NaPi-IIa and interacting partners
NaPi-IIa and interacting partners
N Hernando, S. M Gisler, S Pribanic, N Deliot, P Capuano, C. A Wagner, O. W Moe, J Biber and H Murer
1 Institute of Physiology, Zurich University, CH-8057, Zurich, Switzerland
2 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA
Abstract
Regulation of renal proximal tubular reabsorption of phosphate (Pi) is one of the critical steps in Pi homeostasis. Experimental evidence suggests that this regulation is achieved mainly by controlling the apical expression of the Na+-dependent Pi cotransporter type IIa (NaPi-IIa) in proximal tubules. Only recently have we started to obtain information regarding the molecular mechanisms that control the apical expression of NaPi-IIa. The first critical observation was the finding that truncation of only its last three amino acid residues has a strong effect on apical expression. A second major finding was the observation that the last intracellular loop of NaPi-IIa contains sequence information that confers parathyroid hormone (PTH) sensitivity. The use of the above domains of the cotransporter in yeast two-hybrid (Y2H) screening allowed the identification of proteins interacting with NaPi-IIa. Biochemical and morphological, as well as functional, analyses have allowed us to obtain insights into the physiological roles of such interactions, although our present knowledge is still far from complete.
, http://www.100md.com
Renal reabsorption of Pi: NaPi-IIa
NaPi-IIa, also known as Npt2a (SLC34A1), is an electrogenic Na+-dependent Pi cotransporter expressed in the brush border membranes (BBM) of renal proximal tubules (Custer et al. 1994; Murer et al. 2004). Its pattern of expression along the proximal tubules matches the major sites of renal Pi reabsorption, and its role as the main mediator of that process has been clearly demonstrated in an Npt2–/– mouse model (Beck et al. 1998). Npt2–/– mice show a higher urinary excretion of Pi and lower Na+–Pi cotransport activity in BBM compared to wild-type animals.
, 百拇医药
NaPi-IIa is regulated by factors known to control the reabsorption of Pi in the kidney (for review see Murer et al. 2000). This regulation is mediated almost exclusively by the control of the number of cotransporters expressed in the BBM. Thus, phosphaturic factors reduce the expression of NaPi-IIa whereas factors that increase renal Pi reabsorption increase BBM NaPi-IIa. Therefore, knowledge of the mechanisms that control apical expression and membrane retrieval of NaPi-IIa is essential to our understanding of how Pi homeostasis is achieved.
, http://www.100md.com
In the last few years, the combined work of many laboratories has strongly suggested that the regulation, membrane expression and to some extent the fate of endocytosed proteins depends, at least in part, on the network of interactions in which a particular protein is engaged. Therefore, deciphering the pattern of interactions of a given protein may help us understand how it is regulated.
Identification of proteins that interact with cytoplasmic domains of NaPi-IIa
, 百拇医药
The first attempts to identify proteins interacting with NaPi-IIa were performed by conventional Y2H screenings. A major limitation of this technique is the requirement for both interacting partners to reach the cell nucleus, in order to activate the corresponding reporter gene. Thus, both the bait and the prey constructs should encode soluble peptides. In the case of integral membrane proteins, this requirement restricts the design as baits to soluble (cytoplasmic) domains of the protein of interest (i.e. NaPi-IIa). Based on hydropathy plots and antibody accessibility both the N- and C-terminal tails of NaPi-IIa face the cytoplasm (Magagnin et al. 1993; Lambert et al. 1999). Therefore both tails, as well as an internal loop potentially involved in parathyroid hormone (PTH) regulation (see the following three subsections), were chosen as baits for conventional Y2H screenings of a mouse kidney cDNA library. A minimal requisite to ascribe physiological meaning to the interaction is that both partners must be expressed within the same tissue and subcellular compartment in vivo. Therefore, expression within renal proximal tubules of all NaPi-IIa partners described below was confirmed. A summary of these interactions is shown in Fig. 1.
, http://www.100md.com
The expression of all interacting partners in renal proximal tubules was confirmed either by immunoblots, immunohistochemistry and/or RT-PCR. The abbreviations are: PTH-R, PTH receptors; PLC, phospholipase C; PDZ, PDZ domains; MERM, merlin-ezrin-radixin-moesin family; MERM-BD, MERM-binding domain; PKA, protein kinase A; D-AKAP2, dual A-kinase anchoring protein 2. The PDZ domains of NHERF1 and PDZK1 are indicated by rectangles. Interactions between proteins are illustrated either by showing direct contacts of a cartoon-form of each protein or by arrows.
, 百拇医药
Proteins that interact with the N-terminal tail of NaPi-IIa. Y2H screening indicated that the N-terminal cytoplasmic tail of NaPi-IIa interacts with MAP17 (Pribanic et al. 2003a) as well as with VILIP-3, a visinin-like protein (Pribanic et al. 2003b).
MAP17 is a 17 kDa membrane protein associated with various human carcinomas, but also expressed in normal kidneys. Interestingly, MAP17 also interacts with PDZK1 (also known as CLAMP, CAP70 or Diphor1), a PDZ protein found to associate with the C-terminal tail of NaPi-IIa. Yeast trap assays and GST pull-down experiments suggested that MAP17 interacts only with the last (4th) PDZ domain of PDZK1 (Pribanic et al. 2003a). RT-PCR of microdissected tubules indicated the presence of MAP17 mRNA in the S1 and S3 segments of both superficial and deep nephrons. At the protein level, renal expression of MAP17 is restricted to the cortex, with similar intensity in the proximal tubules of superficial and juxtamedullar nephrons. The highest intensity is observed in S1 segments and gradually decreases towards the S3 segments (Pribanic et al. 2003a).
, http://www.100md.com
VILIP-3 is a member of the neuronal calcium sensors family (Spilker et al. 2000). This protein family is characterized by calcium binding, myristoylation, and calcium-dependent membrane association. However, very little is known about their function. RT-PCR from microdissected nephron segments was also used to analyse the expression of VILIP-3 mRNA in kidney (Pribanic et al. 2003b). This study detected VILIP-3 in all segments from both superficial and deep nephrons. By immunofluorescence, VILIP-3 protein is observed in proximal and distal segments as well as in glomeruli. Interestingly, in contrast to distal tubules where VILIP-3 is distributed throughout the cytoplasm, in proximal tubules it is associated with the BBM.
, http://www.100md.com
Proteins that interact with the last intracellular loop of NaPi-IIa. We found several years ago that NaPi-IIb, a second member of the NaPi-II family, is PTH insensitive (Karim-Jimenez et al. 2000). Based on chimera studies, the signal responsible for hormonal sensitivity was identified. This signal, located on the last intracellular loop, consists of two positively charged residues in NaPi-IIa (KR) that are replaced by uncharged residues in NaPi-IIb (NI). The PTH sensitivity of the cotransporters can be reverted by swapping just these two isoform-specific residues. Y2H screening performed in our laboratory failed to identify interacting candidates that could provide hints about the molecular mechanism underlying the above findings (S. Pribanic, unpublished observations). However, the KR motif has been recently found to interact with PEX19 (Ito et al. 2004), a protein involved in the binding and trafficking of peroxisomal proteins (Jones et al. 2004). PEX19 mRNA is expressed in kidneys and in opossum kidney (OK) cells (Ito et al. 2004). In kidney, protein expression has been suggested in cytosolic and BBM fractions. PEX19 binds to wild-type NaPi-IIa but not to a mutant in which the KR motif is replaced by NI residues (Ito et al. 2004). Transfection of PEX19 in OK cells stimulates NaPi-IIa endocytosis even in the absence of PTH, suggesting a role in the internalization of the cotransporter.
