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. 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. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCRG protein-coupled receptorPTHparathyroid hormonebPTHbovine PTHPTH-(1–34)Nle8,18,Tyr34bPTH-(1–34)NH2PTH-(7–34)Nle8,18,d-Trp12,Tyr34bPTH-(7–34)NH2PTHrPPTH-related proteinhPTHhuman PTHhPTH1-RchPTH receptor subtype 1β-Arr2β-Arrestin2Bpa1-PTHrP-(1–36)p-benzoylphenylalanine1,Ile5,Arg11,13,Tyr36PTHrP-(1–36)NH2Fluo-PTH-(1–34)Nle8,18,Lys13(N ε-5-carboxymethylfluorescein),l-2-Nal23, Arg26,27,Tyr34bPTH-(1–34)NH2Fluo-PTH-(7–34)Nle8,18,d-2-Nal12,Lys13(N ε-5-carboxymethylfluorescein),l-2-Nal23,Arg26,27,Tyr34bPTH- (7Barak L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 8Ashworth R., Yu, R. Nelson E.J. Dermer S. Gershengorn M.C. Hinkle P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 512-516Crossref PubMed Scopus (98) Google Scholar, 9Schlador M.L. Nathanson N.M. J. Biol. Chem. 1997; 272: 18882-18890Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Hein L. Meinel L. Pratt R.E. Dzau V.J. Koblika B.K. Mol. Endocrinol. 1997; 11: 1266-1277Crossref PubMed Scopus (217) Google Scholar, 11Zhang J. Ferguson S.S.G. Barak L.S. Bodduluri S.R. Laporte S.A. Law P.Y. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7157-7162Crossref PubMed Scopus (466) Google Scholar, 12Awaji T. Hirasawa A. Kataoka M. Shinoura H. Nakayama Y. Sugawara T. Izumi S. Tsujimoto G. Mol. Endocrinol. 1998; 12: 1099-1111PubMed Google Scholar, 13Roettger B.F. Rentsch R.U. Hadac E.M. Hellen E.H. Burghardt T.P. Miller L.J. J. Cell Biol. 1995; 130: 579-590Crossref PubMed Scopus (54) Google Scholar, 14Raposo G. Dunia I. Delavier-Klutchko C. Kaveri S. Strosberg A.D. Benedetti E.L. Eur. J. Cell Biol. 1989; 50: 340-352PubMed Google Scholar, 15Segre G.V. Goldring S.R. Trends Endocrinol. Metab. 1993; 4: 309-314Abstract Full Text PDF PubMed Scopus (303) Google Scholar, 16Bisello A. Adams A.E. Mierke D.F. Pellegrini M. Rosenblatt M. Suva L.J. Chorev M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, R. A. C. Rosenblatt M. Suva L.J. Chorev M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, M. H. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, H. R.E. PubMed Scopus Google Scholar, M. Y. Hadac E.M. Miller L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T.P. Trends Sci. 1995; Full Text PDF PubMed Scopus Google Scholar, C. Law C. Trends Sci. 1997; Full Text PDF PubMed Scopus Google Scholar, S. T.P. 1995; PubMed Scopus Google Scholar, of Scholar, H. T. J. G.V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. A. 1989; PubMed Scopus Google Scholar, M. S. E.M. S. Chorev M. Rosenblatt M. PubMed Scopus Google Scholar, S. G. Cell 1997; Scopus Google Scholar, A. 1998; PubMed Scopus Google Scholar, T. M. Mol. Endocrinol. 1998; 12: PubMed Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, T. Y. J. PubMed Scopus Google fluorescent kinase kinase 12-myristate receptor subtype a of that a of of the of of to and to is by receptor and G receptor internalization and of receptor L. M. A. Trends Sci. Full Text PDF PubMed Scopus Google Scholar, S.S.G. Caron M.G. Cell Biol. 1998; PubMed Scopus Google Scholar, 1998; PubMed Scopus Google In a for agonist-activated involves mobilization of by internalization clathrin-coated for the receptor M. Koblika B.K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, S.S.G. Zhang J. Barak L.S. Caron M.G. Sci. 1998; PubMed Scopus Google of to (7Barak L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 8Ashworth R., Yu, R. Nelson E.J. Dermer S. Gershengorn M.C. Hinkle P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 512-516Crossref PubMed Scopus (98) Google Scholar, 9Schlador M.L. Nathanson N.M. J. Biol. Chem. 1997; 272: 18882-18890Abstract Full Text Full Text PDF PubMed Scopus (72) Google but although the receptor is rapidly internalized in to agonist the receptor is not L. Meinel L. Pratt R.E. Dzau V.J. Koblika B.K. Mol. Endocrinol. 1997; 11: 1266-1277Crossref PubMed Scopus (217) Google the receptor agonist β-arrestin mobilization and receptor not J. Ferguson S.S.G. Barak L.S. Bodduluri S.R. Laporte S.A. Law P.Y. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7157-7162Crossref PubMed Scopus (466) Google in to the for receptor to protein kinase C (PKC) activation internalization of the in the of T. Hirasawa A. Kataoka M. Shinoura H. Nakayama Y. Sugawara T. Izumi S. Tsujimoto G. Mol. Endocrinol. 1998; 12: 1099-1111PubMed Google whereas endocytosis of involves clathrin-coated L. M. A. Trends Sci. Full Text PDF PubMed Scopus Google internalization of receptor and receptors independently of clathrin-coated B.F. Rentsch R.U. Hadac E.M. Hellen E.H. Burghardt T.P. Miller L.J. J. Cell Biol. 1995; 130: 579-590Crossref PubMed Scopus (54) Google Scholar, 14Raposo G. Dunia I. Delavier-Klutchko C. Kaveri S. Strosberg A.D. Benedetti E.L. Eur. J. Cell Biol. 1989; 50: 340-352PubMed Google G protein-coupled receptor parathyroid hormone PTH PTH-related protein human PTH receptor subtype 1 ε-5-carboxymethylfluorescein),l-2-Nal23, ε-5-carboxymethylfluorescein),l-2-Nal23,Arg26,27,Tyr34]bPTH- (7Barak L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 8Ashworth R., Yu, R. Nelson E.J. Dermer S. Gershengorn M.C. Hinkle P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 512-516Crossref PubMed Scopus (98) Google Scholar, 9Schlador M.L. Nathanson N.M. J. Biol. Chem. 1997; 272: 18882-18890Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Hein L. Meinel L. Pratt R.E. Dzau V.J. Koblika B.K. Mol. Endocrinol. 1997; 11: 1266-1277Crossref PubMed Scopus (217) Google Scholar, 11Zhang J. Ferguson S.S.G. Barak L.S. Bodduluri S.R. Laporte S.A. Law P.Y. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7157-7162Crossref PubMed Scopus (466) Google Scholar, 12Awaji T. Hirasawa A. Kataoka M. Shinoura H. Nakayama Y. Sugawara T. Izumi S. Tsujimoto G. Mol. Endocrinol. 1998; 12: 1099-1111PubMed Google Scholar, 13Roettger B.F. Rentsch R.U. Hadac E.M. Hellen E.H. Burghardt T.P. Miller L.J. J. Cell Biol. 1995; 130: 579-590Crossref PubMed Scopus (54) Google Scholar, 14Raposo G. Dunia I. Delavier-Klutchko C. Kaveri S. Strosberg A.D. Benedetti E.L. Eur. J. Cell Biol. 1989; 50: 340-352PubMed Google Scholar, 15Segre G.V. Goldring S.R. Trends Endocrinol. Metab. 1993; 4: 309-314Abstract Full Text PDF PubMed Scopus (303) Google Scholar, 16Bisello A. Adams A.E. Mierke D.F. Pellegrini M. Rosenblatt M. Suva L.J. Chorev M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, R. A. C. Rosenblatt M. Suva L.J. Chorev M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, M. H. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, H. R.E. PubMed Scopus Google Scholar, M. Y. Hadac E.M. Miller L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T.P. Trends Sci. 1995; Full Text PDF PubMed Scopus Google Scholar, C. Law C. Trends Sci. 1997; Full Text PDF PubMed Scopus Google Scholar, S. T.P. 1995; PubMed Scopus Google Scholar, of Scholar, H. T. J. G.V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. A. 1989; PubMed Scopus Google Scholar, M. S. E.M. S. Chorev M. Rosenblatt M. PubMed Scopus Google Scholar, S. G. Cell 1997; Scopus Google Scholar, A. 1998; PubMed Scopus Google Scholar, T. M. Mol. Endocrinol. 1998; 12: PubMed Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, T. Y. J. PubMed Scopus Google green fluorescent protein human protein kinase A protein kinase C phorbol 12-myristate 13-acetate PTH receptor subtype 1 is the internalization and intracellular trafficking of a of G.V. Goldring S.R. Trends Endocrinol. Metab. 1993; 4: 309-314Abstract Full Text PDF PubMed Scopus (303) Google ligands of receptors for parathyroid hormone and PTH-related protein and of the and the is rapidly A. Adams A.E. Mierke D.F. Pellegrini M. Rosenblatt M. Suva L.J. Chorev M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, R. A. C. Rosenblatt M. Suva L.J. Chorev M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, M. H. