Figure 8. Colocalization of β-Arrestin2-GFP with Internalized wt and S(483–498)A Mutant PTHRs HEK-wt PTHR and HEK-S(483–498)A PTHR cells were transiently transfected with a plasmid containing the cDNA for β-arrestin-2-GFP. Cells were treated or not (basal) with 1 μm bPTH(1–34) for the indicated times, fixed, and immunostained with a mouse antiopossum PTHR monoclonal antibody and rhodamine-conjugated goat antimouse IgG. Another set of cells was treated for 30 min with 1 μm bPTH(1–34), acid washed, and incubated at 37 C for 30 min, 1 h, or 4 h to allow receptor recycling. Shown are representative confocal microscopy images of PTHR receptor immunofluorescence (red) and β-arrestin-2-GFP fluorescence (green). Colocalization (yellow) of the receptor with β-arrestin-2-GFP is shown in the overlay. All experiments were performed in the presence of cycloheximide. From: Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and β-Arrestin Mol Endocrinol. 2002;16(12):2720-2732. doi:10.1210/me.2002-0049 Mol Endocrinol | Copyright © 2002 by The Endocrine Society

Figure 7. Role of PTHR Phosphorylation in Recycling Recycling was studied for PTHRs that either lack phosphorylation sites (S(483–498)A) or contain acidic residues to mimic phosphorylation (S(483–498)D/E). These mutant PTHRs were stably expressed in HEK293, and recycling was examined after PTH-induced internalization, using the procedure described in Fig. 2 (A) and Fig. 3 (B). In A, the PTHRs were visualized using a donkey antimouse IgG-fluorescein-conjugated secondary antibody (S(483–498)D/E) or a goat antimouse IgG-rhodamine conjugated secondary antibody (S(483–498)A) followed by confocal microscopy. In B, the percent recycling was calculated as follows: [(bound ligand after recycling − bound ligand before recycling)/(internalized ligand)] × 100. All experiments were performed in the presence of cycloheximide. Data represent the mean ± se of three independent experiments. From: Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and β-Arrestin Mol Endocrinol. 2002;16(12):2720-2732. doi:10.1210/me.2002-0049 Mol Endocrinol | Copyright © 2002 by The Endocrine Society

Figure 6. Effect of Okadaic Acid on PTHR Dephosphorylation Dephosphorylation experiments were performed on HEK-wt PTHR cells using the procedure described in Fig. 4, in the absence or continuous presence of 1 μm okadaic acid. Results are the mean ± se of three independent experiments. From: Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and β-Arrestin Mol Endocrinol. 2002;16(12):2720-2732. doi:10.1210/me.2002-0049 Mol Endocrinol | Copyright © 2002 by The Endocrine Society

Figure 5. Effect of Okadaic Acid and Calyculin A on PTHR Recycling The effects of 1 μm okadaic acid or 2 nm calyculin A on PTHR recycling was assessed in HEK-wt PTHR cells, using the procedure described in Fig. 3. Results are the mean ± se of five independent experiments with okadaic acid, and four independent experiments with calyculin A. From: Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and β-Arrestin Mol Endocrinol. 2002;16(12):2720-2732. doi:10.1210/me.2002-0049 Mol Endocrinol | Copyright © 2002 by The Endocrine Society

Figure 4. Kinetics of PTHR Dephosphorylation HEK-wt PTHR cells were labeled for 2 h in phosphate-free media containing 100 μCi [³²P]-orthophosphoric acid at 37 C. Cells were treated for 30 min at room temperature with 240 nm bPTH(1–34). After removal of the agonist by acid washes, cells were incubated at 37 C for the time indicated and then solubilized. PTHRs were immunoprecipitated and resolved by SDS-PAGE. ³²P incorporation was quantified from autoradiograms using the Bio-Rad Laboratories, Inc. GS-363 Phosphorimager system. Results are expressed as the increase in phosphorylation of the PTHR in cells exposed to PTH (minus basal phosphorylation), expressed as a percentage of that seen at the time of removal of PTH. Results are the mean ± se of six independent experiments. From: Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and β-Arrestin Mol Endocrinol. 2002;16(12):2720-2732. doi:10.1210/me.2002-0049 Mol Endocrinol | Copyright © 2002 by The Endocrine Society