, 百拇医药
Proteins that interact with the C-terminal tail of NaPi-IIa. Apical expression of NaPi-IIa is partially prevented upon truncation of the last three C-terminal residues (TRL). After transfection in OK cells, the TRL mutant fails to reach and/or remain at the plasma membrane and accumulates in the cytoplasm, indicating that these residues are required for proper apical expression (Karim-Jimenez et al. 2001). The TRL sequence resembles a class-I PSD-95, Discs-large, ZO-1 (PDZ) binding domain. PDZ domains represent a family of protein–protein interacting motifs that are involved in many cellular processes (Nourry et al. 2003). PDZ proteins often contain other interacting domains (SH3, MAGUK, LRR, WW or PH) and can form homo- or heterodimers, such that they have the capacity to scaffold large protein complexes.
, http://www.100md.com
A Y2H screening of a mouse kidney library using the C-terminal tail of NaPi-IIa as prey identified a large number of potential partners (Gisler et al. 2001, 2003b). Not surprisingly, several of these proteins are PDZ proteins, among them the NHE3 regulatory factors NHERF1 and NHERF2 (also known as EBP50 and E3KARP, respectively) as well as PDZK1 and PDZK2 (the latter also known as IKEPP). Immunostaining of kidney sections showed that NHERF1 and PDZK1 are expressed in the BBM of proximal tubules, where they overlap with NaPi-IIa (Gisler et al. 2001); therefore, interaction between these proteins in vivo is ‘topologically’ possible. NHERF2 and PDZK2 are also expressed in proximal tubules but they are located subapically (Gisler et al. 2001; Wade et al. 2003); this suggests that such interactions are not possible at steady state, when NaPi-IIa resides in the BBM, although they could theoretically take place with NaPi-IIa molecules in transit to/from the plasma membrane. Given the capacity of both NHERF1/2 and PDZK1 to form homo- and heterodimers, it is possible to imagine an apical–subapical network of PDZ-based protein interactions.
, 百拇医药
NHERF1 and NHERF2 contain two PDZ domains (Weinman et al. 1995) and a C-terminal region that interacts with the merlin-ezin-radixin-moesin (MERM) family of cytoskeletal proteins (Yun et al. 1998; Murthy et al. 1998). The capability of ezrin to bind PKA as well widens the range of processes potentially regulated through association with NHERF1. Overlay assays as well as GST pull-downs indicated that NHERF1 interacts through its first PDZ domain with the C-terminal tail of NaPi-IIa in a TRL-dependent manner (Gisler et al. 2001). However, TRL-independent interactions have also been reported (Lederer et al. 2003). PDZK1 and PDZK2 encompass four PDZ domains (Kocher et al. 1999; Gisler et al. 2001). Interaction with NaPi-IIa takes place between their third PDZ domain and the TRL sequence of the cotransporter (Gisler et al. 2001). PDZK1 also interacts with myosin VIIb (S. Pribanic, unpublished observations) and dual A-kinase anchoring protein 2 (D-AKAP2) (Gisler et al. 2003a), interactions which could be related to cytoskeletal attachment and intracellular signalling, respectively. In addition, PDZK1 forms heterodimers with NHERF1 and probably self-homodimerizes (Gisler et al. 2003b).
, 百拇医药
In most cases, the above interactions were further confirmed by several assays, independently of yeast, such as overlay assays, GST pull-downs in BBM isolated from mouse kidney cortex as well as in OK cell lysates, co-immunoprecipitation in OK cells using anti-NaPi-IIa and anti-NHERF1 antibodies.
Role of these interactions
Membrane anchoring and/or trafficking. The specific expression of proteins at either the apical or basolateral membrane can be the consequence of two processes: specific sorting and targeting from the trans Golgi network or selective retention at one membrane post targeting. PDZ-mediated membrane anchoring has been reported for a number of proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) (Moyer et al. 2000) or the multidrug resistance-associated protein MRP2 (Harris et al. 2001; for review see Hernando et al. 2004). The role of NHERF1 as a membrane anchor probably depends on its capacity to associate with the actin cytoskeleton through its MERM-binding domain. Based on in vitro and in vivo studies, apical expression of NaPi-IIa depends, at least partially, on the interaction with NHERF1. Thus, expression of NaPi-IIa is impaired in OK cells upon the introduction of a dominant-negative NHERF1 construct that contains only the first PDZ domain (Hernando et al. 2002). Furthermore, animals deficient in NHERF1 have a reduced expression of NaPi-IIa in BBM and an increased accumulation of the cotransporter in a subapical compartment (Shenolikar et al. 2002). These animals also show phosphaturia and an impaired up-regulation of NaPi-IIa in BBM in response to a low phosphate diet (Cunningham et al. 2004).
, 百拇医药
Apical expression of NaPi-IIa is also impaired in OK cells after transfection of a dominant-negative form of PDZK1 that contains only the third PDZ domain (Hernando et al. 2002). The expression of the cotransporter is not affected upon ablation of the PDZK1 gene in mice (Kocher et al. 2003). However, NaPi-IIa abundance in BBM is reduced in PDZK1-deficient animals fed a high Pi diet compared to wild-type (Capuano et al. 2005), suggesting that PDZK1 may be involved in stabilizing NaPi-IIa at the plasma membrane, at least under high Pi dietary conditions.
, 百拇医药
Results obtained from transfection studies using OK cells also indicated that MAP17 is required for apical localization of PDZK1 (Pribanic et al. 2003a). However, hepatic overexpression of MAP17 in mice resulted in a reduction of PDZK1 in liver (Silver et al. 2003). Similar to the PDZK1-deficient mice, MAP17 transgenic mice show a deficiency of the high-density lipoprotein (HDL) receptor SR-BI and therefore increased plasma HDL. However, the precise role of MAP17 in NaPi-IIa expression and/or regulation remains unknown.
, 百拇医药
Intracellular signalling. NHERF2 and most probably NHERF1 couple PTH receptors (PTH-R) to the PLC–PKC pathway (Mahon et al. 2002). Thus, NHERF1 and NHERF2 can associate in vitro with PTH-R and phospholipase C beta 1 (PLC1). PTH treatment of cells that express NHERF2 and PTH-R activates PLC1 and inhibits adenylyl cyclase. Therefore, the presence of NHERF may determine the activation of intracellular signalling upon binding of PTH to PTH-R. Interestingly, we found that both 1–34 PTH (a fragment that activates PKA and PKC) and 3–34 PTH (a fragment that activates only PKC) activate PLC in kidney slices from wild-type but not from NHERF1–/– mice (P. Capuano, unpublished observations). Moreover, 3–34 PTH down-regulates NaPi-IIa in wild-type but not in NHERF1–/– animals. Therefore, NHERF1 seems to couple PTH-R to the PLC–PKC pathway. This role of NHERF1 as a molecular switch of intracellular signalling may explain previous observations that in renal proximal tubules apical PTH-R (through association with NHERF1) signals mostly through PLC–PKC whereas basolateral PTH-R (in the absence of NHERF1) activates both PKA and PKC pathways (Traebert et al. 2000).