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, H. R.E. PubMed Scopus Google Scholar, M. Y. Hadac E.M. Miller L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the receptor occupancy, signaling, and in of that for T.P. Trends Sci. 1995; Full Text PDF PubMed Scopus Google Scholar, C. Law C. Trends Sci. 1997; Full Text PDF PubMed Scopus Google Scholar, S. T.P. 1995; PubMed Scopus Google by of ligands and and and PTH is the of and and a in of Activation of the parathyroid hormone receptor subtype 1 by PTH kinase A and the and H. T. J. G.V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. A. 1989; PubMed Scopus Google Scholar, M. S. E.M. S. Chorev M. Rosenblatt M. PubMed Scopus Google In PTH G protein-coupled receptor kinase S. G. Cell 1997; Scopus Google Scholar, A. 1998; PubMed Scopus Google Scholar, T. M. Mol. Endocrinol. 1998; 12: PubMed Google of is to receptor endocytosis not that internalization agonist via clathrin-coated J. Cell Biol. PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google of receptor to PTH stimulation to receptor to the cell PubMed Scopus Google and in the of the in cells Y. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google that in the receptor to the internalization receptor protein kinase activation to a in internalization of the in a human cell T. M. Mol. Endocrinol. 1998; 12: PubMed Google Scholar, T. Y. J. PubMed Scopus Google the receptor signaling, and endocytosis in the PTH to of by fluorescence microscopy is a for the of internalization and trafficking of and receptors in cells. In the and fluorescence-labeled PTH agonists and epitope-tagged human in to independently the trafficking of receptor and and to the receptor occupancy, activation of specific intracellular and endocytosis of by a fusion protein (7Barak L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google the of in endocytosis and trafficking of the hPTH1-Rc. were were were and were and were and were phorbol 12-myristate 13-acetate were and by the and R. A. C. Rosenblatt M. Suva L.J. Chorev M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google in 1 of in the of of or and were by of the the was for of to the was of and and were by R. A. C. Rosenblatt M. Suva L.J. Chorev M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google and of and were used for the and were and C. Adams A.E. Suva L.J. Rosenblatt M. Chorev M. 1995; PubMed Scopus Google were by and human cells and HEK-293 cells stably transfected the hPTH1-Rc and and or a C-Tag-hPTH1-Rc were in M. S. E.M. S. Chorev M. Rosenblatt M. PubMed Scopus Google Scholar, M. Adams A.E. S. R. Chorev M. Rosenblatt M. Suva L.J. PubMed Scopus Google transfected HEK-293 cells and were in and to were C. Adams A.E. Suva L.J. Rosenblatt M. Chorev M. 1995; PubMed Scopus Google Chorev M. Rosenblatt M. PubMed Scopus Google were by and was M. Adams A.E. S. R. Chorev M. Rosenblatt M. Suva L.J. PubMed Scopus Google transfected HEK-293 cells and were in and to activation by PTH agonists and of activation by PTH was in the of 1 C. Adams A.E. Suva L.J. Rosenblatt M. Chorev M. 1995; PubMed Scopus Google was by the Y. C. M. PubMed Scopus Google In the of or staurosporine cells were for at 37 °C in the of to PTH stimulation of in intracellular by and was in HEK-293 cells stably transfected hPTH1-Rc M. C. A. R. Chorev M. Rosenblatt M. Suva L.J. PubMed Scopus Google In to real-time microscopy fluorescence-labeled PTH cells were and for were in and in in were fluorescence-labeled PTH agonists or for and the was by were 1 of and the cell was at 37 °C in a for the of the of fluorescent ligands was by the at 37 °C PTH in the of fluorescent PTH ligands was were for was In cells were or or of or and of PKC or In cells were the for at 37 in the Subsequently, were and cells were the fluorescent ligands were the and at 37 °C for the of the was used of PKC, was but were the fluorescent ligands and at 37 °C for the of the was a for and were a and the in were at the cell was used for of fluorescence-labeled In to real-time fluorescence microscopy of β-arrestin mobilization and cells were transfected of (7Barak L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google of after were and ligands at 37 °C and in the was hPTH1-Rc A.E. M. C. R. Chorev M. Rosenblatt M. Suva