Dissociation between parathyroid hormone‐stimulated cAMP and calcium increase in UMR‐106‐01 cells

Merritt, Bradley S.; Yamaguchi, Dean T.; Green, Jacob; Muallem, Shmuel

doi:10.1002/jcp.1041520311

Cited by 12 publications

(13 citation statements)

References 44 publications

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“…No shortterm desensitization in PTH/PTHrP receptor was observed in LLCPK-1 cells. Although classic desensitization of PTH/PTHrP receptor has been reported previously (13)(14)(15)(16)(17)(18)(19)(20)(21)(22), it required long treatment with PTH. Additionally, it was reported that different levels of expression of ␤-adrenergic receptor kinase in lung cell lines resulted in a remarkable difference in short-term desensitization of the ␤ 2 -adrenergic receptor (47).…”

Section: Discussionmentioning

confidence: 95%

“…PTHrP regulates bone development and differentiation by interacting with the PTH/PTHrP receptor found on chondrocytes and bone stromal cells (3)(4)(5)(6). Binding of PTH or PTHrP to the PTH/PTHrP receptor stimulates the accumulation of several intracellular second messengers, such as cAMP and IP3 (2,7), increases the phosphorylation of the PTH/PTHrP receptor on several serine residues within its carboxylterminal tail (8,9), stimulates receptor internalization (10)(11)(12), and causes desensitization of the PTH/PTHrP receptor-second messenger system (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) It has been shown that agonist-stimulated phosphorylation of the PTH/PTHrP receptor is not dependent on PKA or PKC (9,24). Similarly, activation of PKA and PKC is neither necessary nor required for agonist-stimulated receptor internalization (12,25).…”

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confidence: 98%

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

Tawfeek

Qian

Abou‐Samra

2002

Molecular Endocrinology

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We have previously shown that agonist-dependent phosphorylation of the PTH/PTHrP receptor occurs on its carboxyl-terminal tail. Using site-directed mutagenesis, phosphopeptide mapping, and direct sequencing of cyanogen bromide-cleaved fragments of phosphoreceptors, we report here that PTH-dependent phosphorylation occurs on the serine residues at positions 491, 492, 493, 495, 501, and 504, and that the serine residue at position 489 is required for phosphorylation. When these seven sites were mutated to alanine residues, the mutant receptor was no longer phosphorylated after PTH stimulation. The phosphorylation-deficient receptor, stably expressed in LLCPK-1 cells, was impaired in PTH-dependent internalization and showed an increased sensitivity to PTH stimulation; the EC(50) for PTH-stimulated cAMP accumulation was decreased by 7-fold. Furthermore, PTH stimulation of the phosphorylation-deficient PTH/PTHrP receptor caused a sustained elevation in intracellular cAMP levels. These data indicate that agonist-dependent phosphorylation of the PTH/PTHrP receptor plays an important role in receptor function.

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Section: Discussionmentioning

confidence: 95%

mentioning

confidence: 98%

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

Tawfeek

Qian

Abou‐Samra

2002

Molecular Endocrinology

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“…The presence of a functional PC2 in primary cilia suggests that Ca 2ϩ enters the cell, at least in part, through ciliary channels. A functional cAMP pathway in primary cilia suggests a contribution of cAMP-mediated signals to balance and counter Ca 2ϩ surges (3,13,20) and to regulate function in this organelle by controlling the Ca 2ϩ responses, which likely contribute to microtubular stability and thus ciliary length. In summary, herein, we provide the first experimental evidence for the presence of ciliary V2R and a functional cAMP pathway in primary cilia of renal epithelial cells.…”

Section: Discussionmentioning

confidence: 99%

Vasopressin receptor-mediated functional signaling pathway in primary cilia of renal epithelial cells

Raychowdhury

Ramos²,

Zhang³

et al. 2009

American Journal of Physiology-Renal Physiology

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The primary cilium of renal epithelial cells is a nonmotile sensory organelle, implicated in mechanosensory transduction signals. Recent studies from our laboratory indicate that renal epithelial primary cilia display abundant channel activity; however, the presence and functional role of specific membrane receptors in this organelle are heretofore unknown. Here, we determined a functional signaling pathway associated with the type 2 vasopressin receptor (V2R) in primary cilia of renal epithelial cells. Besides their normal localization on basolateral membrane, V2R was expressed in primary cilia of LLC-PK(1) renal epithelial cells. The presence of V2R in primary cilia was determined by spontaneous fluorescence of a V2R-gfp chimera and confirmed by immunocytochemical analysis of wild-type LLC-PK(1) cells stained with anti-V2R antibodies and in LLC-PK(1) cells overexpressing the V2R-Flag, with anti-Flag antibody. Ciliary V2R colocalized with adenylyl cyclase (AC) type V/VI in all cell types tested. Functional coupling of the receptors with AC was confirmed by measurement of cAMP production in isolated cilia and by testing AVP-induced cation-selective channel activity either in reconstituted lipid bilayers or subjected to membrane-attached patch clamping. Addition of either 10 microM AVP (trans) or forskolin (cis) in the presence but not the absence of ATP (1 mM, cis) stimulated cation-selective channel activity in ciliary membranes. This channel activity was reduced by addition of the PKA inhibitor PKI. The data provide the first demonstration for the presence of V2R in primary cilia of renal epithelial cells, and a functional cAMP-signaling pathway, which targets ciliary channel function and may help control the sensory function of the primary cilium.