, http://www.100md.com
Upon PTH treatment, NaPi-IIa is targeted to lysosomes (Lotscher et al. 1996) whereas NHERF1 and PDZK1 remain attached to the BBM (Deliot et al. 2005). There is a reduction in the amount of NaPi-IIa that co-immunoprecipitates with NHERF1 in OK cells treated with PTH (in the presence of leupeptin) (Deliot et al. 2005). The above findings suggest that PTH regulates the association between NaPi-IIa and NHERF1. NHERF1 is constitutively phosphorylated and several serine residues have been identified as the target for different kinases (Hall et al. 1999; He et al. 2001; Raghuram et al. 2003). We found that PTH, as well as independent pharmacological activation of PKA and PKC, leads to an increased phosphorylation of NHERF1 in kidney (Deliot et al. 2005). One can hypothesize that phosphorylation could trigger the dissociation of NaPi-IIa–NHERF1 complexes, although this remains to be seen experimentally. Similar to NHERF1, PDZK1 is a phosphoprotein in kidney and PTH leads to an increase in its phosphorylation state (N. Deliot, unpublished observations). It will be interesting to study whether this change in phosphorylation represents a mechanism of regulation of NaPi-IIa–PDZK1 interaction.
, http://www.100md.com
Interactions at the level of full-length cotransporter: split-ubiquitin Y2H
Recently, a split-ubiquitin Y2H system was developed in order to detect interactions at the level of integral membrane proteins (Stagljar et al. 1998). This system is based on the capacity of the severed C- (Cub) and N-terminal (Nub) halves of ubiquitin (Ub) to reassemble and reconstitute a functional protein (Johnsson & Varshavsky, 1994). This reassembly is prevented by mutation of an isoleucine to glycine near the N-terminus (NubI to NubG). However, as part of bait/prey fusion proteins, Cub and NubG associate and reconstitute a functional Ub if the bait/prey moieties interact with each other (Stagljar et al. 1998). The reconstituted Ub is then recognized by ubiquitin-specific proteases which cleave a transcription factor (TF) fused C-terminal to Cub. The released TF can then translocate to the nucleus and activate the reporter gene as in the conventional Y2H.
, 百拇医药
In the available split-ubiquitin Y2H system, the integral membrane bait protein is C-terminal to the Cub-TF cassette and the prey is fused to NubG. Because many proteins contain interacting motifs at their C-termini, we inverted the orientation of the parental vector in order to liberate the C-terminus of the bait protein (i.e. NaPi-IIa) from the Cub-TF cassette (S. M. Gisler, unpublished observations). Based on immunofluorescence and biochemical experiments, the TF-Cub-NaPi-IIa fusion protein is expressed in the yeast membrane. Furthermore, 32P uptakes performed in yeast transformed with the TF-Cub-fused cotransporter confirmed intact functional activity (S. M. Gisler, unpublished observations). Based on quantitative galactosidase activity, full-length NaPi-IIa interacts with PDZK1, NHERF1 and NHERF2. Thus, the interactions identified by conventional Y2H are reproduced (and confirmed) at the level of the full-length, membrane-inserted and functionally active cotransporter. In all three cases, interaction was prevented by truncation of the TRL PDZ-binding motif of NaPi-IIa. In addition, we found that full-length NaPi-IIa can form homodimers in yeast, independently of its cytoplasmic C-terminus. This finding was corroborated by co-immunoprecipitation from oocytes injected with cRNAs encoding for HA- and myc-tagged NaPi-IIa.
, 百拇医药
Conclusions and perspectives
Analysis of the pattern of protein interactions with different segments of NaPi-IIa has provided useful information regarding the molecular mechanisms that control its apical expression/retrieval as well as its hormonal regulation. These interactions were identified by classical yeast two-hybrid screening, using discrete domains of the cotransporter as baits. All interacting partners were expressed in renal proximal tubules and interactions were confirmed in several assays such as GST pull-downs, overlay assays or co-immunoprecipitations. In addition, many of these interactions have been reproduced by a new approach that allows the use of full-length, membrane-inserted proteins as baits.
, 百拇医药
The functional significance of these interactions is still being unveiled. The phenotypes of the PDZK1 and NHERF1-deficient mice with regard to the expression and/or regulation of NaPi-IIa indicate that these two PDZ-containing proteins do not have redundant roles. Moreover, based on the phenotype of the NHERF1-deficient mice, both NHERF isoforms also have specific roles. Therefore, despite their high degree of homology, NHERF1 and NHERF2 do not seem to functionally overlap. An issue that remains to be experimentally addressed is the relative affinities of all these PDZ proteins for NaPi-IIa in vivo, as well as the effect that the binding of a particular protein may have on the association of a second one.
, 百拇医药
Footnotes
This report was presented at The Journal of Physiology Symposium on PDZ domain scaffolding proteins and their functions in polarized cells, San Diego, CA, USA, 4 April 2005. It was commissioned by the Editorial Board and reflects the views of the authors.
References
Beck L, Karaplis AC, Amizuka N, Hewson AS, Ozawa H & Tenenhouse HS (1998). Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities. Proc Natl Acad Sci U S A 95, 5372–5377.
, 百拇医药
Capuano P, Bacic D, Stange G, Hernando N, Kaissling B, Pal R, Kocher O, Biber J, Wagner CA & Murer H (2005). Expression and regulation of the renal Na/Phosphate cotransporter NaPi-IIa in a mouse model deficient for the PDZ protein PDZK1. Pflugers Arch 449, 392–402.
Cunningham R, Steplock D, Wang F, Huang HEX, Shenolikar S & Weinman EJ (2004). Defective PTH regulation of NHE3 activity and phosphate adaptation in cultured NHERF-1–/– renal proximal tubule cells. J Biol Chem 279, 37815–37821.
, 百拇医药
Custer M, Lotscher M, Biber J, Murer H & Kaissling B (1994). Expression of Na-P(i) cotransport in rat kidney: localization by RT-PCR and immunohistochemistry. Am J Physiol 266, F767–F774.
Deliot N, Hernando N, Horst-Liu Z, Capuano P, Bacic D, Wagner CA, O'Brien S, Biber J & Murer H (2005). PTH treatment induces dissociation of NaPi-IIa/NHERF1 complexes. Am J Physiol Cell Physiol; PMID: 15788483.
Gisler SM, Madjdpour C, Pribanic S, Bacic D, Taylor SS, Biber J & Murer H (2003a). PDZK1. II. An anchoring site for the PKA-binding protein D-AKAP2 in renal proximal tubular cells. Kidney Int 64, 1746–1754.
, 百拇医药
Gisler SM, Pribanic S, Bacic D, Forrer P, Gantenbein A, Sabourin LA, Tsuji A, Zhao ZS, Manser E, Biber J & Murer H (2003b). PDZK1: I. A major scaffolder in brush borders of proximal tubular cells. Kidney Int 64, 1733–1745.
Gisler SM, Stagljar I, Traebert M, Bacic D, Biber J & Murer H (2001). Interaction of the type IIa Na/Pi cotransporter with PDZ proteins. J Biol Chem 276, 9206–9213.
Hall RA, Spurney RF, Premont RT, Rahman N, Blitzer JT, Pitcher JA & Lefkowitz RJ (1999). G protein-coupled receptor kinase 6A phosphorylates the Na+/H+ exchanger regulatory factor via a PDZ domain-mediated interaction. J Biol Chem 274, 24328–24334.
