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ABSTRACT: Parathyroid hormone (PTH) regulates calcium homeostasis and bone remodeling through its cognitive receptor (PTHR). We describe here a PTHR isoform harboring an in-frame 42-bp deletion of exon 14 (Δe14-PTHR) that encodes transmembrane domain 7. Δe14-PTHR was detected in human kidney and buccal epithelial cells. We characterized its topology, cellular localization, and signaling, as well as its interactions with PTHR. The C-terminus of the Δe14-PTHR is extracellular, and cell surface expression is strikingly reduced compared with the PTHR. Δe14-PTHR displayed impaired trafficking and accumulated in endoplasmic reticulum. Signaling and activation of cAMP and ERK by Δe14-PTHR was decreased significantly compared with PTHR. Δe14-PTHR acts as a functional dominant-negative by suppressing the action of PTHR. Cells cotransfected with both receptors exhibit markedly reduced PTHR cell membrane expression, colocalization with Δe14-PTHR in endoplasmic reticulum, and diminished cAMP activation and ERK phosphorylation in response to challenge with PTH. Δe14-PTHR forms heterodimers with PTHR, which may account for cytoplasmic retention of PTHR in the presence of Δe14-PTHR. Analysis of the PTHR heteronuclear RNA suggests that base-pair complementarity in introns surrounding exon 14 causes exon skipping and accounts for generation of the Δe14-PTHR isoform. Thus Δe14-PTHR is a poorly functional receptor that acts as a dominant-negative of PTHR trafficking and signaling and may contribute to PTH resistance.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 01/2011; 26(1):143-55. · 6.04 Impact Factor
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ABSTRACT: Bone growth and remodeling depend upon the opposing rates of bone formation and resorption. These functions are regulated by intrinsic seven transmembrane-spanning receptors, the parathyroid hormone receptor (PTH1R) and frizzled (FZD), through their respective ligands, parathyroid hormone (PTH) and Wnt. FZD activation of canonical beta-catenin signaling requires the adapter protein Dishevelled (Dvl). We identified a Dvl-binding motif in the PTH1R. Here, we report that the PTH1R activates the beta-catenin pathway by directly recruiting Dvl, independent of Wnt or LRP5/6. PTH1R coimmunoprecipitated with Dvl. Deleting the carboxyl-terminal PTH1R PDZ-recognition domain did not abrogate PTH1R-Dvl interactions; nor did truncating the receptor at position 480. However, further deletion eliminating the putative Dvl recognition domain abolished PTH1R interactions with Dvl. PTH activated beta-catenin in a time- and concentration-dependent manner and translocated beta-catenin to the nucleus. beta-Catenin activation was inhibited by Dvl2 dominant negatives and by short hairpin RNA sequences targeted against Dvl2. PTH-induced osteoclastogenesis was also inhibited by Dvl2 dominant negative mutants. These findings demonstrate that G protein-coupled receptors other than FZD directly activate beta-catenin signaling, thereby mimicking many of the functions of the canonical Wnt-FZD pathway. The distinct modes whereby FZD and PTH1R activate beta-catenin control convergent or divergent effects on osteoblast differentiation, and osteoclastogenesis may arise from PTH1R-induced second messenger phosphorylation.
Journal of Biological Chemistry 03/2010; 285(19):14756-63. · 4.77 Impact Factor
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ABSTRACT: Transgenic mice lacking calcium channel beta3 subunits (CaVbeta3) were used to determine the involvement of a multimeric calcium channel in mediating stimulated renal calcium absorption. We measured the ability of calcium channel beta3 subunit-null (CaVbeta3-/-) and wild-type (CaVbeta3+/+) mice to increase renal calcium absorption in response to the calcium-sparing diuretic chlorothiazide (CTZ). Control rates of fractional sodium excretion were comparable in CaVbeta3-/- and CaVbeta3+/+ mice and CTZ increased sodium excretion similarly in both groups. CTZ enhanced calcium absorption only in wild-type CaVbeta3+/+ mice. This effect was specific for diuretics acting on distal tubules because both CaVbeta3-/- and CaVbeta3+/+ mice responded comparably to furosemide. The absence of beta3 subunits resulted in compensatory increases of TrpV5 calcium channels, the plasma membrane Ca-ATPase, NCX1 Na/Ca exchanger protein, and calbindin-D9k but not calbindin-D28k. We conclude that TrpV5 mediates basal renal calcium absorption and that a multimeric calcium channel that includes CaVbeta3 mediates stimulated calcium transport.
