Interaction of MAP17 with NHERF3/4 induces translocation of the renal Na/Pi IIa transporter to the trans-Golgi

2007 ◽  
Vol 292 (1) ◽  
pp. F230-F242 ◽  
Author(s):  
Miguel A. Lanaspa ◽  
Héctor Giral ◽  
Sophia Y. Breusegem ◽  
Nabil Halaihel ◽  
Goretti Baile ◽  
...  
Keyword(s):  
Pdz Domain ◽  
Phosphate Transport ◽  
Interacting Proteins ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Kinase C ◽  
Important Intermediate ◽  
Pdz Protein ◽  
Golgi Network ◽  
Two Hybrid

The function of the NaPiIIa renal sodium-phosphate transporter is regulated through a complex network of interacting proteins. Several PDZ domain-containing proteins interact with its COOH terminus while the small membrane protein MAP17 interacts with its NH2 end. To elucidate the function of MAP17, we identified its interacting proteins using both bacterial and mammalian two-hybrid systems. Several PDZ domain-containing proteins, including the four NHERF proteins, as well as NaPiIIa and NHE3, were found to bind to MAP17. The interactions of MAP17 with the NHERF proteins and with NaPiIIa were further analyzed in opossum kidney (OK) cells. Expression of MAP17 alone had no effect on the NaPiIIa apical membrane distribution, but coexpression of MAP17 and NHERF3 or NHERF4 induced internalization of NaPiIIa, MAP17, and the PDZ protein to the trans-Golgi network (TGN). This effect was not observed when MAP17 was cotransfected with NHERF1/2 proteins. Inhibition of protein kinase C (PKC) prevented expression of the three proteins in the TGN. Activation of PKC in OK cells transfected only with MAP17 induced complete degradation of MAP17 and NaPiIIa. When lysosomal degradation was prevented, both proteins accumulated in the TGN. When the dopamine D1-like receptor was activated with fenoldopam, both NaPiIIa and MAP17 also accumulated in the TGN. Finally, cotransfection of MAP17 and NHERF3 prevented the adaptive upregulation of phosphate transport activity in OK cells in response to low extracellular phosphate. Therefore, the interaction between MAP17, NHERF3/4, and NaPiIIa in the TGN could be an important intermediate or alternate path in the internalization of NaPiIIa.

A dual mechanism for regulation of kidney phosphate transport by parathyroid hormone

1987 ◽  
Vol 253 (2) ◽  
pp. E221-E227 ◽  
Author(s):  
J. A. Cole ◽  
S. L. Eber ◽  
R. E. Poelling ◽  
P. K. Thorne ◽  
L. R. Forte
Keyword(s):  
Protein Kinase C ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Rank Order ◽  
Phosphate Transport ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Kinase C ◽  
Camp Formation

Regulation of phosphate transport by parathyroid hormone (PTH) was investigated in continuous lines of kidney cells. Phosphate transport was reduced by PTH-(1-34) at physiological concentrations (EC50 5 X 10(-11) M), whereas much higher concentrations were required to stimulate cAMP formation (EC50 1 X 10(-8) M) in opossum kidney (OK) cells. The PTH analogue [Nle]PTH-(3-34) also inhibited phosphate transport but did not enhance cAMP formation. Instead, [Nle]PTH-(3-34) was a competitive antagonist of PTH-(1-34) at cyclase-coupled receptors. PTH-(7-34) had no effect on phosphate transport or cAMP formation. Phorbol esters or mezerein were potent inhibitors of phosphate transport but did not affect cAMP synthesis. Their potencies paralleled the rank-order potency of these agents as activators of protein kinase c in other systems. Maximally effective concentrations of PTH-(1-34) and mezerein did not produce additive inhibition of phosphate transport in OK cells. Phorbol esters stimulated phosphate transport in JTC-12 cells, but PTH-(1-34) had no effect. We concluded that PTH regulates OK cell phosphate transport by interacting with two classes of receptors, and transmembrane-signaling mechanisms. Physiological levels of PTH-(1-34) may regulate phosphate transport by activation of protein kinase c, whereas higher concentrations appear to activate adenylate cyclase.

scholarly journals Inorganic Phosphate Modulates the Expression of the NaPi-2a Transporter in thetrans-Golgi Network and the Interaction with PIST in the Proximal Tubule

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Miguel A. Lanaspa ◽  
Yupanqui A. Caldas ◽  
Sophia Y. Breusegem ◽  
Ana Andrés-Hernando ◽  
Christina Cicerchi ◽  
...  
Keyword(s):  
Inorganic Phosphate ◽  
Culture Medium ◽  
Pdz Domain ◽  
Proximal Tubules ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Pi Homeostasis ◽  
Pi Transport ◽  
Endosomal Transport ◽  
Golgi Network