, 百拇医药
Harris MJ, Kuwano M, Webb M & Board PG (2001). Identification of the apical membrane-targeting signal of the multidrug resistance-associated protein 2 (MRP2/cMOAT). J Biol Chem 276, 20876–20881.
He J, Lau AG, Yaffe MB & Hall RA (2001). Phosphorylation and cell cycle-dependent regulation of Na+/H+ exchanger regulatory factor-1 by Cdc2 kinase. J Biol Chem 276, 41559–41565.
Hernando N, Deliot N, Gisler SM, Lederer E, Weinman EJ, Biber J & Murer H (2002). PDZ–domain interactions and apical expression of type IIa Na/P(i) cotransporters. Proc Natl Acad Sci U S A 99, 11957–11962.
, http://www.100md.com
Hernando N, Wagner CA, Gisler SM, Biber J & Murer H (2004). PDZ proteins and proximal ion transport. Curr Opin Nephrol Hypertens 13, 569–574.
Ito M, Iidawa S, Izuka M, Haito S, Segawa H, Kuwahata M, Ohkido I, Ohno H & Miyamoto K-I (2004). Interaction of a farnesylated protein with renal type IIa Na/Pi co-transporter in response to parathyroid hormone and dietary phosphate. Biochem J 377, 607–616.
Johnsson N & Varshavsky A (1994). Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci U S A 91, 10340–10344.
, 百拇医药
Jones JM, Morrell JC & Gould SJ (2004). PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins. J Cell Biol 2004, 57–67.
Karim-Jimenez Z, Hernando N, Biber J & Murer H (2001). Molecular determinants for apical expression of the renal type IIa Na+/Pi-cotransporter. Pflugers Arch 442, 782–790.
Karim-Jimenez Z, Hernando N, Biber J & Murer H (2000). A dibasic motif involved in PTH-induced downregulation of the type IIa NaPi-cotransporter. Proc Natl Acad Sci U S A 97, 12896–12901.
, 百拇医药
Kocher O, Comella N, Gilchrist A, Pal R, Tognazzi K, Brown LF & Knoll JH (1999). PDZK1, a novel PDZ domain-containing protein up-regulated in carcinomas and mapped to chromosome 1q21, interacts with cMOAT (MRP2), the multidrug resistance-associated protein. Lab Invest 79, 1161–1170.
Kocher O, Pal R, Roberts M, Cirovic C & Gilchrist A (2003). Targeted disruption of the PDZK1 gene by homologous recombination. Mol Cell Biol 23, 1175–1180.
, 百拇医药
Lambert G, Traebert M, Hernando N, Biber J & Murer H (1999). Studies on the topology of the renal type II NaPi-cotransporter. Pflugers Arch 437, 972–978.
Lederer ED, Khundmiri SJ & Weinman EJ (2003). Role of NHERF-1 in regulation of the activity of Na–K ATPase and sodium–phosphate co-transport in epithelial cells. J Am Soc Nephrol 14, 1711–1719.
Lotscher M, Kaissling B, Biber J, Murer H, Kempson SA & Levi M (1996). Regulation of rat renal Na/Pi-cotransporter by parathyroid hormone: immunohistochemistry. Kidney Int 49, 1010–1011.
, http://www.100md.com
Magagnin S, Werner A, Markovich D, Sorribas V, Stange G, Biber J & Murer H (1993). Expression cloning of human and rat renal cortex Na/Pi-cotransport. Proc Natl Acad Sci U S A 90, 5979–5983.
Mahon MJ, Donowitz M, Yun CC & Segre GV (2002). Na+/H+ exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling. Nature 417, 858–861.
Moyer BD, Duhaime M, Shaw C, Denton J, Reynolds D, Karlson KH, Pfeiffer J, Wang S, Mickle JE, Milewski M, Cutting GR, Guggino WB, Li M & Stanton BA (2000). The PDZ-interacting domain of cystic fibrosis transmembrane conductance regulator is required for functional expression in the apical plasma membrane. J Biol Chem 275, 27069–27074.
, http://www.100md.com
Murer H, Forster IC & Biber J (2004). The sodium phosphate cotransporter family SLC34. Pflugers Arch 447, 763–767.
Murer H, Hernando N, Forster I & Biber J (2000). Proximal tubular phosphate reabsorption: molecular mechanisms. Physiol Rev 80, 1373–1409.
Murthy A, Gonzalez-Agosti C, Cordero E, Pinney D, Candia C, Solomon F, Gusella J & Ramesh V (1998). NHE-RF, a regulatory cofactor for Na+–H+ exchange, is a common interactor for merlin and ERM (MERM) proteins. J Biol Chem 273, 1273–1276.
, 百拇医药
Nourry C, Grant SGN & Borg JP (2003). PDZ domain proteins: plug and play. Sci STKE 179, 1–12.
Pribanic S, Gisler SM, Bacic D, Madjdpour C, Hernando N, Sorribas V, Gantenbein A, Biber J & Murer H (2003a). Interactions of MAP17 with the NaPi-IIa/PDZK1 protein complex in renal proximal tubular cells. Am J Physiol Renal Physiol 285, F784–F791.
Pribanic S, Loffing J, Madjdpour C, Bacic D, Gisler S, Braunewell KH, Biber J & Murer H (2003b). Expression of visinin-like protein-3 (VILIP-3) in mouse kidney. Nephron Physiol 95, 76–82.
, 百拇医药
Raghuram V, Hormuth H & Foskett JK (2003). A kinase-regulated mechanism controls CFTR channel gating by disrupting bivalent PDZ domain interactions. Proc Natl Acad Sci U S A 100, 9620–9625.
Shenolikar S, Voltz JW, Minkoff CM, Wade JB & Weinman EJ (2002). Targeted disruption of the mouse NHERF-1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type IIa and renal phosphate wasting. Proc Natl Acad Sci U S A 99, 11470–11475.
, http://www.100md.com
Silver DI, Wang N, Vogel S, (2003). Identification of small Pdzk1-associated protein, Dd96/Map17 as a regulator for Pdzk1 and plasma high density lipoprotein levels. J Biol Chem 278, 28528–28532.
Spilker C, Richter K, Smalla KH, Manahan-Vaughan D, Gundelfinger ED & Braunewell KH (2000). The neuronal EF-hand calcium-binding protein visinin-like protein-3 is expressed in cerebellar Purkinje cells and shows a calcium-dependent membrane association. Neuroscience 96, 121–129.
, 百拇医药
Stagljar I, Korostensky C, Johnsson N & te Heesen S (1998). A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A 95, 5187–5192.
Traebert M, Volkl H, Biber J, Murer H & Kaissling B (2000). Luminal and contraluminal action of 1–34 and 3–34 PTH peptides on renal type IIa Na-P(i) cotransporter. Am J Physiol Renal Physiol 278, F792–F798.
Wade JB, Liu J, Coleman RA, Cunningham R, Steplock DA, Lee-Kwon W et al. (2003). Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse. Am J Physiol Cell Physiol 285, C1494–C1503.
, 百拇医药
Weinman EJ, Steplock D, Wang Y & Shenolikar S (1995). Characterization of a protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na+–H+ exchanger. J Clin Invest 95, 2143–2149.
