Noncanonical Sequences Involving NHERF1 Interaction with NPT2A Govern Hormone-Regulated Phosphate Transport: Binding Outside the Box.

Na+/H+ exchange factor-1 (NHERF1), a multidomain PDZ scaffolding phosphoprotein, is required for the type II sodium-dependent phosphate cotransporter (NPT2A)-mediated renal phosphate absorption. Both PDZ1 and PDZ2 domains are involved in NPT2A-dependent phosphate uptake. Though harboring identical core-binding motifs, PDZ1 and PDZ2 play entirely different roles in hormone-regulated phosphate transport. PDZ1 is required for the interaction with the C-terminal PDZ-binding sequence of NPT2A (-TRL). Remarkably, phosphocycling at Ser290 distant from PDZ1, the penultimate step for both parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) regulation, controls the association between NHERF1 and NPT2A. PDZ2 interacts with the C-terminal PDZ-recognition motif (-TRL) of G Protein-coupled Receptor Kinase 6A (GRK6A), and that promotes phosphorylation of Ser290. The compelling biological puzzle is how PDZ1 and PDZ2 with identical GYGF core-binding motifs specifically recognize distinct binding partners. Binding determinants distinct from the canonical PDZ-ligand interactions and located "outside the box" explain PDZ domain specificity. Phosphorylation of NHERF1 by diverse kinases and associated conformational changes in NHERF1 add more complexity to PDZ-binding diversity.

Canonical and Noncanonical Sites Determine NPT2A Binding Selectivity to NHERF1 PDZ1.

Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain. We sought to define the structural determinants responsible for the specificity of interaction between NHERF1 PDZ domains and NPT2A. By performing all-atom/explicit-solvent molecular dynamics (MD) simulations in combination with biological mutagenesis, fluorescent polarization (FP) binding assays, and isothermal titration calorimetry (ITC), we found that in addition to canonical interactions of residues at 0 and -2 positions, Arg at the -1 position of NPT2A plays a critical role in association with Glu43 and His27 of PDZ1 that are absent in PDZ2. Experimentally introduced mutation in PDZ1 (Glu43Asp and His27Asn) decreased binding to NPT2A. Conversely, introduction of Asp183Glu and Asn167His mutations in PDZ2 promoted the formation of favorable interactions yielding micromolar KDs. The results describe novel determinants within both the PDZ domain and outside the canonical PDZ-recognition motif that are responsible for discrimination of NPT2A between two PDZ domains. The results challenge general paradigms for PDZ recognition and suggest new targets for drug development.

Identification of the first sodium binding site of the phosphate cotransporter NaPi-IIa (SLC34A1).

Transporters of the SLC34 family (NaPi-IIa,b,c) catalyze uptake of inorganic phosphate (Pi) in renal and intestinal epithelia. The transport cycle requires three Na(+) ions and one divalent Pi to bind before a conformational change enables translocation, intracellular release of the substrates, and reorientation of the empty carrier. The electrogenic interaction of the first Na(+) ion with NaPi-IIa/b at a postulated Na1 site is accompanied by charge displacement, and Na1 occupancy subsequently facilitates binding of a second Na(+) ion at Na2. The voltage dependence of cotransport and presteady-state charge displacements (in the absence of a complete transport cycle) are directly related to the molecular architecture of the Na1 site. The fact that Li(+) ions substitute for Na(+) at Na1, but not at the other sites (Na2 and Na3), provides an additional tool for investigating Na1 site-specific events. We recently proposed a three-dimensional model of human SLC34a1 (NaPi-IIa) including the binding sites Na2, Na3, and Pi based on the crystal structure of the dicarboxylate transporter VcINDY. Here, we propose nine residues in transmembrane helices (TM2, TM3, and TM5) that potentially contribute to Na1. To verify their roles experimentally, we made single alanine substitutions in the human NaPi-IIa isoform and investigated the kinetic properties of the mutants by voltage clamp and (32)P uptake. Substitutions at five positions in TM2 and one in TM5 resulted in relatively small changes in the substrate apparent affinities, yet at several of these positions, we observed significant hyperpolarizing shifts in the voltage dependence. Importantly, the ability of Li(+) ions to substitute for Na(+) ions was increased compared with the wild-type. Based on these findings, we adjusted the regions containing Na1 and Na3, resulting in a refined NaPi-IIa model in which five positions (T200, Q206, D209, N227, and S447) contribute directly to cation coordination at Na1.

