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Regulation of parathyroid hormone type 1 receptor dynamics, traffic, and signaling by the Na+/H+ exchanger regulatory factor-1 in rat osteosarcoma ROS 17/2.8 cells.

ABSTRACT: The effects of the expression of the Na+/H+ exchanger regulatory factor-1 (NHERF1) on the distribution, dynamics, and signaling properties of the PTH type 1 receptor (PTH1R) were studied in rat osteosarcoma cells ROS 17/2.8. NHERF1 had a dramatic effect on the subcellular distribution of PTH1R, promoting a substantial relocation of the receptor to regions of the plasma membrane located in very close proximity to cytoskeletal fibers. Direct interactions of NHERF1 with the PTH1R and the cytoskeleton were required for these effects, because they were abolished by 1) PTH1R mutations that impair NHERF1 binding, and 2) NHERF1 mutations that impair binding to the PTH1R or the cytoskeleton. NHERF1 reduced significantly the diffusion of the PTH1R by a mechanism that was also dependent on a direct association of NHERF1 with the PTH1R and the cytoskeleton. NHERF1 increased ligand-dependent production of cAMP and induced ligand-dependent rises in intracellular calcium. These effects on calcium were due to increased calcium uptake, as they were blocked by calcium channel inhibitors and by the addition of EGTA to the medium. These calcium effects were abolished by protein kinase A inhibition but phospholipase C inhibition was without effect. Based on these analyses, we propose that, in ROS cells, the presence of NHERF1 induces PTH-dependent calcium signaling by a cAMP-mediated mechanism that involves local protein kinase A-dependent activation of calcium channels.

SUBMITTER: Wheeler D

PROVIDER: S-EPMC2366176 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Regulation of parathyroid hormone type 1 receptor dynamics, traffic, and signaling by the Na+/H+ exchanger regulatory factor-1 in rat osteosarcoma ROS 17/2.8 cells.

Wheeler David D Garrido Jose Luis JL Bisello Alessandro A Kim Yung Kyu YK Friedman Peter A PA Romero Guillermo G

Molecular endocrinology (Baltimore, Md.) 20080117 5

The effects of the expression of the Na+/H+ exchanger regulatory factor-1 (NHERF1) on the distribution, dynamics, and signaling properties of the PTH type 1 receptor (PTH1R) were studied in rat osteosarcoma cells ROS 17/2.8. NHERF1 had a dramatic effect on the subcellular distribution of PTH1R, promoting a substantial relocation of the receptor to regions of the plasma membrane located in very close proximity to cytoskeletal fibers. Direct interactions of NHERF1 with the PTH1R and the cytoskelet ...[more]

PMID: 18202147

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Project description:It is known that parathyroid hormone (PTH) activates the cyclic AMP (cAMP) signalling pathway in osteoblasts. In recent years it has been suggested that an elevation of the intracellular free Ca2+ concentration ([Ca2+]i) may also be involved in the regulation of osteoblast function by PTH. However, this remains controversial. Here we investigated the effect of PTH on the [Ca2+]i of ROS 17/2.8 cells and normal human osteoblasts. The [Ca2+]i was measured in single aequorin-injected cells and in suspensions of cells loaded with fura-2. Human PTH-(1-38)-peptide (1-300 nM) had no effect on the [Ca2+]i in single aequorin-injected ROS 17/2.8 cells (n = 17) measured at various times after injection (1-20 h), or in suspensions of fura-2-loaded ROS 17/2.8 cells (n = 9). Ionomycin (1 microM) increased the [Ca2+]i in fura-2-loaded and single aequorin-injected ROS 17/2.8 cells by 285 +/- 60 nM (n = 9) and 312 +/- 99 nM (n = 6) respectively, indicating that both methods detect changes in [Ca2+]i with equal sensitivity. In contrast, human PTH-(1-38) (10-100 nM) markedly stimulated cAMP accumulation in ROS 17/2.8 cells. In single aequorin-injected normal human osteoblasts there was no change in the [Ca2+]i in response to 100 nM human PTH-(1-38) or 100 nM bovine PTH-(1-84) (n = 18). In contrast, in suspensions of normal human osteoblasts loaded with fura-2, an increase in [Ca2+]i in response to human PTH-(1-38) (100 nM) was found (60 +/- 28 nM; n = 6). Considerable variation in the magnitude of the response was observed between individual preparations and donors. These data indicate that PTH activates cAMP accumulation without affecting [Ca2+]i in ROS 17/2.8 cells and that PTH causes a rise in [Ca2+]i only in a small subset of normal human osteoblasts. We suggest that the Ca2+ response to PTH in osteoblasts is limited by the state of differentiation of the cells, and may be due either to the presence of a distinct Ca2(+)-mobilizing receptor or to a cAMP-mediated Ca2+ response.

Project description:Disorders related to parathyroid hormone (PTH) resistance and PTH signaling pathway impairment are historically classified under the term of pseudohypoparathyroidism (PHP). The disease was first described and named by Fuller Albright and colleagues in 1942. Albright hereditary osteodystrophy (AHO) is described as an associated clinical entity with PHP, characterized by brachydactyly, subcutaneous ossifications, round face, short stature and a stocky build. The classification of PHP is further divided into PHP-Ia, pseudo-PHP (pPHP), PHP-Ib, PHP-Ic and PHP-II according to the presence or absence of AHO, together with an in vivo response to exogenous PTH and the measurement of Gs? protein activity in peripheral erythrocyte membranes in vitro. However, PHP classification fails to differentiate all patients with different clinical and molecular findings for PHP subtypes and classification become more complicated with more recent molecular characterization and new forms having been identified. So far, new classifications have been established by the EuroPHP network to cover all disorders of the PTH receptor and its signaling pathway. Inactivating PTH/PTH-related protein signaling disorder (iPPSD) is the new name proposed for a group of these disorders and which can be further divided into subtypes - iPPSD1 to iPPSD6. These are termed, starting from PTH receptor inactivation mutation (Eiken and Blomstrand dysplasia) as iPPSD1, inactivating Gs? mutations (PHP-Ia, PHP-Ic and pPHP) as iPPSD2, loss of methylation of GNAS DMRs (PHP-Ib) as iPPSD3, PRKAR1A mutations (acrodysostosis type 1) as iPPSD4, PDE4D mutations (acrodysostosis type 2) as iPPSD5 and PDE3A mutations (autosomal dominant hypertension with brachydactyly) as iPPSD6. iPPSDx is reserved for unknown molecular defects and iPPSDn+1 for new molecular defects which are yet to be described. With these new classifications, the aim is to clarify the borders of each different subtype of disease and make the classification according to molecular pathology. The iPPSD group is designed to be expandable and new classifications will readily fit into it as necessary.

Dataset Information

Regulation of parathyroid hormone type 1 receptor dynamics, traffic, and signaling by the Na+/H+ exchanger regulatory factor-1 in rat osteosarcoma ROS 17/2.8 cells.

Publications

Regulation of parathyroid hormone type 1 receptor dynamics, traffic, and signaling by the Na+/H+ exchanger regulatory factor-1 in rat osteosarcoma ROS 17/2.8 cells.

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