFP Mobilization and Trafficking in Living Cells
In order to perform real-time fluorescence microscopy of ␤-arrestin mobilization and trafficking, C-21 cells grown on glass coverslips (as described above) were transfected with 0.3 g of ␤-Arr2-GFPp(S65T)-N3 plasmid (7) (a generous gift of Dr. Marc Caron, Duke University). Twenty-four hours after transfection, coverslips were prepared (as described above) and incubated with ligands at 37°C using concentrations and durations as detailed in the figure legends. Fluorescence was viewed as described above.

Generation of a C-terminal Epitope-tagged hPTH1-Rc (C-Tag-hPTH1-Rc)
The full-length hPTH1-Rc cDNA, previously cloned into pcDNA1 (40), was amplified by polymerase chain reaction using primers designed to delete the stop codon (GenBank TM accession number L04308; nucleotides 26 -42, forward, and 1790 -1807, reverse). The 2.0-kb polymerase chain reaction product was sub-cloned into the TOPO® ligation site of the mammalian expression vector pCDNA3.1/V5/His (Invitrogen, Carlsbad, CA), in frame with the 3Ј-V5 and 3Ј-His 6 tag sequences followed by a stop codon. Integrity and orientation of the final construct were verified by dideoxynucleotide sequencing. Plasmid pCDNA3-C-Tag-hPTH1-Rc was transfected into HEK-293 cells using Fugene® (Roche Molecular Biochemicals), and stable transfectants were selected with G418 (800 g/ml). Because preliminary experiments in COS-7 cells transiently transfected with this plasmid demonstrated a 4 -6-fold increase of cAMP levels in response to 100 nM PTH-(1-34), G418-resistant clones were screened for expression of the C-Tag-hPTH1-Rc by adenylyl cyclase assay. Stable expression of the C-Tag-hPTH1-Rc was further confirmed by Western blot using mouse anti-V5 or anti-His 6 monoclonal antibodies (diluted 1:100 to 1:1000, Invitrogen, Carlsbad, CA) and horseradish peroxidase-conjugated secondary antibody (Roche Molecular Biochemicals). The specificity of both antibodies for the epitope-tagged receptor was assessed by using as controls PTH1-Rc negative HEK-293 and C-21 cells, which express ϳ400,000 hPTH1-Rc/cell.

Localization of the C-Tag-hPTH1-Rc in Fixed Cells by Indirect Immunofluorescence
HEK-293 cells stably expressing the C-Tag-hPTH1-Rc (HPTD8 cells) were grown to 50 -60% confluency on glass coverslips as described above. Cells were then treated as detailed in the figure legends, transferred to ice, rinsed in PBS, and fixed and permeabilized in neat methanol for 15 min at Ϫ20°C. Alternatively, fixation and permeabilization were performed using a 2% paraformaldehyde/0.2% Triton X-100 solution for 30 min on ice. Blocking was performed by incubating the cells for 10 min at room temperature in 4% goat serum. Primary antibody (mouse anti-V5 mAb), diluted 1:100 in PBS/1% bovine serum albumin, was applied to the specimens for 1 h at room temperature, followed by three washes with the same buffer. Rhodamine-tagged secondary antibody (goat anti-mouse (HϩL) IgG, preadsorbed against human serum (Jackson Immunoresearch, West Grove, PA)) was diluted 1:300 and applied in the same conditions as the primary antibody. Coverslips were then rinsed in PBS and mounted for immunofluorescence microscopy, which was performed as described above.
In some experiments, in order to prevent de novo protein synthesis, cells were preincubated 2 h with cycloheximide (25 g/ml) at 37°C and maintained in the presence of cycloheximide during and after treatment with PTH.
Cellular Distribution of Fluorescent PTH Agonist and Antagonist- Fig. 1 illustrates real-time monitoring of the cellular distribution of the agonist Rho-PTH-  or the antagonist Rho-PTH-(7-34) in living C-21 cells by fluorescence microscopy. In cells incubated with either ligand (10 -100 nM, 5 min on ice to prevent spontaneous internalization) and extensively washed to remove unbound ligand, fluorescence was initially well delineated on the cell membrane ( Fig. 1, a and e). The ligand distribution was hPTH1-Rc specific: it was inhibited by preincubation (30 min at 37°C) with nonfluorescent PTH-(7-34) (Fig. 1i) or PTH-(1-34) (not shown) and was undetectable in nontransfected HEK-293 cells (Fig. 1j). No change in ligand distribution occurred in cells maintained at 4°C (data not shown). However, within minutes after raising the temperature to 37°C, the agonist Rho-PTH-(1-34) formed clusters on the cell membrane and subsequently was internalized (Fig. 1, b  and c). After 50 min, there was coalescence of the intracellular fluorescence, with complete disappearance of the ligand from the cell surface (Fig. 1d). Analogous results were obtained with Fluo-PTH-(1-34) (data not shown).