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“…Several strategies were employed to assess which, if any, of these signal cascades induced by PTH-(1-34) is involved in the suppression of apatite production in UMR cultures. One approach employed a PTH peptide antagonist, Nle 8,18 -Tyr 34 -PTH-(3-34) amide (PTH-(3-34)), which binds to the PTH receptor with high affinity (46,47) but does not produce a cAMP response needed to activate PKA at doses up to 10 nM (43)(44)(45)(46)(47)(48)(49). When added to cultures at concentrations up to 10 nM, PTH-(3-34) did not suppress apatite deposition in UMR cultures (Fig.…”

Section: Pth-(1-34) Potently Suppresses Apatite Deposition In Umrmentioning

confidence: 99%

“…1, A and B). The 10 -20% suppression in apatite content of UMR cultures treated with a 100 nM concentration of the antagonist peptide may be the result of a slight stimulation of PKA at this high dose (43,48), a low affinity stimulation of other signaling mechanisms (44,45,49), 2 or a minor agonist contaminant in the peptide preparation (46,47). These data indicate that mere binding of ligand to the PTH receptor is not sufficient to achieve a significant inhibition of apatite deposition in UMR cultures.…”

Section: Pth-(1-34) Potently Suppresses Apatite Deposition In Umrmentioning

confidence: 99%

Reversible Suppression of in Vitro Biomineralization by Activation of Protein Kinase A

Wang

Martin

Lembke

et al. 2000

Journal of Biological Chemistry

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Parathyroid hormone (PTH-(1-34)) potently suppresses apatite deposition in osteoblastic cultures. These inhibitory effects are mediated through signaling events following PTH receptor binding. Using both selective inhibitors and activators of protein kinase A (PKA), this study shows that a transient activation of PKA is sufficient to account for PTH's inhibition of apatite deposition. This inhibition is not a result of reduced cell proliferation, reduced alkaline phosphatase activity, increased collagenase production, or lowering medium pH. Rather, data suggest a functional relationship between matrix assembly and apatite deposition in vitro. Bone sialoprotein (BSP) and apatite co-localize in the extracellular matrix of mineralizing cultures, with matrix deposition of BSP temporally preceding that of apatite. Transient activation of PKA by either PTH-(1-34) or short term cAMP analog treatment blocks the deposition of BSP in the extracellular matrix without a significant reduction in the total amount of BSP synthesized and secreted. This effect is reversible after allowing the cultures to recover in the absence of PKA activators for several days. Thus, a transient activation of PKA may suppress mineral deposition in vitro as a consequence of altering the assembly of an extracellular matrix permissive for apatite formation.The need to better understand the biological nature of bone mineralization is illustrated by the fact that a properly mineralized endoskeleton is crucial for vertebrate survival (1, 2). To date, several biomineralization theories have been formulated, and each advances a mechanistic model to explain bone formation (3). Lately, these models have emphasized the role of several bone-specific, noncollagenous proteins (4) as promoting the tightly regulated processes of apatite crystal nucleation and growth in an extracellular environment. One such candidate is bone sialoprotein (BSP), 1 a sulfated, phosphorylated matrix glycoprotein (5-8) with a restricted pattern of expression within mineralizing tissues (9 -16), which co-localizes with the smallest detectable foci of newly forming mineralized matrix in osteoid (17).Parathyroid hormone (PTH) is a calciotropic hormone that can stimulate either formation or resorption of bone in vivo depending on such factors as dosage, delivery modality, and treatment frequency (18 -21). In vitro, a continuous exposure to PTH results in the suppression of biomineralization in primary cultures of calvarial osteoblasts (22), calvarial explants (23), or growth plate chondrocytes (24, 25). However, the molecular mechanisms mediating PTH's inhibitory effect on in vitro biomineralization reactions have not been elucidated yet. The first 34 amino acid residues in PTH contain most of the biological activity of the full-length, natural hormone including ligand-receptor binding and signal transduction functions (26, 27). Two major plasma membrane reactions occur as a result of PTH's binding to its receptor that then initiate three intracellular signaling cascades. First, there...

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Dissociation between parathyroid hormone‐stimulated cAMP and calcium increase in UMR‐106‐01 cells

Cited by 12 publications

References 44 publications

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

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

Vasopressin receptor-mediated functional signaling pathway in primary cilia of renal epithelial cells

Reversible Suppression of in Vitro Biomineralization by Activation of Protein Kinase A

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