Proximal tubular handling of phosphate: A molecular perspective
2006; Elsevier BV; Volume: 70; Issue: 9 Linguagem: Inglês
10.1038/sj.ki.5001813
ISSN1523-1755
AutoresIan C. Forster, Nati Hernando, Jürg Biber, Heini Murer,
Tópico(s)Magnesium in Health and Disease
ResumoMembers of the SLC34 gene family of solute carriers encode for three Na+-dependent phosphate (Pi) cotransporter proteins, two of which (NaPi-IIa/SLC34A1 and NaPi-IIc/SLC34A3) control renal reabsorption of Pi in the proximal tubule of mammals, whereas NaPi-IIb/SCLC34A2 mediates Pi transport in organs other than the kidney. The Pi 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 Pi is regulated by hormonal and non-hormonal factors. Changes in urinary excretion of Pi 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 Pi homeostasis. Members of the SLC34 gene family of solute carriers encode for three Na+-dependent phosphate (Pi) cotransporter proteins, two of which (NaPi-IIa/SLC34A1 and NaPi-IIc/SLC34A3) control renal reabsorption of Pi in the proximal tubule of mammals, whereas NaPi-IIb/SCLC34A2 mediates Pi transport in organs other than the kidney. The Pi 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 Pi is regulated by hormonal and non-hormonal factors. Changes in urinary excretion of Pi 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 Pi homeostasis. Homeostasis of Pi in higher organisms depends on the coordinated transport of Pi across intestinal and renal epithelia. Transport of Pi across the apical membrane is mediated by the three members of the SLC34 family of solute carriers.1.Murer H. Forster I. Biber J. The sodium phosphate cotransporter family SLC34.Pflugers Arch. 2004; 447: 763-767Crossref PubMed Scopus (245) Google Scholar NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) are specifically expressed in the brush border membrane (BBM) of renal proximal tubules. NaPi-IIb (SLC34A2) has a broader pattern of expression and it is highly abundant in the BBM of small intestine. In both epithelia, the basolateral exit of Pi is mediated by a transporter that remains unidentified. In the proximal tubule it has been proposed that a Na+-dependent electroneutral anion exchanger is at least partially responsible for Pi exit.2.Barac-Nieto M. Alfred M. Spitzer A. Basolateral phosphate transport in renal proximal-tubule-like OK cells.Exp Biol Med. 2002; 227: 626-631PubMed Google Scholar NaPi-IIa and NaPi-IIc mediate the reabsorption of Pi from the primary urine by using the free energy provided by the electrochemical gradient for Na+. NaPi-IIa is electrogenic and transports divalent Pi preferentially. It functions with a strict Na+:Pi stoichiometry of 3:1, which results in the net inward movement of one positive charge per cotransport cycle.3.Forster I.C. Loo D.D. Eskandari S. Stoichiometry and Na+ binding cooperativity of rat and flounder renal type II Na+-Pi cotransporters.Am J Physiol. 1999; 276: F644-F649PubMed Google Scholar NaPi-IIc, in contrast, is electroneutral and exhibits a 2:1 stoichiometry4.Segawa H. Kaneko I. Takahashi A. et al.Growth-related renal type II Na/Pi cotransporter.J Biol Chem. 2002; 277: 19665-19672Crossref PubMed Scopus (236) Google Scholar,5.Bacconi A. Virkki L.V. Biber J. et al.Renouncing electroneutrality is not free of charge: switching on electrogenicity in a Na+-coupled phosphate cotransporter.Proc Natl Acad Sci USA. 2005; 102: 12606-12611Crossref PubMed Scopus (62) Google Scholar (Figure 1). In mice, NaPi-IIa is the protein mainly responsible for Pi reabsorption in the adult kidney, whereas NaPi-IIc appears to be more important in weaning animals. Indeed, the phenotype of NaPi-IIa knockout mice first suggested that this cotransporter is responsible for the bulk of renal Pi reabsorption with a very small percentage potentially attributed to NaPi-IIc.6.Beck L. Karaplis A.C. Amizuka N. et al.Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities.Proc Natl Acad Sci USA. 1998; 95: 5372-5377Crossref PubMed Scopus (482) Google Scholar However, recent data indicate that in humans, NaPi-IIc may have a previously unpredicted importance. The expression of NaPi-IIa and NaPi-IIc is regulated to adapt the renal reabsorption of Pi to the organism needs. Thus, the phosphaturic effect associated with parathyroid hormone (PTH) is due to the membrane retrieval of both cotransporters, whereas in conditions of Pi deprivation their expression is increased.4.Segawa H. Kaneko I. Takahashi A. et al.Growth-related renal type II Na/Pi cotransporter.J Biol Chem. 2002; 277: 19665-19672Crossref PubMed Scopus (236) Google Scholar, 7.Levi M. Lotscher M. Sorribas V. et al.Cellular mechanisms of acute and chronic adaptation of rat renal Pi transporter to alterations in dietary Pi.Am J Physiol. 1994; 267: F900-F908PubMed Google Scholar, 8.Keusch I. Traebert M. Lotscher M. et al.Parathyroid hormone and dietary phosphate provoke a lysosomal routing of the proximal tubular Na/Pi-cotransporter type II.Kidney Int. 1998; 54: 1224-1232Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 9.Segawa H. Yamanaka S. Ito M. et al.Internalization of renal type IIc Na-Pi cotransporter in response to a high-phosphate diet.Am J Physiol Renal Physiol. 2005; 288: F587-F596Crossref PubMed Scopus (65) Google Scholar The following sections summarize our present state of knowledge of the regulatory and pathophysiological roles of NaPi-IIa in renal Pi handling as well as its mechanism and structure–function relations. Many hormonal and non-hormonal factors regulate renal reabsorption of Pi (for review, see Murer et al.10.Murer H. Hernando N. Forster I. Biber J. Regulation of Na/Pi transporter in the proximal tubule.Annu Rev Physiol. 2003; 65: 531-542Crossref PubMed Scopus (126) Google Scholar). The effect of PTH and dietary Pi on NaPi-IIa has been the subject of detailed investigation. These studies suggest that NaPi-IIa regulation depends on its shuttling to/from the BBM. This contrasts with many other transporters, which activity is modulated by modification of the transport protein itself (e.g. phosphorylation, dimerization etc). This means that the body's requirements for a higher Pi reabsorption (i.e. after low Pi-diet) are met by increasing the expression of NaPi-IIa7.Levi M. Lotscher M. Sorribas V. et al.Cellular mechanisms of acute and chronic adaptation of rat renal Pi transporter to alterations in dietary Pi.Am J Physiol. 1994; 267: F900-F908PubMed Google Scholar, 11.Lotscher M. Kaissling B. Biber J. et al.Role of microtubules in the rapid regulation of renal phosphate transport in response to acute alterations in dietary phosphate content.J Clin Invest. 1997; 99: 1302-1312Crossref PubMed Scopus (110) Google Scholar, 12.Pfister M.F. Hilfiker H. Forgo J. et al.Cellular mechanisms involved in the acute adaptation of OK cell Na/Pi-cotransport to high- or low-Pi medium.Pflugers Arch. 1998; 435: 713-719Crossref PubMed Scopus (64) Google Scholar and NaPi-IIc4.Segawa H. Kaneko I. Takahashi A. et al.Growth-related renal type II Na/Pi cotransporter.J Biol Chem. 2002; 277: 19665-19672Crossref PubMed Scopus (236) Google Scholar at the BBM. For NaPi-IIa, acute upregulation is independent of changes in transcription or translation. Therefore, the increased expression of NaPi-IIa must be owing to either the stabilization of the transporter at the BBM or to an increased rate of insertion at the membrane. Experimental data supports this dual mechanism. Thus, dietary-induced upregulation depends on the presence of scaffolding proteins,13.Weinman E.J. Boddeti A. Cunningham R. et al.NHERF-1 is required for renal adaptation to a low-phosphate diet.Am J Physiol Renal Physiol. 2003; 285: F1225-F1232Crossref PubMed Scopus (49) Google Scholar suggesting a stabilization action, and on the microtubule network,11.Lotscher M. Kaissling B. Biber J. et al.Role of microtubules in the rapid regulation of renal phosphate transport in response to acute alterations in dietary phosphate content.J Clin Invest. 1997; 99: 1302-1312Crossref PubMed Scopus (110) Google Scholar suggesting an increased rate of insertion. This latter mechanism requires the presence of an intracellular pool of NaPi-IIa ready to be shuttled to the membrane. Immunostainings of kidneys from rats fed acutely a low Pi-diet have indeed revealed the presence of NaPi-IIa in the Golgi apparatus, although this pool is not detected with all immunostaining protocols.11.Lotscher M. Kaissling B. Biber J. et al.Role of microtubules in the rapid regulation of renal phosphate transport in response to acute alterations in dietary phosphate content.J Clin Invest. 