L.J. 1995; PubMed Scopus Google was by to the and was the of the in the and by a and of the were by was transfected HEK-293 cells and were in cells transfected a of in to were for of the C-Tag-hPTH1-Rc by of the C-Tag-hPTH1-Rc was by or to and of for the epitope-tagged receptor was by HEK-293 and HEK-293 cells stably the C-Tag-hPTH1-Rc were to were in the to in and and in for at and were a for was by the cells for at in in was to the for 1 h at by the human was and in the the were in and for was In in to protein cells were h at 37 °C and in the of and after agonists and and and the of parathyroid hormone were by by of the of of or of was by and by of the of PTH in cells is in I. and agonists for the hPTH1-Rc. and than of and also to the hPTH1-Rc and results were in of the fluorescence-labeled PTH in HEK-293 cells stably the hPTH1-Rc of stimulation of of stimulation of in a 1 real-time of the of the agonist or the in cells by fluorescence In cells to and to fluorescence was the cell membrane a and was hPTH1-Rc was by at 37 PTH-(7–34) 1 or and was in HEK-293 cells in occurred in cells at °C not after the to 37 the agonist the cell membrane and was internalized and was of the intracellular of the the cell 1 results were not a at PTH was to the of cells In to the the the to 37 °C internalization and the of fluorescence results were not a fluorescent internalized were in the after at 37 °C 1 was of internalization or intracellular of the fluorescence not that the of internalized a of hPTH1-Rc. the of to the to and to the of the In to trafficking of the a hPTH1-Rc and at the C terminus (C-Tag-hPTH1-Rc) was and stimulation of in HEK-293 cells stably transfected the C-Tag-hPTH1-Rc receptor to hPTH1-Rc at a in HEK-293 cells M. S. E.M. S. Chorev M. Rosenblatt M. PubMed Scopus Google Scholar, M. Adams A.E. S. R. Chorev M. Rosenblatt M. Suva L.J. PubMed Scopus Google cells and were used to the receptor by fluorescence a the of the C-Tag-hPTH1-Rc and In the of agonist the C-Tag-hPTH1-Rc was to the cell and, to the of or L. M. A. Trends Sci. Full Text PDF PubMed Scopus Google Scholar, S.S.G. Caron M.G. Cell Biol. 1998; PubMed Scopus Google Scholar, 1998; PubMed Scopus Google Scholar, M. Koblika B.K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, S.S.G. Zhang J. Barak L.S. Caron M.G. Sci. 1998; PubMed Scopus Google Scholar, L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 8Ashworth R., Yu, R. Nelson E.J. Dermer S. Gershengorn M.C. Hinkle P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 512-516Crossref PubMed Scopus (98) Google Scholar, 9Schlador M.L. Nathanson N.M. J. Biol. Chem. 1997; 272: 18882-18890Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Hein L. Meinel L. Pratt R.E. Dzau V.J. Koblika B.K. Mol. Endocrinol. 1997; 11: 1266-1277Crossref PubMed Scopus (217) Google Scholar, 11Zhang J. Ferguson S.S.G. Barak L.S. Bodduluri S.R. Laporte S.A. Law P.Y. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7157-7162Crossref PubMed Scopus (466) Google Scholar, 12Awaji T. Hirasawa A. Kataoka M. Shinoura H. Nakayama Y. Sugawara T. Izumi S. Tsujimoto G. Mol. Endocrinol. 1998; 12: 1099-1111PubMed Google Scholar, 13Roettger B.F. Rentsch R.U. Hadac E.M. Hellen E.H. Burghardt T.P. Miller L.J. J. Cell Biol. 1995; 130: 579-590Crossref PubMed Scopus (54) Google Scholar, 14Raposo G. Dunia I. Delavier-Klutchko C. Kaveri S. Strosberg A.D. Benedetti E.L. Eur. J. Cell Biol. 1989; 50: 340-352PubMed Google Scholar, 15Segre G.V. Goldring S.R. Trends Endocrinol. Metab. 1993; 4: 309-314Abstract Full Text PDF PubMed Scopus (303) Google Scholar, 16Bisello A. Adams A.E. Mierke D.F. Pellegrini M. Rosenblatt M. Suva L.J. Chorev M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, R. A. C. Rosenblatt M. Suva L.J. Chorev M. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, M. H. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, H. R.E. PubMed Scopus Google Scholar, M. Y. Hadac E.M. Miller L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T.P. Trends Sci. 1995; Full Text PDF PubMed Scopus Google Scholar, C. Law C. Trends Sci. 1997; Full Text PDF PubMed Scopus Google Scholar, S. T.P. 1995; PubMed Scopus Google Scholar, of Scholar, H. T. J. G.V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. A. 1989; PubMed Scopus Google Scholar, M. S. E.M. S. Chorev M. Rosenblatt M. PubMed Scopus Google Scholar, S. G. Cell 1997; Scopus Google Scholar, A. 1998; PubMed Scopus Google Scholar, T. M. Mol. Endocrinol. 1998; 12: PubMed Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, T. Y. J. PubMed Scopus Google internalization of the removal of the the a of receptors to the cell membrane was and h results were in cells and and In cells for h in the of in to were the cell membrane and in the and were internalized after PTH and to the cell membrane was and after h and in receptor were in the for the of to the was h after of the in cells. In the of in cells h at was and after h of the In to endocytosis of fluorescent agonist the hPTH1-Rc was by clathrin-coated vesicles, cells were in to of J. Cell Biol. 1993; PubMed Scopus Google internalization of was not in cells the and of the clathrin-coated internalization intracellular of the fluorescence of ligands was after at 37 °C indicate that fluorescent PTH at its intracellular via clathrin-coated vesicles. were in to the of and PKC activation internalization of the hPTH1-Rc and its Endocytosis of the agonist in cells was blocked by the PKC inhibitor staurosporine the the inhibitor was results of the of cell were in stably a of hPTH1-Rc not staurosporine in cells and and In the of intracellular stimulated by was in cells in the and of staurosporine that activation of the pathway in intracellular in the internalization of whereas PKC a in cells the were the PKC or Endocytosis of the was by and was for the agonist internalization of the was by staurosporine internalized co-localized that endocytosis of the PTH also occurred clathrin-coated not In of cells did not internalization cells the C-Tag-hPTH1-Rc also were but hPTH1-Rc ligand. In the receptor the cell that internalization in the of not to in the internalization of 1998; PubMed Scopus Google Scholar, S.S.G. Zhang J. Barak L.S. Caron M.G. Sci. 1998; PubMed Scopus Google Scholar, and J. Barak L.S. Laporte S.A. Caron M.G. Ferguson G J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and or not the of β-Arr2-GFP in to hPTH1-Rc In cells the fluorescence was the receptor activation by and β-Arr2-GFP was rapidly to the cell membrane in the of β-Arr2-GFP the β-Arr2-GFP co-localized at the cell membrane and after removal of the β-Arr2-GFP in the not In the PTH-(7–34) that intracellular A. M. and M. stimulated of of the was to the receptor occupancy, activation of and and endocytosis of the hPTH1-Rc and its of internalization L. M. A. Trends Sci. Full Text PDF PubMed Scopus Google Scholar, S.S.G. Caron M.G. Cell Biol. 1998; PubMed Scopus Google Scholar, 1998; PubMed Scopus Google Scholar, M. Koblika B.K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, S.S.G. Zhang J. Barak L.S. Caron M.G. Sci. 1998; PubMed Scopus Google Scholar, L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 8Ashworth R., Yu, R. Nelson E.J. Dermer S. Gershengorn M.C. Hinkle P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 512-516Crossref PubMed Scopus (98) Google Scholar, 9Schlador M.L. Nathanson N.M. J. Biol. Chem. 1997; 272: 18882-18890Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Hein L. Meinel L. Pratt R.E. Dzau V.J. Koblika B.K. Mol. Endocrinol. 1997; 11: 1266-1277Crossref PubMed Scopus (217) Google Scholar, 11Zhang J. Ferguson S.S.G. Barak L.S. Bodduluri S.R. Laporte S.A. Law P.Y. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7157-7162Crossref PubMed Scopus (466) Google Scholar, 12Awaji T. Hirasawa A. Kataoka M. Shinoura H. Nakayama Y. Sugawara T. Izumi S. Tsujimoto G. Mol. Endocrinol. 1998; 12: 1099-1111PubMed Google Scholar, 13Roettger B.F. Rentsch R.U. Hadac E.M. Hellen E.H. Burghardt T.P. Miller L.J. J. Cell Biol. 1995; 130: 579-590Crossref PubMed Scopus (54) Google Scholar, 14Raposo G. Dunia I. Delavier-Klutchko C. Kaveri S. Strosberg A.D. Benedetti E.L. Eur. J. Cell Biol. 1989; 50: 340-352PubMed Google and endocytosis of the hPTH1-Rc and T. M. Mol. Endocrinol. 