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 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.
This work was supported in part by Grant RO1-DK47940 from the NIDDK, National Institutes Health (to M. R.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a postdoctoral fellowship grant from the Swiss National Science Foundation and the Foundation des Bourses de Recherche en Medecine et Biologie.