The SLC34 family of sodium-dependent phosphate transporters.

The SLC34 family of sodium-driven phosphate cotransporters comprises three members: NaPi-IIa (SLC34A1), NaPi-IIb (SLC34A2), and NaPi-IIc (SLC34A3). These transporters mediate the translocation of divalent inorganic phosphate (HPO4 (2-)) together with two (NaPi-IIc) or three sodium ions (NaPi-IIa and NaPi-IIb), respectively. Consequently, phosphate transport by NaPi-IIa and NaPi-IIb is electrogenic. NaPi-IIa and NaPi-IIc are predominantly expressed in the brush border membrane of the proximal tubule, whereas NaPi-IIb is found in many more organs including the small intestine, lung, liver, and testis. The abundance and activity of these transporters are mostly regulated by changes in their expression at the cell surface and are determined by interactions with proteins involved in scaffolding, trafficking, or intracellular signaling. All three transporters are highly regulated by factors including dietary phosphate status, hormones like parathyroid hormone, 1,25-OH2 vitamin D3 or FGF23, electrolyte, and acid-base status. The physiological relevance of the three members of the SLC34 family is underlined by rare Mendelian disorders causing phosphaturia, hypophosphatemia, or ectopic organ calcifications.

Phosphate transport kinetics and structure-function relationships of SLC34 and SLC20 proteins.

Transport of inorganic phosphate (P(i)) is mediated by proteins belonging to two solute carrier families (SLC20 and SLC34). Members of both families transport P(i) using the electrochemical gradient for Na(+). The role of the SLC34 members as essential players in mammalian P(i) homeostasis is well established, whereas that of SLC20 proteins is less well defined. The SLC34 family comprises the following three isoforms that preferentially cotransport divalent P(i) and are expressed in epithelial tissue: the renal NaPi-IIa and NaPi-IIc are responsible for reabsorbing P(i) in the proximal tubule, whereas NaPi-IIb is more ubiquitously expressed, including the small intestine, where it mediates dietary P(i) absorption. The SLC20 family comprises two members (PiT-1, PiT-2) that preferentially cotransport monovalent P(i) and are expressed in epithelial as well as nonepithelial tissue. The transport kinetics of members of both families have been characterized in detail using heterologous expression in Xenopus oocytes. For the electrogenic NaPi-IIa/b, and PiT-1,-2, conventional electrophysiological techniques together with radiotracer methods have been applied, as well as time-resolved fluorometric measurements that allow new insights into local conformational changes of the protein during the cotransport cycle. For the electroneutral NaPi-IIc, conventional tracer uptake and fluorometry have been used to elucidate its transport properties. The 3-D structures of these proteins remain unresolved and structure-function studies have so far concentrated on defining the topology and identifying sites of functional importance.

Analysis of different complexes of type IIa sodium-dependent phosphate transporter in rat renal cortex using blue-native polyacrylamide gel electrophoresis.

Type IIa sodium-dependent phosphate transporter (NaPi-IIa) can be localized in the apical plasma membrane of renal proximal tubule to carry out a rate-limiting step of phosphate reabsorption. For the apical localization, NaPi-IIa is required to form a macromolecular complex with some adaptor proteins such as Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) and ezrin. However, the detail of macromolecular complex containing NaPi-IIa in the apical membrane of the renal proximal tubular cells has not been clarified. In this study, we identified at least four different complexes (220, 480, 920, 1,100 kDa) containing NaPi-IIa by using blue-native polyacrylamide gel electrophoresis. Interestingly, LC-MS/MS analysis and immunoprecipitation analysis reveal that megalin is a component of larger complexes (920 and 1,100 kDa). In addition, NaPi-IIa can be heterogeneously co-localized with ezrin and megalin on the apical membrane of renal proximal tubuler cells by fluorescence microscopy analysis. These results suggest that NaPi-IIa can form some different complexes on the apical plasma membrane of renal proximal tubular cells.

Proximal tubular handling of phosphate: A molecular perspective.