1998; 12: PubMed Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, T. Y. J. PubMed Scopus Google In the and for hPTH1-Rc internalization Our to trafficking in the was to of fluorescence microscopy is the intracellular trafficking of PTH agonists and that a fluorescent the of the receptor by microscopy a epitope-tagged hPTH1-Rc is to in the of a fusion protein (7Barak L.S. Ferguson S.S.G. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (397) Google in to activation and the of of C-Tag-hPTH1-Rc in stably transfected HEK-293 cells that receptors the cell and to a lesser intracellularly. and activation of the receptor a and of fluorescent complexes the cell by of receptor internalization by and of internalized PTH agonist-hPTH1-Rc complexes that the of receptor endocytosis is by clathrin-coated J. Cell Biol. PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google to indicate that the internalized fluorescent is to the membrane. is to ligands for L. M. A. Trends Sci. Full Text PDF PubMed Scopus Google PTH to for In removal of the the internalized hPTH1-Rc to the cell membrane 1 by and of the internalized hPTH1-Rc in the of inhibitor of protein in PubMed Scopus Google that the receptors monitored to the cell the and of receptor to the cell membrane in the of for protein a in of hPTH1-Rc or a of to stimulation of cells a mobilization to and of to the cell by to the cytoplasm. and of β-Arr2-GFP receptor activation of that not at the of of the but also in the intracellular trafficking of hPTH1-Rc. to for the and but and receptors J. Barak L.S. Laporte S.A. Caron M.G. Ferguson G J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that the trafficking of is by the activation of J. Barak L.S. Laporte S.A. Caron M.G. Ferguson G J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that β-Arr2-GFP rapidly in the after removal of PTH agonist that not in the of the internalized hPTH1-Rc to the cell membrane. PTH-(7–34) not internalization of the hPTH1-Rc a of the fluorescent the of a pathway for and receptor the of fluorescent that to the receptor but that not internalized a for the of the functional receptor occupancy, signaling, and agonist stimulation of the hPTH1-Rc the protein kinase A and C H. T. J. G.V. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. A. 1989; PubMed Scopus Google Scholar, M. S. E.M. S. Chorev M. Rosenblatt M. PubMed Scopus Google the of intracellular in and receptor endocytosis was Our indicate that is for internalization of of by the not endocytosis of the fluorescent and stimulation of the pathway by not the membrane of fluorescent In PKC activation by in a specific PKC activation by thrombin, internalization of hPTH1-Rc. PKC activation not to receptor did not of or the cell of by staurosporine of or PKC blocked internalization. the of the PKC pathway in hPTH1-Rc endocytosis was independently by the of the pathway intracellular to that hPTH1-Rc internalization by and activation of the PKC In to and T. M. Mol. Endocrinol. 1998; 12: PubMed Google Scholar, T. Y. J. PubMed Scopus Google that protein kinase and, PKC, is in endocytosis of the agonist-activated the for to in the receptor or the cell S. PubMed Scopus Google Scholar, PubMed Scopus Google of internalization of hPTH1-Rc and the of PTH receptor to internalized that of the receptor that a for for internalization to receptor activation and internalization in the of receptor for B.F. R. C. J. Miller L.J. Mol. 1997; Google and subtype A 1998; PubMed Scopus Google in the of or protein kinase In in not activation of and by the PTH to and also indicate that receptor activation and G protein for In conclusion, a of that to and independently endocytosis and intracellular trafficking of the human and PTH ligands in cells fluorescence Our results the hPTH1-Rc occupancy, and the PKC and β-arrestin in the hPTH1-Rc receptor and to the of by of PTH for the fluorescence microscopy and for and of the also Caron for the β-Arr2-GFP