Inorganic phosphate (Pi) homeostasis is maintained by the tight regulation of renal Pi excretion versus reabsorption rates that are in turn modulated by adjusting the number of Pi transporters (mainly NaPi-2a) in the proximal tubules. In response to some hormones and a high dietary Pi content, NaPi-2a is endocytosed and degraded in the lysosomes; however, we show here that some NaPi-2a molecules are targeted to thetrans-Golgi network (TGN) during the endocytosis. In the TGN, NaPi-2a interacts with PIST (PDZ-domain protein interacting specifically with TC10), a TGN-resident PDZ-domain-containing protein. The extension of the interaction is proportional to the expression of NaPi-2a in the TGN, and, consistent with that, it is increased with a high Pi diet. When overexpressed in opossum kidney (OK) cells, PIST retains NaPi-2a in the TGN and inhibits Na-dependent Pi transport. Overexpression of PIST also prevents the adaptation of OK cells to a low Pi culture medium. Our data supports the view that NaPi-2a is subjected to retrograde trafficking from the plasma membrane to the TGN using one of the machineries involved in endosomal transport and explains the reported expression of NaPi-2a in the TGN.

cAMP-dependent and -independent downregulation of type II Na-Pi cotransporters by PTH

1999 ◽  
Vol 276 (5) ◽  
pp. F720-F725 ◽  
Author(s):  
Markus F. Pfister ◽  
Jutka Forgo ◽  
Urs Ziegler ◽  
Jürg Biber ◽  
Heini Murer
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Phospholipase C ◽  
Type Ii ◽  
Peptide Fragments ◽  
Regulatory Pathways ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Kinase C ◽  
Proximal Tubular

Parathyroid hormone (PTH) leads to the inhibition of Na-Pi cotransport activity and to the downregulation of the number of type II Na-Pi cotransporters in proximal tubules, as well as in opossum kidney (OK) cells. PTH is known also to lead to an activation of adenylate cyclase and phospholipase C in proximal tubular preparations, as well as in OK cells. In the present study, we investigated the involvement of these two regulatory pathways in OK cells in the PTH-dependent downregulation of the number of type II Na-Pi cotransporters. We have addressed this issue by using pharmacological activators of protein kinase A (PKA) and protein kinase C (PKC), i.e., 8-bromo-cAMP (8-BrcAMP) and β-12- O-tetradecanoylphorbol 13-acetate (β-TPA), respectively, as well as by the use of synthetic peptide fragments of PTH that activate adenylate cyclase and/or phospholipase C, i.e., PTH-(1–34) and PTH-(3–34), respectively. Our results show that PTH signal transduction via cAMP-dependent, as well as cAMP-independent, pathways leads to a membrane retrieval and degradation of type II Na-Pi cotransporters and, thereby, to the inhibition of Na-Picotransport activity. Thereby, the cAMP-independent regulatory pathway leads only to partial effects (∼50%).

Clonal sublines that are morphologically and functionally distinct from parental OK cells

1989 ◽  
Vol 256 (4) ◽  
pp. F672-F679 ◽  
Author(s):  
J. A. Cole ◽  
L. R. Forte ◽  
W. J. Krause ◽  
P. K. Thorne
Keyword(s):  
Cell Line ◽  
Phosphate Transport ◽  
Parental Line ◽  
Apical Surface ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Sodium Dependent ◽  
Clonal Lines ◽  
P Cells ◽  
Camp Formation

Three clonal subpopulations of opossum kidney (OK) cells were derived from the parental line. The distribution of apical microvilli suggested that the OK cell line was heterogeneous. The clonal OK sublines appeared homogeneous as reflected by microvilli, which were uniformly distributed on the apical surface. Parathyroid hormone (PTH), forskolin (FSK), and prostaglandin E1 (PGE1) increased adenosine 3',5'-cyclic monophosphate (cAMP) formation in OK cells and all of the clones. PTH inhibited sodium-dependent phosphate transport in parental cells and in OK/B and OK/P clones with maximal effects appearing at 4, 2, and 1 h, respectively. PTH had no effect on phosphate transport in OK/H cells. FSK inhibited phosphate transport in parental cells and OK/B and OK/P clones but was relatively ineffective in OK/H cells. PGE1 decreased phosphate transport in OK/B and OK/P cells but was ineffective in the parental line and in OK/H cells. Phorbol 12-myristate 13-acetate, a potent inhibitor of phosphate transport in the parental OK cell line, had little effect in the clonal sublines. These clonal lines have remained phenotypically stable for 10 passages and should prove useful in studying the regulation of phosphate transport by PTH as well as addressing the question of whether PTH receptor subclasses exist which couple to cAMP and/or calcium effector systems in kidney cells.