Members of the SLC34 gene family of solute carriers encode for three Na+-dependent phosphate (P i) cotransporter proteins, two of which (NaPi-IIa/SLC34A1 and NaPi-IIc/SLC34A3) control renal reabsorption of P i in the proximal tubule of mammals, whereas NaPi-IIb/SCLC34A2 mediates P i transport in organs other than the kidney. The P i transport mechanism has been extensively studied in heterologous expression systems and structure-function studies have begun to reveal the intricacies of the transport cycle at the molecular level using techniques such as cysteine scanning mutagenesis, and voltage clamp fluorometry. Moreover, sequence differences between the three types of cotransporters have been exploited to obtain information about the molecular determinants of hormonal sensitivity and electrogenicity. Renal handling of P i is regulated by hormonal and non-hormonal factors. Changes in urinary excretion of P i are almost invariably mirrored by changes in the apical expression of NaPi-IIa and NaPi-IIc in proximal tubules. Therefore, understanding the mechanisms that control the apical expression of NaPi-IIa and NaPi-IIc as well as their functional properties is critical to understanding how an organism achieves P i homeostasis.

Topology of the type IIa Na+/P(i) cotransporter.

The type IIa Na(+)/P(i) cotransporter (NaPi-IIa) plays a key role in the reabsorption of inorganic phosphate (P(i)) in the renal proximal tubule. The rat NaPi-IIa isoform is a protein of 637 residues for which different algorithms predict 8-12 transmembrane domains (TMDs). Epitope tagging experiments demonstrated that both the N and the C termini of NaPi-IIa are located intracellularly. Site-directed mutagenesis revealed two N-glycosylation sites in a large putative extracellular loop. Results from structure-function studies suggested the assembly of two similar opposed regions that possibly constitute part of the substrate translocation pathway for one phosphate ion together with three sodium ions. Apart from these topological aspects, other structural features of NaPi-IIa are not known. In this study, we have addressed the topology of NaPi-IIa using in vitro transcription/translation of HK-M0 and HK-M1 fusion vectors designed to test membrane insertion properties of cDNA sequences encoding putative NaPi-IIa TMDs. Based on the results of in vitro transcription/translation analyses, we propose a model of NaPi-IIa comprising 12 TMDs, with both N and C termini orientated intracellularly and a large hydrophilic extracellular loop between the fifth and sixth TMDs. The proposed model is in good agreement with the prediction of the NaPi-IIa structure obtained by the hidden Markov algorithm HMMTOP.

Role of membrane microdomains in PTH-mediated down-regulation of NaPi-IIa in opossum kidney cells.

BACKGROUND: Parathyroid hormone (PTH) rapidly down-regulates type IIa sodium-dependent phosphate transporter (NaPi-IIa) via an endocytic pathway. Since the relationship between PTH signaling and NaPi-IIa endocytosis has not been explored, we investigated the role of membrane microdomains in this process. METHODS: We examined the submembrane localization of NaPi-IIa in opossum kidney (OK-N2) cells that stably expressed human NaPi-IIa, and searched for a PTH-induced specific phosphorylating substrate on their membrane microdomains by immunoblotting with specific antibody against phospho substrates of protein kinases. RESULTS: We found that NaPi-IIa was primarily localized in low-density membrane (LDM) domains of the plasma membrane; PTH reduced the levels of immunoreactive NaPi-IIa in these domains. Furthermore, PTH activated both protein kinase A (PKA) and protein kinase Calpha (PKCa) and increased the phosphorylation of 250 kD and 80 kD substrates; this latter substrate was identified as ezrin, which a member of the ezrin-radixin-moesin (ERM) protein family. In response to PTH, ezrin was phosphorylated by both PKA and PKC. Dominant negative ezrin blocked the reduction in NaPi-IIa expression in the LDM domains that was induced by PTH. CONCLUSION: These data suggest that NaPi-IIa and PTH-induced phosphorylated proteins that include ezrin are compartmentalized in LDM microdomains. This compartmentalization may play an important role in the down-regulation of NaPi-IIa via endocytosis.

Functionally important residues in the predicted 3(rd) transmembrane domain of the type IIa sodium-phosphate co-transporter (NaPi-IIa).

The type IIa Na(+)/P(i), cotransporter (NaPi-IIa) mediates electrogenic transport of three Na(+) and one divalent P(i) ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and P(i) transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of P(i) transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the P(i)-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na(+)-dependent P(i) transport. Since electrogenic NaPi-II transports 3 Na(+)/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na(+) binding sites in NaPi-IIa.