Yun CH, Lamprecht G, Forster DV & Sidor A (1998). NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border Na+/H+ exchanger NHE3 and the cytoskeletal protein ezrin. J Biol Chem 273, 25856–25863., 百拇医药
N Hernando, S. M Gisler, S Pribanic, N Deliot, P Capuano, C. A Wagner, O. W Moe, J Biber and H Murer
1 Institute of Physiology, Zurich University, CH-8057, Zurich, Switzerland
2 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA
Abstract
Regulation of renal proximal tubular reabsorption of phosphate (Pi) is one of the critical steps in Pi homeostasis. Experimental evidence suggests that this regulation is achieved mainly by controlling the apical expression of the Na+-dependent Pi cotransporter type IIa (NaPi-IIa) in proximal tubules. Only recently have we started to obtain information regarding the molecular mechanisms that control the apical expression of NaPi-IIa. The first critical observation was the finding that truncation of only its last three amino acid residues has a strong effect on apical expression. A second major finding was the observation that the last intracellular loop of NaPi-IIa contains sequence information that confers parathyroid hormone (PTH) sensitivity. The use of the above domains of the cotransporter in yeast two-hybrid (Y2H) screening allowed the identification of proteins interacting with NaPi-IIa. Biochemical and morphological, as well as functional, analyses have allowed us to obtain insights into the physiological roles of such interactions, although our present knowledge is still far from complete.
, http://www.100md.com
Renal reabsorption of Pi: NaPi-IIa
NaPi-IIa, also known as Npt2a (SLC34A1), is an electrogenic Na+-dependent Pi cotransporter expressed in the brush border membranes (BBM) of renal proximal tubules (Custer et al. 1994; Murer et al. 2004). Its pattern of expression along the proximal tubules matches the major sites of renal Pi reabsorption, and its role as the main mediator of that process has been clearly demonstrated in an Npt2–/– mouse model (Beck et al. 1998). Npt2–/– mice show a higher urinary excretion of Pi and lower Na+–Pi cotransport activity in BBM compared to wild-type animals.
, 百拇医药
NaPi-IIa is regulated by factors known to control the reabsorption of Pi in the kidney (for review see Murer et al. 2000). This regulation is mediated almost exclusively by the control of the number of cotransporters expressed in the BBM. Thus, phosphaturic factors reduce the expression of NaPi-IIa whereas factors that increase renal Pi reabsorption increase BBM NaPi-IIa. Therefore, knowledge of the mechanisms that control apical expression and membrane retrieval of NaPi-IIa is essential to our understanding of how Pi homeostasis is achieved.
, http://www.100md.com
In the last few years, the combined work of many laboratories has strongly suggested that the regulation, membrane expression and to some extent the fate of endocytosed proteins depends, at least in part, on the network of interactions in which a particular protein is engaged. Therefore, deciphering the pattern of interactions of a given protein may help us understand how it is regulated.
Identification of proteins that interact with cytoplasmic domains of NaPi-IIa
, 百拇医药
The first attempts to identify proteins interacting with NaPi-IIa were performed by conventional Y2H screenings. A major limitation of this technique is the requirement for both interacting partners to reach the cell nucleus, in order to activate the corresponding reporter gene. Thus, both the bait and the prey constructs should encode soluble peptides. In the case of integral membrane proteins, this requirement restricts the design as baits to soluble (cytoplasmic) domains of the protein of interest (i.e. NaPi-IIa). Based on hydropathy plots and antibody accessibility both the N- and C-terminal tails of NaPi-IIa face the cytoplasm (Magagnin et al. 1993; Lambert et al. 1999). Therefore both tails, as well as an internal loop potentially involved in parathyroid hormone (PTH) regulation (see the following three subsections), were chosen as baits for conventional Y2H screenings of a mouse kidney cDNA library. A minimal requisite to ascribe physiological meaning to the interaction is that both partners must be expressed within the same tissue and subcellular compartment in vivo. Therefore, expression within renal proximal tubules of all NaPi-IIa partners described below was confirmed. A summary of these interactions is shown in Fig. 1.
, http://www.100md.com
The expression of all interacting partners in renal proximal tubules was confirmed either by immunoblots, immunohistochemistry and/or RT-PCR. The abbreviations are: PTH-R, PTH receptors; PLC, phospholipase C; PDZ, PDZ domains; MERM, merlin-ezrin-radixin-moesin family; MERM-BD, MERM-binding domain; PKA, protein kinase A; D-AKAP2, dual A-kinase anchoring protein 2. The PDZ domains of NHERF1 and PDZK1 are indicated by rectangles. Interactions between proteins are illustrated either by showing direct contacts of a cartoon-form of each protein or by arrows.
, 百拇医药
Proteins that interact with the N-terminal tail of NaPi-IIa. Y2H screening indicated that the N-terminal cytoplasmic tail of NaPi-IIa interacts with MAP17 (Pribanic et al. 2003a) as well as with VILIP-3, a visinin-like protein (Pribanic et al. 2003b).
MAP17 is a 17 kDa membrane protein associated with various human carcinomas, but also expressed in normal kidneys. Interestingly, MAP17 also interacts with PDZK1 (also known as CLAMP, CAP70 or Diphor1), a PDZ protein found to associate with the C-terminal tail of NaPi-IIa. Yeast trap assays and GST pull-down experiments suggested that MAP17 interacts only with the last (4th) PDZ domain of PDZK1 (Pribanic et al. 2003a). RT-PCR of microdissected tubules indicated the presence of MAP17 mRNA in the S1 and S3 segments of both superficial and deep nephrons. At the protein level, renal expression of MAP17 is restricted to the cortex, with similar intensity in the proximal tubules of superficial and juxtamedullar nephrons. The highest intensity is observed in S1 segments and gradually decreases towards the S3 segments (Pribanic et al. 2003a).
, http://www.100md.com
VILIP-3 is a member of the neuronal calcium sensors family (Spilker et al. 2000). This protein family is characterized by calcium binding, myristoylation, and calcium-dependent membrane association. However, very little is known about their function. RT-PCR from microdissected nephron segments was also used to analyse the expression of VILIP-3 mRNA in kidney (Pribanic et al. 2003b). This study detected VILIP-3 in all segments from both superficial and deep nephrons. By immunofluorescence, VILIP-3 protein is observed in proximal and distal segments as well as in glomeruli. Interestingly, in contrast to distal tubules where VILIP-3 is distributed throughout the cytoplasm, in proximal tubules it is associated with the BBM.
, http://www.100md.com
Proteins that interact with the last intracellular loop of NaPi-IIa. We found several years ago that NaPi-IIb, a second member of the NaPi-II family, is PTH insensitive (Karim-Jimenez et al. 2000). Based on chimera studies, the signal responsible for hormonal sensitivity was identified. This signal, located on the last intracellular loop, consists of two positively charged residues in NaPi-IIa (KR) that are replaced by uncharged residues in NaPi-IIb (NI). The PTH sensitivity of the cotransporters can be reverted by swapping just these two isoform-specific residues. Y2H screening performed in our laboratory failed to identify interacting candidates that could provide hints about the molecular mechanism underlying the above findings (S. Pribanic, unpublished observations). However, the KR motif has been recently found to interact with PEX19 (Ito et al. 2004), a protein involved in the binding and trafficking of peroxisomal proteins (Jones et al. 2004). PEX19 mRNA is expressed in kidneys and in opossum kidney (OK) cells (Ito et al. 2004). In kidney, protein expression has been suggested in cytosolic and BBM fractions. PEX19 binds to wild-type NaPi-IIa but not to a mutant in which the KR motif is replaced by NI residues (Ito et al. 2004). Transfection of PEX19 in OK cells stimulates NaPi-IIa endocytosis even in the absence of PTH, suggesting a role in the internalization of the cotransporter.