Brefeldin A inhibits phosphate transport in opossum kidney cells

1993 ◽  
Vol 264 (1) ◽  
pp. C40-C47 ◽  
Author(s):  
A. W. Capparelli ◽  
M. C. Heng ◽  
L. Li ◽  
O. D. Jo ◽  
N. Yanagawa
Keyword(s):  
Protein Synthesis ◽  
Golgi Apparatus ◽  
Phosphate Uptake ◽  
Brefeldin A ◽  
Phosphate Transport ◽  
Transport Processes ◽  
Free Medium ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Phosphate Deprivation

Brefeldin A (BFA) is a fungal metabolite that blocks the transport processes between the endoplasmic reticulum and the Golgi apparatus. In the present study, we have tested the effect of BFA on phosphate transport in a kidney epithelial cell line, opossum kidney (OK) cells. Electron microscopy showed that exposure of OK cells to BFA caused a rapid and reversible disorganization of Golgi apparatus. Addition of BFA also caused a time (2-8 h)- and dose (1-10 micrograms/ml)-dependent inhibition of Na(+)-dependent cell phosphate uptake. The inhibition of cell phosphate uptake by BFA was reversible and was associated with a decrease in the maximum velocity of phosphate transport. Both the inhibition and the stimulation of cell phosphate uptake by parathyroid hormone and insulin, respectively, were not affected by BFA. BFA at 1 microgram/ml concentration did not affect protein synthesis as determined by [3H]leucine incorporation but diminished the adaptive increase in cell phosphate uptake in response to 2 or 8 h of incubation in nominally phosphate-free medium. On the other hand, inhibition of protein synthesis by cycloheximide (5 microM) abolished the adaptive increase in cell phosphate uptake in response to 8 but not 2 h of incubation in nominally phosphate-free medium, indicating the existence of an early response to phosphate deprivation, which does not require new protein synthesis but is sensitive to the effect of BFA. In summary, results of these studies show that, in OK cells, BFA inhibits phosphate uptake and curtails the adaptive response to phosphate deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Extracts from tumors causing oncogenic osteomalacia inhibit phosphate uptake in opossum kidney cells

2001 ◽  
Vol 169 (3) ◽  
pp. 613-620 ◽  
Author(s):  
KB Jonsson ◽  
M Mannstadt ◽  
A Miyauchi ◽  
IM Yang ◽  
G Stein ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Phosphate Transport ◽  
P Uptake ◽  
Low Molecular Weight ◽  
Kidney Cells ◽  
Camp Accumulation ◽  
Oncogenic Osteomalacia ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Hypophosphatemic Osteomalacia

In oncogenic osteomalacia (OOM), a tumor produces an unknown substance that inhibits phosphate reabsorption in the proximal tubules. This causes urinary phosphate wasting and, as a consequence, hypophosphatemic osteomalacia. To characterize this poorly understood biological tumor activity we generated aqueous extracts from several OOM tumors. Extracts from three of four tumors inhibited, dose- and time-dependently, (32)P-orthophosphate uptake by opossum kidney (OK) cells; maximum inhibition was about 45% of untreated control. Further characterization revealed that the factor is resistant to heat and several proteases, and that it has a low molecular weight. The tumor extracts also stimulated cAMP accumulation in OK cells, but not in osteoblastic ROS 17/2.8 and UMR106 cells, or in LLC-PK1 kidney cells expressing the parathyroid hormone (PTH)/PTH-related peptide receptor or the PTH-2 receptor. HPLC separation of low molecular weight fractions of the tumor extracts revealed that the flow-through of all three positive tumor extracts inhibited (32)P uptake and stimulated cAMP accumulation in OK cells. Additionally, a second peak with inhibitory activity on phosphate transport, but without cAMP stimulatory activity, was identified in the most potent tumor extract. We have concluded that several low molecular weight molecules with the ability to inhibit phosphate transport in OK cells can be found in extracts from OOM tumors. It remains uncertain, however, whether these are related to the long-sought phosphaturic factor responsible for the phosphate wasting seen in OOM patients.

Na+/H+ Exchanger-Regulatory Factor 1 Mediates Inhibition of Phosphate Transport by Parathyroid Hormone and Second Messengers by Acting at Multiple Sites in Opossum Kidney Cells

2003 ◽  
Vol 17 (11) ◽  
pp. 2355-2364 ◽  
Author(s):  
Matthew J. Mahon ◽  
Judith A. Cole ◽  
Eleanor D. Lederer ◽  
Gino V. Segre
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylyl Cyclase ◽  
Sodium Phosphate ◽  
Regulatory Factor ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Kinase C ◽  
Phosphate Cotransport ◽  
Sodium Phosphate Cotransport