Following a 5-min preincubation at 4°C, receptor-bound PTH antagonist Rho-PTH-(7-34) was observed to be evenly distributed on the surface of cells (Fig. 1, e-h). In contrast to the findings with the agonist, increasing the temperature to 37°C produced no internalization and the pattern of fluorescence remained essentially unchanged. Analogous results were obtained with Fluo-PTH-(7-34) (data not shown). Only a few isolated fluorescent dots, representing internalized Rho-PTH-(7-34), were detectable in the immediate submembranal region after 50 min at 37°C (Fig. 1h). After prolonged observation (2 h), there was no evidence of further internalization or intracellular coalescence of the fluorescence (data not shown), suggesting that the observed small fraction of internalized Rho-PTH-(7-34) probably represents a slow, constitutive cycling of hPTH1-Rc. Increasing the concentration of Rho-PTH-(7-34) (from 100 to 1000 nM), the preincubation temperature (from 4 to 25°C) and duration (from 5 to 10 min) had no further effects on the cellular distribution of the antagonist.
HPTD8 cells fixed and permeabilized with methanol were used to follow the receptor by indirect fluorescence using a monoclonal antibody directed against the epitope-Tag of the C-Tag-hPTH1-Rc and rhodamine-labeled anti-mouse IgG as secondary antibody (Fig. 2). In the absence of agonist ligand, the C-Tag-hPTH1-Rc was localized to the cell surface, and, less extensively, to the cytoplasm (Fig. 2a). One hour of incubation with 100 nM PTH-(1-34) (Fig. 2b) or PTHrP (1-34) (not shown) induced extensive internalization of the receptor. After removal of the ligand from the medium, a progressive relocalization of receptors to the cell membrane was visualized and completed within 1-2 h (Fig. 2c). Identical results were obtained in HPTD8 cells fixed and permeabilized with paraformaldehyde/Triton X-100 and treated as described above (not shown). In cells preincubated for 2 h in the presence of cycloheximide (25 g/ml) in order to inhibit de novo protein synthesis, C-Tag-hPTH1-Rcs were initially expressed on the cell membrane and less markedly in the cytoplasm, and they were internalized after PTH treatment (Fig. 2, d and e, respectively). However, under these conditions, relocalization to the cell membrane was delayed and incomplete even after 2 h (Fig. 2, f  and g).
These changes in membranal receptor population were in full accordance with the total binding capacity for the radioiodinated PTH-(1-34) analog. Time-dependent recovery of radiolabeled PTH-(1-34) binding capacity (almost back to the initial value) was observed 2 h after wash-out of the ligand (Fig. 3) in HPTD8 cells. In contrast, the recovery of radioligand binding in cycloheximide-treated HPTD8 cells (2 h at 25 g/ml) was reduced and incomplete even after 2 h following wash-out of the ligand (Fig. 3).
Clathrin-mediated Internalization of PTH with the hPTH1-Rc-In order to assess whether endocytosis of fluorescent agonist associated with the hPTH1-Rc was specifically mediated by clathrin-coated vesicles, C-21 cells were incubated with Rho-PTH-(1-34) in hypertonic medium (0.45 M sucrose). These conditions have been reported to selectively disrupt clathrin-coating of endosomes (41). Under these conditions, internalization of Rho-PTH-(1-34) was completely prevented (data not shown). Furthermore, in HPTD8 cells (expressing the C-Tag-hPTH1-Rc) co-incubated with Rho-PTH-(1-34) and fluorescein-conjugated transferrin (a marker of the clathrin-coated pit-mediated internalization pathway), intracellular co-localization of the fluorescence of both ligands was observed after 15 min at 37°C (Fig. 4). These data indicate that fluorescent PTH arrived at its intracellular locale via clathrin-coated vesicles.