, 百拇医药
Proteins that interact with the C-terminal tail of NaPi-IIa. Apical expression of NaPi-IIa is partially prevented upon truncation of the last three C-terminal residues (TRL). After transfection in OK cells, the TRL mutant fails to reach and/or remain at the plasma membrane and accumulates in the cytoplasm, indicating that these residues are required for proper apical expression (Karim-Jimenez et al. 2001). The TRL sequence resembles a class-I PSD-95, Discs-large, ZO-1 (PDZ) binding domain. PDZ domains represent a family of protein–protein interacting motifs that are involved in many cellular processes (Nourry et al. 2003). PDZ proteins often contain other interacting domains (SH3, MAGUK, LRR, WW or PH) and can form homo- or heterodimers, such that they have the capacity to scaffold large protein complexes.
, http://www.100md.com
A Y2H screening of a mouse kidney library using the C-terminal tail of NaPi-IIa as prey identified a large number of potential partners (Gisler et al. 2001, 2003b). Not surprisingly, several of these proteins are PDZ proteins, among them the NHE3 regulatory factors NHERF1 and NHERF2 (also known as EBP50 and E3KARP, respectively) as well as PDZK1 and PDZK2 (the latter also known as IKEPP). Immunostaining of kidney sections showed that NHERF1 and PDZK1 are expressed in the BBM of proximal tubules, where they overlap with NaPi-IIa (Gisler et al. 2001); therefore, interaction between these proteins in vivo is ‘topologically’ possible. NHERF2 and PDZK2 are also expressed in proximal tubules but they are located subapically (Gisler et al. 2001; Wade et al. 2003); this suggests that such interactions are not possible at steady state, when NaPi-IIa resides in the BBM, although they could theoretically take place with NaPi-IIa molecules in transit to/from the plasma membrane. Given the capacity of both NHERF1/2 and PDZK1 to form homo- and heterodimers, it is possible to imagine an apical–subapical network of PDZ-based protein interactions.
, 百拇医药
NHERF1 and NHERF2 contain two PDZ domains (Weinman et al. 1995) and a C-terminal region that interacts with the merlin-ezin-radixin-moesin (MERM) family of cytoskeletal proteins (Yun et al. 1998; Murthy et al. 1998). The capability of ezrin to bind PKA as well widens the range of processes potentially regulated through association with NHERF1. Overlay assays as well as GST pull-downs indicated that NHERF1 interacts through its first PDZ domain with the C-terminal tail of NaPi-IIa in a TRL-dependent manner (Gisler et al. 2001). However, TRL-independent interactions have also been reported (Lederer et al. 2003). PDZK1 and PDZK2 encompass four PDZ domains (Kocher et al. 1999; Gisler et al. 2001). Interaction with NaPi-IIa takes place between their third PDZ domain and the TRL sequence of the cotransporter (Gisler et al. 2001). PDZK1 also interacts with myosin VIIb (S. Pribanic, unpublished observations) and dual A-kinase anchoring protein 2 (D-AKAP2) (Gisler et al. 2003a), interactions which could be related to cytoskeletal attachment and intracellular signalling, respectively. In addition, PDZK1 forms heterodimers with NHERF1 and probably self-homodimerizes (Gisler et al. 2003b).
, 百拇医药
In most cases, the above interactions were further confirmed by several assays, independently of yeast, such as overlay assays, GST pull-downs in BBM isolated from mouse kidney cortex as well as in OK cell lysates, co-immunoprecipitation in OK cells using anti-NaPi-IIa and anti-NHERF1 antibodies.
Role of these interactions
Membrane anchoring and/or trafficking. The specific expression of proteins at either the apical or basolateral membrane can be the consequence of two processes: specific sorting and targeting from the trans Golgi network or selective retention at one membrane post targeting. PDZ-mediated membrane anchoring has been reported for a number of proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) (Moyer et al. 2000) or the multidrug resistance-associated protein MRP2 (Harris et al. 2001; for review see Hernando et al. 2004). The role of NHERF1 as a membrane anchor probably depends on its capacity to associate with the actin cytoskeleton through its MERM-binding domain. Based on in vitro and in vivo studies, apical expression of NaPi-IIa depends, at least partially, on the interaction with NHERF1. Thus, expression of NaPi-IIa is impaired in OK cells upon the introduction of a dominant-negative NHERF1 construct that contains only the first PDZ domain (Hernando et al. 2002). Furthermore, animals deficient in NHERF1 have a reduced expression of NaPi-IIa in BBM and an increased accumulation of the cotransporter in a subapical compartment (Shenolikar et al. 2002). These animals also show phosphaturia and an impaired up-regulation of NaPi-IIa in BBM in response to a low phosphate diet (Cunningham et al. 2004).
, 百拇医药
Apical expression of NaPi-IIa is also impaired in OK cells after transfection of a dominant-negative form of PDZK1 that contains only the third PDZ domain (Hernando et al. 2002). The expression of the cotransporter is not affected upon ablation of the PDZK1 gene in mice (Kocher et al. 2003). However, NaPi-IIa abundance in BBM is reduced in PDZK1-deficient animals fed a high Pi diet compared to wild-type (Capuano et al. 2005), suggesting that PDZK1 may be involved in stabilizing NaPi-IIa at the plasma membrane, at least under high Pi dietary conditions.
, 百拇医药
Results obtained from transfection studies using OK cells also indicated that MAP17 is required for apical localization of PDZK1 (Pribanic et al. 2003a). However, hepatic overexpression of MAP17 in mice resulted in a reduction of PDZK1 in liver (Silver et al. 2003). Similar to the PDZK1-deficient mice, MAP17 transgenic mice show a deficiency of the high-density lipoprotein (HDL) receptor SR-BI and therefore increased plasma HDL. However, the precise role of MAP17 in NaPi-IIa expression and/or regulation remains unknown.
, 百拇医药
Intracellular signalling. NHERF2 and most probably NHERF1 couple PTH receptors (PTH-R) to the PLC–PKC pathway (Mahon et al. 2002). Thus, NHERF1 and NHERF2 can associate in vitro with PTH-R and phospholipase C beta 1 (PLC1). PTH treatment of cells that express NHERF2 and PTH-R activates PLC1 and inhibits adenylyl cyclase. Therefore, the presence of NHERF may determine the activation of intracellular signalling upon binding of PTH to PTH-R. Interestingly, we found that both 1–34 PTH (a fragment that activates PKA and PKC) and 3–34 PTH (a fragment that activates only PKC) activate PLC in kidney slices from wild-type but not from NHERF1–/– mice (P. Capuano, unpublished observations). Moreover, 3–34 PTH down-regulates NaPi-IIa in wild-type but not in NHERF1–/– animals. Therefore, NHERF1 seems to couple PTH-R to the PLC–PKC pathway. This role of NHERF1 as a molecular switch of intracellular signalling may explain previous observations that in renal proximal tubules apical PTH-R (through association with NHERF1) signals mostly through PLC–PKC whereas basolateral PTH-R (in the absence of NHERF1) activates both PKA and PKC pathways (Traebert et al. 2000).