Canadian Journal of Physiology and Pharmacology 07/2009; 87(7):522-30. · 1.95 Impact Factor
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ABSTRACT: PTH regulates renal calcium homeostasis by actions on the distal nephron. PTH-induced calcium transport in mouse distal convoluted tubule (DCT) cells requires activation of ERK1/2. ERK activation by beta-adrenergic receptors occurs in a biphasic manner and involves receptor internalization. An early rapid phase is beta-arrestin (betaAr) independent, whereas prolonged activation is betaAr dependent. We characterized PTH-stimulated ERK activation and the involvement of receptor internalization and betaAr dependence. In DCT cells, PTH transiently activated ERK maximally at 5 min and then returned to baseline. betaAr dependence of PTH receptor (PTH1R)-mediated ERK stimulation was assessed using mouse embryonic fibroblasts (MEFs) from betaAr1- and -2-null mice. In wild-type MEFs, PTH(1-34)-stimulated ERK activation peaked after 5 min, was 50% maximal after 15 min, and then recovered to 80% of maximal stimulation by 30 min. In MEFs null for betaAr1 and -2, PTH-stimulated ERK activation peaked by 5 min and returned to baseline. The effect was identical in betaAr2-null MEFs. In betaAr1-null MEFs, ERK exhibited delayed activation and remained elevated. PTH-stimulated ERK activation and receptor endocytosis were not inhibited by the clathrin-binding domain of betaAr1 [Ar(319-418)]. Coexpression of the sodium proton exchanger regulatory factor 1 (NHERF1) with Ar(319-418) blocked PTH1R internalization. We conclude that PTH-stimulated ERK activation in DCT cells proceeds with a rapid but transient phase that may involve betaAr1. Furthermore, the betaAr-dependent late phase of ERK activation by PTH requires the participation of betaAr2 and PTH1R internalization.
Endocrinology 09/2007; 148(8):4073-9. · 4.46 Impact Factor
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ABSTRACT: The sodium-hydrogen exchange regulatory factor 1 (NHERF-1/EBP50) interacts with the C terminus of several G protein-coupled receptors (GPCRs). We examined the role of NHERF-1 and the cytoskeleton on the distribution, dynamics, and trafficking of the beta(2)-adrenergic receptor (beta(2)AR; a type A receptor), the parathyroid hormone receptor (PTH1R; type B), and the calcium-sensing receptor (CaSR; type C) using fluorescence recovery after photobleaching, total internal reflection fluorescence, and image correlation spectroscopy. beta(2)AR bundles were observed only in cells that expressed NHERF-1, whereas the PTH1R was localized to bundles that parallel stress fibers independently of NHERF-1. The CaSR was never observed in bundles. NHERF-1 reduced the diffusion of the beta(2)AR and the PTH1R. The addition of ligand increased the diffusion coefficient and the mobile fraction of the PTH1R. Isoproterenol decreased the immobile fraction but did not affect the diffusion coefficient of the beta(2)AR. The diffusion of the CaSR was unaffected by NHERF-1 or the addition of calcium. NHERF-1 reduced the rate of ligand-induced internalization of the PTH1R. This phenomenon was accompanied by a reduction of the rate of arrestin binding to PTH1R in ligand-exposed cells. We conclude that some GPCRs, such as the beta(2)AR, are attached to the cytoskeleton primarily via the binding of NHERF-1. Others, such as the PTH1R, bind the cytoskeleton via several interacting proteins, one of which is NHERF-1. Finally, receptors such as the CaSR do not interact with the cytoskeleton in any significant manner. These interactions, or the lack thereof, govern the dynamics and trafficking of the receptor.
Journal of Biological Chemistry 09/2007; 282(34):25076-87. · 4.77 Impact Factor
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ABSTRACT: The PTH receptor (PTH1R) activates multiple signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/2). The role of epidermal growth factor receptor (EGFR) transactivation in ERK1/2 activation by PTH in distal kidney cells, a primary site of PTH action, was characterized. ERK1/2 phosphorylation was stimulated by PTH and blocked by the EGFR inhibitor, AG1478. Upon PTH stimulation, metalloprotease cleavage of membrane-bound heparin-binding fragment (HB-EGF) induced EGFR transactivation of ERK. Conditioned media from PTH-treated distal kidney cells activated ERK in HEK-293 cells. AG1478 added to HEK-293 cells ablated transactivation by conditioned media. HB-EGF directly activated ERK1/2 in HEK-293 cells. Pretreatment of distal kidney cells with the metalloprotease inhibitor GM-6001 abolished transactivation of ERK1/2 by PTH. The role of the PTH1R COOH terminus in PTX-sensitive ERK1/2 activation was characterized in HEK-293 cells transfected with wild-type PTH1R, with a PTH1R mutated at its COOH terminus, or with PTH1R truncated at position 480. PTH stimulated ERK by wild-type, mutated and truncated PTH1Rs 21-, 27- and 57-fold, respectively. Thus, the PTH1R COOH terminus exerts an inhibitory effect on ERK activation. EBP50, a scaffolding protein that binds to the PDZ recognition domain of the PTH1R, impaired PTH but not isoproterenol or calcitonin-induced ERK activation. Pertussis toxin inhibited PTH-stimulated ERK1/2 by mutated and truncated PTH1Rs and abolished ERK1/2 activation by wild-type PTH1R. We conclude that ERK phosphorylation in distal kidney cells by PTH requires PTH1R activation of G(i), which leads to stimulation of metalloprotease-mediated cleavage of HB-EGF and transactivation of the EGFR and is regulated by EBP50.