Effect of PKA and PKC on Endocytosis-The following experiments were carried out in order to examine the role of PKA and PKC activation on internalization of the hPTH1-Rc and its ligands. Endocytosis of the agonist Rho-PTH-(1-34) in C-21 cells (Fig. 5a) was completely blocked by the PKC inhibitor staurosporine (5 M) (Fig. 5b). Under the same experimental conditions, the PKA inhibitor H8 (50 M) was ineffective (Fig.  5c). These results are independent of the number of hPTH1-Rcs on cell surface, because identical findings were obtained in C-20 cells, which stably express a 10-fold lower level of hPTH1-Rc (data not shown). Importantly, staurosporine (5 M) significantly increases basal cAMP levels in C-21 cells and markedly potentiates PTH-stimulated cAMP production (EC 50 ϭ 0.28 versus 3.5 nM with and without staurosporine, respectively). In addition, the magnitude of intracellular calcium transients stimulated by 100 nM PTH-(1-34) was similar (150 Ϯ 20 nM) in C-21 cells in the presence and absence of staurosporine (5 M). These observations therefore strongly suggest that neither activation of the cAMP/PKA pathway nor increases in intracellular calcium levels are directly involved in the internalization of agonist-occupied hPTH1-Rc, whereas PKC may play a critical role in this process. To support this hypothesis, C-21 cells incubated with the antagonist Rho-PTH-  were treated with the PKC activator PMA (1 M) or thrombin (1 M). Endocytosis of the otherwise noninternalized Rho-PTH-(7-34) (Fig. 5d) was markedly induced by both PMA (Fig. 5e) and thrombin (Fig. 5f). As was seen for the agonist Rho-PTH-(1-34), internalization of the antagonist was completely inhibited by staurosporine (5 M) (Fig. 5g). Moreover, internalized Rho-PTH-(7-34) co-localized with fluorescein-transferrin, indicating that PKC-activated endocytosis of the PTH antagonist also occurred through clathrin-coated vesicles (data not shown). In contrast, treatment of C-21 cells with forskolin (10 -100 M) did not produce Rho-PTH-(7-34) internalization (Fig. 5h), despite increasing cAMP 20-fold.
HPTD8 cells expressing the C-Tag-hPTH1-Rc also were incubated with PMA (1 M) but without any hPTH1-Rc ligand. In this case, the receptor remained mostly localized on the cell surface, suggesting that no significant internalization occurs in the absence of ligand (data not shown).

DISCUSSION
The objective of the present study was to investigate the relationship among receptor occupancy, activation of PKA-and PKC-dependent second messenger pathways, and endocytosis of the hPTH1-Rc and its ligands. Direct evidence of GCPR internalization has been reported previously (Refs. 1-14 and references therein). However, data concerning endocytosis of the hPTH1-Rc are sparse and mostly indirect (30 -34). In particular, the molecular and biochemical mechanisms responsible for hPTH1-Rc internalization remain undetermined. Our approach to studying trafficking in the PTH1-Rc system was to use three independent lines of investigation based on fluorescence microscopy methodologies. One avenue is based on monitoring the intracellular trafficking of PTH ligands, both agonists and antagonists, that have been labeled with a fluorescent moiety. The second avenue examines the distribution of the receptor by immunofluorescence microscopy using a newly created C-terminal epitope-tagged hPTH1-Rc (C-Tag-hPTH1-Rc). The third avenue is to monitor changes in the cellular distribution of a ␤-arrestin2-GFP fusion protein (7) in relation to PTH1-Rc activation and the movement of PTH-PTH1-Rc complexes.
Immunostaining of C-Tag-hPTH1-Rc in stably transfected HEK-293 cells indicates that unoccupied receptors are present predominantly on the cell surface and to a lesser extent intracellularly. Agonist occupancy and activation of the receptor causes a rapid and temperature-dependent clustering of fluorescent PTH-hPTH1Rc complexes on the cell membrane, followed by endocytosis. Inhibition of agonist-occupied receptor internalization by hypertonic medium and co-localization of internalized PTH agonist-hPTH1-Rc complexes with transferrin confirms that the process of receptor endocytosis is mediated by clathrin-coated pits, as previously suggested (31,32). We found no evidence to indicate that the internalized fluorescent Rho-PTH-(1-34) agonist, which accumulates intracellularly, is recycled to the membrane. It is likely that, similar to other ligands for GPCRs (1), endosomes target PTH to lysosomes for degradation.
In contrast, following removal of PTH-(1-34) from the medium, the internalized hPTH1-Rc relocalizes to the cell membrane within 1 h, as assessed by both immunostaining and radioligand binding. Recycling of the internalized hPTH1-Rc still occurs, even in the presence of cycloheximide, an inhibitor of protein synthesis, in agreement with previous observations (33). These data suggest that the receptors originally monitored are returned to the cell surface. However, the slower rate and lower extent of receptor relocalization to the cell membrane in the presence of cycloheximide suggests some role for protein biosynthesis during recycling. Whether this observation reflects a reduction in de novo synthesis of hPTH1-Rc or a depletion of particular trafficking-adaptor molecules remains to be elucidated.
