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

Abstract

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 clathrincoated 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 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-␤-arrestin2 fusion protein (␤-Arr2-GFP), PTH agonists stimulated ␤-Arr2-GFP mobilization to the cell membrane. Subsequently, fluorescent PTH-(1-34)-hPTH1Rc complexes and ␤-Arr2-GFP co-localized intracellularly. In conclusion, agonist-activated hPTH1-Rc internalization involves ␤-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)1 represent a major class of membrane-bound proteins that mediate a wide variety of biological functions, including expression of the biological actions of various hormones. The responsiveness of GPCRs to extracellular stimuli, and particularly to their natural ligands, is regulated by several mechanisms, including receptor phosphorylation, coupling and uncoupling from G proteins, receptor internalization (endocytosis), and regulation of receptor gene transcription (1-3). In particular, a model for agonist-activated GPCR endocytosis, which involves mobilization of ␤-arrestins followed by agonist-receptor complex internalization through clathrin-coated pits, has been well characterized for the ␤2-adrenergic receptor (4 -6). Some of these key features are common to other GPCRs (7-9), but notable exceptions exist. For example, although the angiotensin receptor AT1 is rapidly internalized in response to agonist stimulation, the homologous AT2 receptor is not (10). Although the -opioid receptor agonist etorphin promotes ␤-arrestin mobilization and receptor internalization, morphin does not (11). Moreover, in contrast to the widely observed requirement for receptor occupancy to enable endocyto...

Differential Conformational Requirements for Activation of G Proteins and the Regulatory Proteins Arrestin and G Protein-coupled Receptor Kinase in the G Protein-coupled Receptor for Parathyroid Hormone (PTH)/PTH-related Protein

“…Using inhibitors (staurosporine) and activators (phorbol 12-myristate 13-acetate) of protein kinase C, it was previously reported that PTHR internalization requires protein kinase C activation (17). Our results, which show that the G q /phospholipase C pathway could be blocked with no outcome on receptor internalization and ␤-arrestin translocation, make it improbable that PTHR-mediated activation of protein kinase C is an obligatory requisite for receptor endocytosis via an arrestin pathway.…”

“…Agonist-induced activation of PTHR results in its phosphorylation by GRK-2 (15,16) and in the rapid internalization of the PTH-receptor complex via an arrestin-dependent pathway (17). Is the same G s protein-receptor active state required for PTH-stimulated activation of G q , GRK, and arrestin?…”

Formation of a Ternary Complex among NHERF1, β-Arrestin, and Parathyroid Hormone Receptor

“…Complex-It has previously been shown that ␤-arrestin2 and NHERF1 interact with intracellular parts of the PTHR (14,24). Although ␤-arrestin2 interaction with the PTHR has been demonstrated in vitro and in living cells, NHERF1 binding to the PTHR C terminus so far has only been shown in in vitro experiments.…”

“…Agonist occupancy of the PTH/PTH-related protein receptor (PTHR) leads to G s -mediated stimulation of adenylyl cyclase, resulting in cAMP production and PKA activation, and G q/11 -mediated phosphatidylinositol-specific phospholipase C␤ stimulation, leading to inositol 1,4,5-trisphosphate formation, calcium mobilization, and PKC activation (10 -12). PTH-induced activation of the PTHR results in rapid phosphorylation of several serine residues in the receptor C terminus with subsequent internalization of the PTH⅐receptor complex via the clathrin-coated pit pathway involving ␤-arrestin2 recruitment (13,14). Our previous work revealed that the G protein-activating conformation of the PTHR and that leading to ␤-arrestin interaction and receptor internalization may differ (15).…”

“…We show that NHERF1 forms a ternary complex with ␤-arrestin2 and PTHR, which increases the association kinetics of arrestin to the receptor. (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), abbreviated as PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), was a kind gift from Dr. Thomas Gardella (Massachusetts General Hospital, Boston, MA). Monoclonal HA.11 anti-hemagglutinin (HA) antibody was purchased from Covance, and anti-HA and anti-FLAG affinity-agarose were from Sigma-Aldrich.…”