Figure 3. Functional Recycling of the PTHR HEK-wt PTHR cells were incubated with or without 1 μm bPTH(1–34) for 30 min at 37 C (+PTH or basal, respectively), followed by removal of the agonist and further incubated at 37 C to allow receptor recycling for the times indicated. These experiments were performed in the presence of cycloheximide to exclude the possible contribution of new receptor synthesis. The number of receptors present at the cell surface was measured by radioligand binding and expressed as the percentage of specific binding. Data represent the mean ± se of nine independent experiments. From: Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and β-Arrestin Mol Endocrinol. 2002;16(12):2720-2732. doi:10.1210/me.2002-0049 Mol Endocrinol | Copyright © 2002 by The Endocrine Society

Figure 2. PTH-Induced Endocytosis and Rapid Recycling of the PTHR HEK-wt PTHR cells were fixed and immunostained using a mouse antiopossum PTHR monoclonal antibody and fluorescein isothiocyanate-conjugated donkey antimouse IgG. Micrographs (magnification, ×100) show PTHR immunolocalization on the cell membrane in untreated cells (basal), internalization after 30 min incubation with 1 μm bPTH(1–34) (PTH), and relocalization to the cell membrane rapidly after removal of the ligand (30 min, 1 h, 2 h, and 4 h recycling). All experiments were performed in the presence of cycloheximide. The results are representative of those obtained in five separate experiments. From: Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and β-Arrestin Mol Endocrinol. 2002;16(12):2720-2732. doi:10.1210/me.2002-0049 Mol Endocrinol | Copyright © 2002 by The Endocrine Society

Figure 1. Effect of PTH Treatment on Total Cellular Expression of the PTHR HEK-wt PTHR cells were treated at 37 C for 30 min with or without 1 μm bPTH(1–34). At the end of the incubation, the cells were subjected to radioligand binding, as described in Materials and Methods (A). HEK-wt PTHR cells were treated at 37 C for either 3 or 24 h in the absence (B) or 3 h in presence of cycloheximide (C) with or without 1 μm bPTH(1–34). Cells were solubilized and cellular proteins were resolved by SDS-PAGE, and Western blots of the PTHR were carried out as described in Materials and Methods. Each lane was loaded with equivalent amounts of total cell protein. The exposed films were scanned and quantified using Bio-Rad Laboratories, Inc. Molecular Analyst Program. Data represent the mean ± se of three or more independent experiments. From: Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and β-Arrestin Mol Endocrinol. 2002;16(12):2720-2732. doi:10.1210/me.2002-0049 Mol Endocrinol | Copyright © 2002 by The Endocrine Society

Figure 9. Role of β-Arrestin in PTHR Recycling HEK293 cells transiently transfected with the wt PTHR alone or with a dominant-negative (dn) form of β-arrestin-1 (β-arrestin-1-V54D) were treated with bPTH(1–34) for 30 min (+PTH). After washing out the agonist, the cells were incubated at 37 C to allow recycling for the times indicated. The cells were then fixed, immunostained, and observed by confocal microscopy as described in Materials and Methods (A). The results are representative of those obtained in three separate experiments. In some cases, cells were solubilized and resolved by SDS-PAGE, transferred and blotted for β-arrestin (B). HEK293 cells transiently transfected with the wt PTHR alone or with β-arrestin-1-V54D (dn-arr1) were treated with 1 μm bPTH(1–34), and the internalization was measured after 10 min of exposure to [¹²⁵I]PTHrP as described in Materials and Methods (B). Data represent the mean ± se of three or more independent experiments. *, P < 0.01. All experiments were performed in the presence of cycloheximide. From: Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and β-Arrestin Mol Endocrinol. 2002;16(12):2720-2732. doi:10.1210/me.2002-0049 Mol Endocrinol | Copyright © 2002 by The Endocrine Society