Agonist stimulation of hPTH1-Rc-expressing cells causes a very rapid mobilization to and clustering of ␤-arrestin2 to the cell membrane, followed later by return to the cytoplasm. The time course and pattern of ␤-Arr2-GFP redistribution following receptor activation coincide with those of PTH-hPTH1Rc complex movement, indicating that ␤-arrestins are involved not only at the time of initiation of the endocytic process but also in the intracellular trafficking of hPTH1-Rc. These observations, analogous to those reported for the angiotensin AT1A and neurotensin receptors, but different from ␤2-adrenergic and other receptors (42), provide further evidence that the cellular trafficking of ␤-arrestins themselves is differentially regulated by the activation of distinct GPCRs (42). The observation that ␤-Arr2-GFP rapidly redistributes evenly in the cytoplasm after removal of PTH agonist suggests that arrestins are not involved in the recycling of the internalized hPTH1-Rc to the cell membrane. Although PTH-(7-34) antagonists do not induce rapid internalization of the hPTH1-Rc nor mobilize ␤-Arr2-GFP, a minor fraction of the fluorescent antagonist accumulates intracellularly over time. These findings are consistent with the existence of a different pathway responsible for slow agonist-and arrestin-independent receptor cycling. Importantly, the availability of fluorescent antagonists that bind to the receptor with high affinity but that are not actively internalized provides a unique reagent for the study of the functional relationship among receptor occupancy, signaling, and ligand-receptor complex endocytosis.
Because agonist stimulation of the hPTH1-Rc activates both the protein kinase A and C pathways (25)(26)(27), the role of these intracellular signaling events in ligand and receptor endocytosis was investigated. Our data indicate that PKA activity is neither sufficient nor required for internalization of ligandoccupied hPTH1-Rc: inhibition of PKA by the agent H8 does not prevent endocytosis of the fluorescent Rho-PTH-(1-34) agonist, and maximal exogenous stimulation of the cAMP/PKA pathway by forskolin does not alter the membrane distribution of fluorescent Rho-PTH-  antagonist. In contrast, exogenous PKC activation by PMA or, in a more specific manner, heterologous PKC activation by thrombin, markedly increases internalization of antagonist-occupied hPTH1-Rc. However, PKC activation per se is not sufficient to promote receptor internalization: pretreatment with PMA did not significantly affect either binding of radiolabeled PTH-  or the cell surface distribution of unoccupied C-Tag-hPTH1-Rc. Furthermore, inhibition by staurosporine of either agonist-, PMA-, or thrombininduced PKC activity completely blocked ligand-receptor complex internalization. Moreover, the essential role of the PKC pathway in hPTH1-Rc endocytosis was independently validated by the inability of Bpa 1 -PTHrP-(1-36), which selectively activates the cAMP/PKA pathway without producing intracellular calcium transients, to induce ␤-arrestin2 mobilization. Taking these data together, we conclude that hPTH1-Rc internaliza- tion requires occupancy by ligand and concomitant activation (not necessarily homologous) of the PKC signaling pathway.
In contrast to our observations, Nissenson and co-workers (30,34) recently reported that protein kinases activity, including PKA, GPCR kinase 2, and, notably, PKC, is only minimally involved in endocytosis of the agonist-activated opossum PTH1-Rc receptor. Although the reasons for such an apparent discrepancy need to be further investigated, they may originate from species-related differences in the receptor or the cell systems studied (43,44).
The lack of internalization of unoccupied hPTH1-Rc and the ability of PTH antagonist-occupied receptor to be internalized suggests that occupancy of the receptor induces conformational changes that may be a prerequisite for endocytosis. The conformational features required for internalization clearly are distinct from those leading to receptor activation and signaling. Antagonist-promoted internalization in the absence of receptor signaling has been observed for CCK (45) and endothelin subtype A (46) receptors, even in the absence of either exogenous or heterologous protein kinase activation. In addition, in staurosporine-treated cells, Rho-PTH-(1-34)-occupied hPTH1-Rcs do not necessarily internalize, despite efficient activation of both G s and G q by the PTH agonist, leading to increased cAMP and cytosolic calcium levels. These observations also indicate that receptor activation and G protein coupling are neither sufficient nor required for ligand-receptor endocytosis.
In conclusion, we have generated a series of novel molecular reagents that enable us to study directly and independently endocytosis and intracellular trafficking of both the human PTH1-Rc and PTH ligands in living cells using fluorescence microscopy. Our results provide new insights into the relationship among hPTH1-Rc occupancy, activation, and signaling (particularly through the PKC pathway), and endocytosis, as well as ␤-arrestin trafficking. These reagents may prove useful in elucidating the molecular mechanisms underlying hPTH1-Rc receptor desensitization and their relevance to the wide spectrum of metabolic outcomes produced by various regimens of PTH treatment.