Phosphorylation of the Receptor for PTH and PTHrP Is Required for Internalization and Regulates Receptor Signaling

Tawfeek, Hesham A.; Qian, Fang; Abou‐Samra, Abdul Badi

doi:10.1210/mend.16.1.0760

Cited by 63 publications

(33 citation statements)

References 54 publications

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“…In cells expressing the PD-PTHR1, the fluorescence was initially weak and diffuse at 5 min, and then some punctae were apparent at dispersed locations by 30 min. The overall results of these in vitro studies are largely consistent with prior studies (5)(6)(7)15), and thus show that the PD-PTHR1 is at least as functional as the WT-PTHR1 in terms of its capacity to bind PTH ligands and activate second-messenger signaling pathways, and exhibits at least some delay in ligand-induced internalization responses.…”

Section: Resultssupporting

confidence: 85%

“…As for most GPCRs, the PTHR1 is phosphorylated on its C-terminal tail following agonist activation, and this phosphorylation promotes the internalization and desensitization responses, in large part by stabilizing interaction with β-arrestins (5-9). Phosphorylationdeficient (PD) mutant PTH receptors that have key serine residues replaced by alanine are thus defective for mediating efficient β-arrestin recruitment, receptor internalization, and signal desensitization responses to PTH challenge (5)(6)(7)(8)(9). Genetically altered knock-in mice expressing such a PD-PTHR1 in place of the endogenous receptor exhibit prolonged blood cAMP elevations in response to injected PTH (1-34) and develop significant hypercalcemia after several days of continuous PTH (1-34) infusion (10).…”

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confidence: 99%

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Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH

Maeda

Okazaki

Baron

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Agonist-induced phosphorylation of the parathyroid hormone (PTH) receptor 1 (PTHR1) regulates receptor signaling in vitro, but the role of this phosphorylation in vivo is uncertain. We investigated this role by injecting "knock-in" mice expressing a phosphorylation-deficient (PD) PTHR1 with PTH ligands and assessing acute biologic responses. Following injection with PTH (1-34), or with a unique, long-acting PTH analog, PD mice, compared with WT mice, exhibited enhanced increases in cAMP levels in the blood, as well as enhanced cAMP production and gene expression responses in bone and kidney tissue. Surprisingly, however, the hallmark hypercalcemic and hypophosphatemic responses were markedly absent in the PD mice, such that paradoxical hypocalcemic and hyperphosphatemic responses were observed, quite strikingly with the long-acting PTH analog. Spot urine analyses revealed a marked defect in the capacity of the PD mice to excrete phosphate, as well as cAMP, into the urine in response to PTH injection. This defect in renal excretion was associated with a severe, PTH-induced impairment in glomerular filtration, as assessed by the rate of FITC-inulin clearance from the blood, which, in turn, was explainable by an overly exuberant systemic hypotensive response. The overall findings demonstrate the importance in vivo of PTH-induced phosphorylation of the PTHR1 in regulating acute ligand responses, and they serve to focus attention on mechanisms that underlie the acute calcemic response to PTH and factors, such as blood phosphate levels, that influence it.calcium homeostasis | family B GPCR | phosphate homeostasis | receptor phosphorylation P arathyroid hormone (PTH) plays a critical role in regulating blood concentrations of ionized calcium by acting via its receptor, the PTH receptor 1 (PTHR1), expressed by target cells of bone and kidney. In response to decreases in blood Ca 2+ levels, PTH thus acts to promote the release of calcium from bone, and the reabsorption of calcium from the glomerular filtrate. PTH also acts to lower blood inorganic phosphate (Pi) by promoting the renal excretion of Pi into the urine. PTH acts in concert with several other calcium-and phosphate-regulating factors, including 1,25(OH) 2 -vitamin-D 3 and FGF23, as part of a tightly regulated homeostatic system of mineral ion control (1). Of note, PTH peptides injected daily at low dose can stimulate new bone formation and are thus in use to treat osteoporosis (2). Because of their calcemic actions, PTH peptides also hold promise as treatments for hypoparathyroidism (3).The PTHR1 is a class B G protein-coupled receptor (GPCR) that signals via the Gαs/cAMP/PKA and Gαq/phopholipase C (PLC)/intracellular calcium (iCa)/PKC pathways (4). In cultured cells, agonist activation of the PTHR1 induces rapid receptor internalization and signal desensitization. As for most GPCRs, the PTHR1 is phosphorylated on its C-terminal tail following agonist activation, and this phosphorylation promotes the internalization and desensitization responses, in large part...