American journal of physiology. Renal physiology 04/2007; 292(3):F1028-34. · 3.68 Impact Factor
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ABSTRACT: Internalization of the PTH type I receptor (PTH1R) is regulated in a cell- and ligand-specific manner. We previously demonstrated that the sodium/proton exchanger regulatory factor type 1 (NHERF1; EBP50) is pivotal in determining the range of peptides that internalize the PTH1R. Antagonist PTH fragments can internalize the PTH1R in some kidney and bone cell models. PTH(7-34), which binds to, but does not activate, the PTH1R, internalizes the PTH1R in kidney distal tubule (DT) cells, where NHERF1 is not expressed. The effect of antagonist PTHrP peptides has not, to this point, been assessed. PTH1R internalization was measured by real-time confocal fluorescence microscopy of DT cells stably expressing 105 EGFP-tagged PTH1R/cell. PTHrP(7-34) internalized the PTH1R in a manner indistinguishable from PTH(7-34). Introduction of NHERF1 into DT cells, however, blocked PTH(7-34)-, but not PTHrP(7-34)-, induced PTH1R internalization. To delineate the sequences within PTHrP that determine whether PTH1R internalization is affected by NHERF1, chimeric PTH/PTHrP fragments were tested for their ability to induce PTH1R internalization. PTH(7-21)/PTHrP (22-34), PTH(7-32)/PTHrP(33-34), and PTH(7-33)/PTHrP(34) at 1 microM each internalized the PTH1R 50-70% in a NHERF1-independent manner. When the C terminus of PTHrP was replaced with homologous amino acids from PTH, NHERF1 inhibited PTH1R internalization. It was determined that simply mutating F34 to A in PTH induced PTH1R internalization in a NHERF1-independent manner. None of the chimeric peptides activated the PTH1R but all effectively competed for 1 nM PTH(1-34) in cyclic AMP assays. In addition, all chimeric peptides competed for radiolabeled PTH(1-34) in binding assays in DT cells. PTH(1- 34) and PTHrP(7-34), but not PTH(7-34), efficiently recruited beta-arrestin1 to plasma membrane PTH1Rs. We, therefore, conclude that PTH(1-34) and PTHrP(7-34) induce a conformational change in the PTH1R that promotes arrestin binding and dissociates NHERF1 from PTH1R internalization.
Endocrine 01/2007; 30(3):343-52. · 1.42 Impact Factor
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ABSTRACT: G protein-coupled receptors (GPCRs) mediate the action of many hormones, cytokines, and sensory and chemical signals. It is generally thought that receptor desensitization and internalization require occupancy and activation of the GPCR. PTH and PTHrP receptor (PTH1R) belongs to GPCR class B and is the major regulator of extracellular calcium homeostasis. Using kidney distal convoluted tubule cells transfected with a human PTH1R/enhanced green fluorescent protein fusion protein, quantitative, real-time fluorescence microscopy was used to analyze receptor internalization. In these cells, which are the target of the calcium-sparing action of PTH, PTH(1-34) activated adenylyl cyclase (AC) and phospholipase C (PLC) and PTH1R endocytosis. PTH(1-31), however, stimulated AC and PLC but not PTH1R endocytosis. Conversely, PTH(7-34) rapidly stimulated PTH1R internalization without activating AC or PLC. PTH(2-34) and (3-34) caused PTH1R internalization intermediate between PTH(1-34) and (7-34). PTH1R sequestration occurred in a dynamin- and clathrin-dependent manner. Directly activating AC inhibited PTH1R internalization in response to PTH(7-34). PTH1R endocytosis was sensitive to protein kinase C inhibition. PTH(1-34), (7-34), and (1-31) evoked PTH1R phosphorylation. Removal of most of the C terminus of the PTH1R eliminated receptor phosphorylation and the cAMP/protein kinase C sensitivity of internalization. PTH(1-34) and (7-34) internalized the truncated PTH1R with identical kinetics, and the response was unaffected by forskolin. Thus, the PTH1R C terminus contains regulatory sequences that are involved in, but not required for, PTH1R internalization. The results demonstrate that receptor activation and internalization can be selectively dissociated.
Endocrinology 07/2004; 145(6):2815-23. · 4.46 Impact Factor
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ABSTRACT: Parathyroid hormone (PTH) regulates extracellular calcium homeostasis through the type 1 PTH receptor (PTH1R) expressed in kidney and bone. The PTH1R undergoes beta-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 beta-arrestin-independent but dynamin-dependent 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.
Journal of Biological Chemistry 11/2003; 278(44):43787-96. · 4.77 Impact Factor