, http://www.100md.com
Upon PTH treatment, NaPi-IIa is targeted to lysosomes (Lotscher et al. 1996) whereas NHERF1 and PDZK1 remain attached to the BBM (Deliot et al. 2005). There is a reduction in the amount of NaPi-IIa that co-immunoprecipitates with NHERF1 in OK cells treated with PTH (in the presence of leupeptin) (Deliot et al. 2005). The above findings suggest that PTH regulates the association between NaPi-IIa and NHERF1. NHERF1 is constitutively phosphorylated and several serine residues have been identified as the target for different kinases (Hall et al. 1999; He et al. 2001; Raghuram et al. 2003). We found that PTH, as well as independent pharmacological activation of PKA and PKC, leads to an increased phosphorylation of NHERF1 in kidney (Deliot et al. 2005). One can hypothesize that phosphorylation could trigger the dissociation of NaPi-IIa–NHERF1 complexes, although this remains to be seen experimentally. Similar to NHERF1, PDZK1 is a phosphoprotein in kidney and PTH leads to an increase in its phosphorylation state (N. Deliot, unpublished observations). It will be interesting to study whether this change in phosphorylation represents a mechanism of regulation of NaPi-IIa–PDZK1 interaction.
, http://www.100md.com
Interactions at the level of full-length cotransporter: split-ubiquitin Y2H
Recently, a split-ubiquitin Y2H system was developed in order to detect interactions at the level of integral membrane proteins (Stagljar et al. 1998). This system is based on the capacity of the severed C- (Cub) and N-terminal (Nub) halves of ubiquitin (Ub) to reassemble and reconstitute a functional protein (Johnsson & Varshavsky, 1994). This reassembly is prevented by mutation of an isoleucine to glycine near the N-terminus (NubI to NubG). However, as part of bait/prey fusion proteins, Cub and NubG associate and reconstitute a functional Ub if the bait/prey moieties interact with each other (Stagljar et al. 1998). The reconstituted Ub is then recognized by ubiquitin-specific proteases which cleave a transcription factor (TF) fused C-terminal to Cub. The released TF can then translocate to the nucleus and activate the reporter gene as in the conventional Y2H.
, 百拇医药
In the available split-ubiquitin Y2H system, the integral membrane bait protein is C-terminal to the Cub-TF cassette and the prey is fused to NubG. Because many proteins contain interacting motifs at their C-termini, we inverted the orientation of the parental vector in order to liberate the C-terminus of the bait protein (i.e. NaPi-IIa) from the Cub-TF cassette (S. M. Gisler, unpublished observations). Based on immunofluorescence and biochemical experiments, the TF-Cub-NaPi-IIa fusion protein is expressed in the yeast membrane. Furthermore, 32P uptakes performed in yeast transformed with the TF-Cub-fused cotransporter confirmed intact functional activity (S. M. Gisler, unpublished observations). Based on quantitative galactosidase activity, full-length NaPi-IIa interacts with PDZK1, NHERF1 and NHERF2. Thus, the interactions identified by conventional Y2H are reproduced (and confirmed) at the level of the full-length, membrane-inserted and functionally active cotransporter. In all three cases, interaction was prevented by truncation of the TRL PDZ-binding motif of NaPi-IIa. In addition, we found that full-length NaPi-IIa can form homodimers in yeast, independently of its cytoplasmic C-terminus. This finding was corroborated by co-immunoprecipitation from oocytes injected with cRNAs encoding for HA- and myc-tagged NaPi-IIa.
, 百拇医药
Conclusions and perspectives
Analysis of the pattern of protein interactions with different segments of NaPi-IIa has provided useful information regarding the molecular mechanisms that control its apical expression/retrieval as well as its hormonal regulation. These interactions were identified by classical yeast two-hybrid screening, using discrete domains of the cotransporter as baits. All interacting partners were expressed in renal proximal tubules and interactions were confirmed in several assays such as GST pull-downs, overlay assays or co-immunoprecipitations. In addition, many of these interactions have been reproduced by a new approach that allows the use of full-length, membrane-inserted proteins as baits.
, 百拇医药
The functional significance of these interactions is still being unveiled. The phenotypes of the PDZK1 and NHERF1-deficient mice with regard to the expression and/or regulation of NaPi-IIa indicate that these two PDZ-containing proteins do not have redundant roles. Moreover, based on the phenotype of the NHERF1-deficient mice, both NHERF isoforms also have specific roles. Therefore, despite their high degree of homology, NHERF1 and NHERF2 do not seem to functionally overlap. An issue that remains to be experimentally addressed is the relative affinities of all these PDZ proteins for NaPi-IIa in vivo, as well as the effect that the binding of a particular protein may have on the association of a second one.
, 百拇医药
Footnotes
This report was presented at The Journal of Physiology Symposium on PDZ domain scaffolding proteins and their functions in polarized cells, San Diego, CA, USA, 4 April 2005. It was commissioned by the Editorial Board and reflects the views of the authors.
References
Beck L, Karaplis AC, Amizuka N, Hewson AS, Ozawa H & Tenenhouse HS (1998). Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities. Proc Natl Acad Sci U S A 95, 5372–5377.
, 百拇医药
Capuano P, Bacic D, Stange G, Hernando N, Kaissling B, Pal R, Kocher O, Biber J, Wagner CA & Murer H (2005). Expression and regulation of the renal Na/Phosphate cotransporter NaPi-IIa in a mouse model deficient for the PDZ protein PDZK1. Pflugers Arch 449, 392–402.
Cunningham R, Steplock D, Wang F, Huang HEX, Shenolikar S & Weinman EJ (2004). Defective PTH regulation of NHE3 activity and phosphate adaptation in cultured NHERF-1–/– renal proximal tubule cells. J Biol Chem 279, 37815–37821.
, 百拇医药
Custer M, Lotscher M, Biber J, Murer H & Kaissling B (1994). Expression of Na-P(i) cotransport in rat kidney: localization by RT-PCR and immunohistochemistry. Am J Physiol 266, F767–F774.
Deliot N, Hernando N, Horst-Liu Z, Capuano P, Bacic D, Wagner CA, O'Brien S, Biber J & Murer H (2005). PTH treatment induces dissociation of NaPi-IIa/NHERF1 complexes. Am J Physiol Cell Physiol; PMID: 15788483.
Gisler SM, Madjdpour C, Pribanic S, Bacic D, Taylor SS, Biber J & Murer H (2003a). PDZK1. II. An anchoring site for the PKA-binding protein D-AKAP2 in renal proximal tubular cells. Kidney Int 64, 1746–1754.
, 百拇医药
Gisler SM, Pribanic S, Bacic D, Forrer P, Gantenbein A, Sabourin LA, Tsuji A, Zhao ZS, Manser E, Biber J & Murer H (2003b). PDZK1: I. A major scaffolder in brush borders of proximal tubular cells. Kidney Int 64, 1733–1745.
Gisler SM, Stagljar I, Traebert M, Bacic D, Biber J & Murer H (2001). Interaction of the type IIa Na/Pi cotransporter with PDZ proteins. J Biol Chem 276, 9206–9213.
Hall RA, Spurney RF, Premont RT, Rahman N, Blitzer JT, Pitcher JA & Lefkowitz RJ (1999). G protein-coupled receptor kinase 6A phosphorylates the Na+/H+ exchanger regulatory factor via a PDZ domain-mediated interaction. J Biol Chem 274, 24328–24334.