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Section: Resultssupporting

confidence: 85%

mentioning

confidence: 99%

Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH

Maeda

Okazaki

Baron

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Strikingly, this PTH1R internalization occurs in a markedly cell-specific fashion and proceeds through a mechanism that is distinct from that induced by PTH agonists. The agonist PTH-(1-34) activated the PTH1R, mobilized ␤-arrestin, and internalized the receptor, as previously shown in HEK-293, COS-7, and LLC-PK 1 cells (11,44,45). PTH-(7-34), a competitive inhibitor of the PTH1R, however, robustly promoted receptor internalization in DCT and ROS cells without accompanying activation or ␤-arrestin-2 translocation.…”

Section: Nherf1 Regulation Of Pth Receptor Internalizationsupporting

confidence: 65%

Activation-independent Parathyroid Hormone Receptor Internalization Is Regulated by NHERF1 (EBP50)

Sneddon

Syme

Bisello

et al. 2003

Journal of Biological Chemistry

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139

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Parathyroid hormone (PTH) regulates extracellular calcium homeostasis through the type 1 PTH receptor (PTH1R) expressed in kidney and bone. The PTH1R undergoes ␤-arrestin/dynamin-mediated endocytosis in response to the biologically active forms of PTH, PTH-(1-34), and PTH-(1-84). We now show that amino-truncated forms of PTH that do not activate the PTH1R nonetheless induce PTH1R internalization in a cell-specific pattern. Activation-independent PTH1R endocytosis proceeds through a distinct arrestin-independent mechanism that is operative in cells lacking the adaptor protein Na/H exchange regulatory factor 1 (NHERF1) (ezrin-binding protein 50). Using a combination of radioligand binding experiments and quantitative, live cell confocal microscopy of fluorescently tagged PTH1Rs, we show that in kidney distal tubule cells and rat osteosarcoma cells, which lack NHERF1, the synthetic antagonist PTH-(7-34) and naturally circulating PTH-(7-84) induce internalization of PTH1R in a ␤-arrestin-independent but dynamindependent manner. Expression of NHERF1 in these cells inhibited antagonist-induced endocytosis. Conversely, expression of dominant-negative forms of NHERF1 conferred internalization sensitivity to PTH-(7-34) in cells expressing NHERF1. Mutation of the PTH1R PDZ-binding motif abrogated interaction of the receptor with NHERF1. These mutated receptors were fully functional but were now internalized in response to PTH-(7-34) even in NHERF1-expressing cells. Removing the NHERF1 ERM domain or inhibiting actin polymerization allowed otherwise inactive ligands to internalize the PTH1R. These results demonstrate that NHERF1 acts as a molecular switch that legislates the conditional efficacy of PTH fragments. Distinct endocytic pathways are determined by NHERF1 that are operative for the PTH1R in kidney and bone cells.Extracellular calcium homeostasis in vertebrate animals is primarily under the endocrine control of the parathyroid hormone (PTH) 1 /type I PTH receptor (PTH1R). The PTH1R, predominantly expressed in kidney and bone cells, belongs to class B of the large superfamily of G protein-coupled receptors (GPCRs) that consists of receptors for peptide hormones and neuropeptides (1). Class B GPCRs are characterized by a common topology and by their ability to couple to multiple signaling pathways via distinct G proteins.PTH is synthesized by the parathyroid glands as a mature peptide of 84 amino acids that is stored in secretory vesicles and dense core granules. Reductions of extracellular calcium levels are detected by the calcium-sensing receptor on parathyroid chief cells and promote the release of PTH, which acts on bone (to increase resorption) and kidney (to augment reabsorption), thereby restoring serum calcium levels. PTH-(1-84) is usually the major form of PTH secreted by the parathyroid glands. However, recent analyses reveal that PTH fragments that are likely to be PTH-(7-84) are also secreted by the parathyroid glands and generated by peripheral metabolism (2, 3). These PTH fragments or their synthetic a...

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“…Absence of the PTH1R COOH terminus may expose sequencespecific components that promote ERK activation. Alternatively, it may remove steric hindrance of signaling elements in the third intracellular loop, or reflect impaired desensitization of this mutant receptor, which lacks agonist-induced phosphorylation sites (17,34). The experiments with mutant receptors were conducted on HEK-293 that do not express endogenous PTH1R.…”

Section: Discussionmentioning

confidence: 99%

Extracellular signal-regulated kinase activation by parathyroid hormone in distal tubule cells

Sneddon

Yang²,

Ba³

et al. 2007

American Journal of Physiology-Renal Physiology

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Phosphorylation of the Receptor for PTH and PTHrP Is Required for Internalization and Regulates Receptor Signaling

Cited by 63 publications

References 54 publications

Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH

Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH

Activation-independent Parathyroid Hormone Receptor Internalization Is Regulated by NHERF1 (EBP50)

Extracellular signal-regulated kinase activation by parathyroid hormone in distal tubule cells

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