Endocytosis of ligand-human parathyroid hormone receptor 1 complexes is protein kinase C-dependent and involves β-arrestin2. Real-time monitoring by fluorescence microscopy

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 i 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 inhibitor N-(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.