, 百拇医药
Harris MJ, Kuwano M, Webb M & Board PG (2001). Identification of the apical membrane-targeting signal of the multidrug resistance-associated protein 2 (MRP2/cMOAT). J Biol Chem 276, 20876–20881.
He J, Lau AG, Yaffe MB & Hall RA (2001). Phosphorylation and cell cycle-dependent regulation of Na+/H+ exchanger regulatory factor-1 by Cdc2 kinase. J Biol Chem 276, 41559–41565.
Hernando N, Deliot N, Gisler SM, Lederer E, Weinman EJ, Biber J & Murer H (2002). PDZ–domain interactions and apical expression of type IIa Na/P(i) cotransporters. Proc Natl Acad Sci U S A 99, 11957–11962.
, http://www.100md.com
Hernando N, Wagner CA, Gisler SM, Biber J & Murer H (2004). PDZ proteins and proximal ion transport. Curr Opin Nephrol Hypertens 13, 569–574.
Ito M, Iidawa S, Izuka M, Haito S, Segawa H, Kuwahata M, Ohkido I, Ohno H & Miyamoto K-I (2004). Interaction of a farnesylated protein with renal type IIa Na/Pi co-transporter in response to parathyroid hormone and dietary phosphate. Biochem J 377, 607–616.
Johnsson N & Varshavsky A (1994). Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci U S A 91, 10340–10344.
, 百拇医药
Jones JM, Morrell JC & Gould SJ (2004). PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins. J Cell Biol 2004, 57–67.
Karim-Jimenez Z, Hernando N, Biber J & Murer H (2001). Molecular determinants for apical expression of the renal type IIa Na+/Pi-cotransporter. Pflugers Arch 442, 782–790.
Karim-Jimenez Z, Hernando N, Biber J & Murer H (2000). A dibasic motif involved in PTH-induced downregulation of the type IIa NaPi-cotransporter. Proc Natl Acad Sci U S A 97, 12896–12901.
, 百拇医药
Kocher O, Comella N, Gilchrist A, Pal R, Tognazzi K, Brown LF & Knoll JH (1999). PDZK1, a novel PDZ domain-containing protein up-regulated in carcinomas and mapped to chromosome 1q21, interacts with cMOAT (MRP2), the multidrug resistance-associated protein. Lab Invest 79, 1161–1170.
Kocher O, Pal R, Roberts M, Cirovic C & Gilchrist A (2003). Targeted disruption of the PDZK1 gene by homologous recombination. Mol Cell Biol 23, 1175–1180.
, 百拇医药
Lambert G, Traebert M, Hernando N, Biber J & Murer H (1999). Studies on the topology of the renal type II NaPi-cotransporter. Pflugers Arch 437, 972–978.
Lederer ED, Khundmiri SJ & Weinman EJ (2003). Role of NHERF-1 in regulation of the activity of Na–K ATPase and sodium–phosphate co-transport in epithelial cells. J Am Soc Nephrol 14, 1711–1719.
Lotscher M, Kaissling B, Biber J, Murer H, Kempson SA & Levi M (1996). Regulation of rat renal Na/Pi-cotransporter by parathyroid hormone: immunohistochemistry. Kidney Int 49, 1010–1011.
, http://www.100md.com
Magagnin S, Werner A, Markovich D, Sorribas V, Stange G, Biber J & Murer H (1993). Expression cloning of human and rat renal cortex Na/Pi-cotransport. Proc Natl Acad Sci U S A 90, 5979–5983.
Mahon MJ, Donowitz M, Yun CC & Segre GV (2002). Na+/H+ exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling. Nature 417, 858–861.
Moyer BD, Duhaime M, Shaw C, Denton J, Reynolds D, Karlson KH, Pfeiffer J, Wang S, Mickle JE, Milewski M, Cutting GR, Guggino WB, Li M & Stanton BA (2000). The PDZ-interacting domain of cystic fibrosis transmembrane conductance regulator is required for functional expression in the apical plasma membrane. J Biol Chem 275, 27069–27074.
, http://www.100md.com
Murer H, Forster IC & Biber J (2004). The sodium phosphate cotransporter family SLC34. Pflugers Arch 447, 763–767.
Murer H, Hernando N, Forster I & Biber J (2000). Proximal tubular phosphate reabsorption: molecular mechanisms. Physiol Rev 80, 1373–1409.
Murthy A, Gonzalez-Agosti C, Cordero E, Pinney D, Candia C, Solomon F, Gusella J & Ramesh V (1998). NHE-RF, a regulatory cofactor for Na+–H+ exchange, is a common interactor for merlin and ERM (MERM) proteins. J Biol Chem 273, 1273–1276.
, 百拇医药
Nourry C, Grant SGN & Borg JP (2003). PDZ domain proteins: plug and play. Sci STKE 179, 1–12.
Pribanic S, Gisler SM, Bacic D, Madjdpour C, Hernando N, Sorribas V, Gantenbein A, Biber J & Murer H (2003a). Interactions of MAP17 with the NaPi-IIa/PDZK1 protein complex in renal proximal tubular cells. Am J Physiol Renal Physiol 285, F784–F791.
Pribanic S, Loffing J, Madjdpour C, Bacic D, Gisler S, Braunewell KH, Biber J & Murer H (2003b). Expression of visinin-like protein-3 (VILIP-3) in mouse kidney. Nephron Physiol 95, 76–82.
, 百拇医药
Raghuram V, Hormuth H & Foskett JK (2003). A kinase-regulated mechanism controls CFTR channel gating by disrupting bivalent PDZ domain interactions. Proc Natl Acad Sci U S A 100, 9620–9625.
Shenolikar S, Voltz JW, Minkoff CM, Wade JB & Weinman EJ (2002). Targeted disruption of the mouse NHERF-1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type IIa and renal phosphate wasting. Proc Natl Acad Sci U S A 99, 11470–11475.
, http://www.100md.com
Silver DI, Wang N, Vogel S, (2003). Identification of small Pdzk1-associated protein, Dd96/Map17 as a regulator for Pdzk1 and plasma high density lipoprotein levels. J Biol Chem 278, 28528–28532.
Spilker C, Richter K, Smalla KH, Manahan-Vaughan D, Gundelfinger ED & Braunewell KH (2000). The neuronal EF-hand calcium-binding protein visinin-like protein-3 is expressed in cerebellar Purkinje cells and shows a calcium-dependent membrane association. Neuroscience 96, 121–129.
, 百拇医药
Stagljar I, Korostensky C, Johnsson N & te Heesen S (1998). A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A 95, 5187–5192.
Traebert M, Volkl H, Biber J, Murer H & Kaissling B (2000). Luminal and contraluminal action of 1–34 and 3–34 PTH peptides on renal type IIa Na-P(i) cotransporter. Am J Physiol Renal Physiol 278, F792–F798.
Wade JB, Liu J, Coleman RA, Cunningham R, Steplock DA, Lee-Kwon W et al. (2003). Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse. Am J Physiol Cell Physiol 285, C1494–C1503.
, 百拇医药
Weinman EJ, Steplock D, Wang Y & Shenolikar S (1995). Characterization of a protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na+–H+ exchanger. J Clin Invest 95, 2143–2149.
Yun CH, Lamprecht G, Forster DV & Sidor A (1998). NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border Na+/H+ exchanger NHE3 and the cytoskeletal protein ezrin. J Biol Chem 273, 25856–25863., 百拇医药