Abstract The opossum kidney (OK) line displays PTH-mediated activation of adenylyl cyclase and phospholipase C and inhibition of phosphate (Pi) uptake via regulation of the type IIa sodium-phosphate cotransporter, consistent with effects in vivo. OKH cells, a subclone of the OK cell line, robustly activates PTH-mediated activation of adenylyl cyclase, but is defective in PTH-mediated inhibition of sodium-phosphate cotransport and signaling via phospholipase C. Compared with wild-type OK cells, OKH cells express low levels of the Na+/H+ exchanger regulatory factor 1 (NHERF-1). Stable expression of NHERF-1 in OKH cells (OKH-N1) rescues the PTH-mediated inhibition of sodium-phosphate cotransport. NHERF-1 also restores the capacity of 8-bromo-cAMP and forskolin to inhibit Pi uptake, but the PTH dose-response for cAMP accumulation and inhibition of Pi uptake differ by 2 orders of magnitude. NHERF-1, in addition, modestly restores phorbol ester-mediated inhibition of Pi uptake, which is much weaker than that elicited by PTH. A poor correlation exists between the inhibition of Pi uptake mediated by PTH (∼60%) and the inhibition mediated by phorbol 12-myristate 13-acetate (∼30%) and the ability of these molecules to activate the protein kinase C-responsive reporter gene. Furthermore, we show that NHERF-1 directly interacts with type IIa cotransporter in OK cells. Although, PTH-mediated inhibition of Pi uptake in OK cells is largely NHERF-1 dependent, the signaling pathway(s) by which this occurs is still unclear. These pathways may involve cooperativity between cAMP- and protein kinase C-dependent pathways or activation/inhibition of an unrecognized NHERF-1-dependent pathway(s).

Modulation of Na+-Pi cotransport in opossum kidney cells by extracellular phosphate

1988 ◽  
Vol 255 (2) ◽  
pp. C155-C161 ◽  
Author(s):  
J. Biber ◽  
J. Forgo ◽  
H. Murer
Keyword(s):  
Actinomycin D ◽  
Phosphate Transport ◽  
Kidney Cells ◽  
Transport Activity ◽  
Similar Extent ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Opossum Kidney Cells ◽  
Dependent Transport ◽  
Posttranscriptional Level

The effect of the extracellular concentration of Pi on the Na+-dependent phosphate transport activity of OK cells was investigated. When incubated with extracellular Pi at concentrations of 200 microM or less, Na+-Pi cotransport increased approximately twofold in OK cells compared with control cells (kept in 0.85 mM Pi), whereas other Na+-dependent transport activities were not affected. After Pi deprivation, Na+-Pi cotransport could be inhibited to a similar extent (80%) by parathyroid hormone (PTH) as in control cells, suggesting that the PTH-sensitive Na+-Pi cotransport activity is also regulated by extracellular Pi. The increase of Na+-Pi cotransport was maximally expressed after 6 h and could be prevented by cycloheximide (70 microM) but not by actinomycin D (0.5-5 g/ml). However, the adaptive response was completely blocked by 3'-deoxyadenosine (cordycepin) at 100 microM. From these data, it is concluded that the upregulation of Na+-Pi cotransport in OK cells due to low extracellular Pi is controlled at a posttranscriptional level.

scholarly journals Dynamics of PTH-induced disassembly of Npt2a/NHERF-1 complexes in living OK cells

2011 ◽  
Vol 300 (1) ◽  
pp. F231-F235 ◽  
Author(s):  
Edward J. Weinman ◽  
Deborah Steplock ◽  
Shirish Shenolikar ◽  
Thomas A. Blanpied
Keyword(s):  
Proximal Tubule ◽  
Time Course ◽  
Apical Surface ◽  
Adapter Protein ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Sequence Of Events ◽  
Kinase C ◽  
Sodium Hydrogen Exchanger ◽  
Rapid Activation

Parathyroid hormone (PTH) inhibits the reabsorption of phosphate in the renal proximal tubule by disrupting the binding of the sodium-dependent phosphate transporter 2A (Npt2a) to the adapter protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), a process initiated by activation of protein kinase C (PKC). To gain additional insights into the dynamic sequence of events, the time course of these responses was studied in living opossum kidney (OK) cells. Using a FRET-based biosensor, we found that PTH activated intracellular PKC within seconds to minutes. In cells expressing GFP-Npt2a and mCherry-NHERF, PTH did not affect the relative abundance of NHERF-1 but there was a significant and time-dependent decrease in the Npt2a/NHERF-1 ratio. The half-time to maximal dissociation was 15 to 20 min. By contrast, PTH had no effect on the fluorescence ratio for GFP-ezrin compared with mCherry-NHERF-1 at the apical surface. These experiments establish that PTH treatment of proximal tubule OK cells leads to rapid activation of PKC with the subsequent dissociation of Npt2a/NHERF-1 complexes. The association of NHERF-1 with Ezrin and their localization at the apical membrane, however, was unperturbed by PTH, thereby enabling the rapid recruitment and membrane reinsertion of Npt2a and other NHERF-1 targets on termination of the hormone response.

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