1997; 99: 1302-1312Crossref PubMed Scopus (110) Google Scholar In contrast, reduced reabsorption of Pi (i.e. upon PTH release or high Pi-diet) is achieved via downregulation of NaPi-IIa8.Keusch I. Traebert M. Lotscher M. et al.Parathyroid hormone and dietary phosphate provoke a lysosomal routing of the proximal tubular Na/Pi-cotransporter type II.Kidney Int. 1998; 54: 1224-1232Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 11.Lotscher M. Kaissling B. Biber J. et al.Role of microtubules in the rapid regulation of renal phosphate transport in response to acute alterations in dietary phosphate content.J Clin Invest. 1997; 99: 1302-1312Crossref PubMed Scopus (110) Google Scholar, 14.Pfister M.F. Lederer E. Forgo J. et al.Parathyroid hormone-dependent degradation of type II Na+/Pi cotransporters.J Biol Chem. 1997; 272: 20125-20130Crossref PubMed Scopus (117) Google Scholar and NaPi-IIc9.Segawa H. Yamanaka S. Ito M. et al.Internalization of renal type IIc Na-Pi cotransporter in response to a high-phosphate diet.Am J Physiol Renal Physiol. 2005; 288: F587-F596Crossref PubMed Scopus (65) Google Scholar at the BBM. PTH-induced downregulation of NaPi-IIa has been extensively studied and the identifiable steps are summarized in Figure 2. Because endocytosed cotransporters do not recycle to the BBM but instead are degraded in lysosomes, recovery of NaPi-IIa basal levels upon PTH removal depends on de novo synthesis. It is therefore clear that apical retention/removal of NaPi-IIa must be a regulated process, beyond the control of protein turnover. We will now describe in detail the steps summarized in Figure 2, integrating what is known about the mechanisms that regulate NaPi-IIa expression with the role of protein complexes. Apical expression of NaPi-IIa is dependent on its last three residues (TRL, see Figure 4a). Truncation of these residues leads to intracellular accumulation of the cotransporter, suggesting an impaired sorting and/or stability of the mutated protein.15.Karim-Jimenez Z. Hernando N. Biber J. Murer H. Molecular determinants for apical expression of the renal type IIa Na+/Pi-cotransporter.Pflugers Arch. 2001; 442: 782-790Crossref PubMed Scopus (59) Google Scholar The TRL sequence represents a PDZ (PSD-95, Discs-large and ZO-1) binding motif that interacts with several PDZ proteins.16.Gisler S.M. Stagljar I. Traebert M. et al.Interaction of the type IIa Na/Pi cotransporter with PDZ proteins.J Biol Chem. 2001; 276: 9206-9213Crossref PubMed Scopus (198) Google Scholar,17.McWilliams R.R. Breusegem S.Y. Brodsky K.F. et al.Shank2E binds NaPi cotransporter at the apical membrane of proximal tubule cells.Am J Physiol Cell Physiol. 2005; 289: C1042-C1051Crossref PubMed Scopus (27) Google Scholar PDZ domains, first described in the early 1990s, comprise 80–100 residues distributed in six β strands and two α helices. They bind to the carboxyl-terminal tail (PDZ-binding motif) of the corresponding ligand (for review, see Nourry et al.18.Nourry C. Grant S.G. Borg J.P. PDZ domain proteins: plug and play!.Sci STKE. 2003; 179: RE7Google Scholar). A conserved sequence between the βA and βB strands of the PDZ domain (GLGF) provides a hydrophobic pocket for ligand binding. Among the PDZ proteins that interact with NaPi-IIa are the Na/H-exchanger regulatory factors NHERF1 (EBP50) and NHERF2 (E3KARP) as well as PDZK1 (NHERF3), PDZK2 (IKEPP, NHERF4), and Shank2E.16.Gisler S.M. Stagljar I. Traebert M. et al.Interaction of the type IIa Na/Pi cotransporter with PDZ proteins.J Biol Chem. 2001; 276: 9206-9213Crossref PubMed Scopus (198) Google Scholar,17.McWilliams R.R. Breusegem S.Y. Brodsky K.F. et al.Shank2E binds NaPi cotransporter at the apical membrane of proximal tubule cells.Am J Physiol Cell Physiol. 2005; 289: C1042-C1051Crossref PubMed Scopus (27) Google Scholar NHERF1 and NHERF2 are two related proteins each containing two PDZ domains and a C-terminal Merlin-Ezin-Radixin-Moesin-binding domain.19.Weinman E.J. Steplock D. Wang Y. Shenolikar S. Characterization of a protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na+-H+ exchanger.J Clin Invest. 1995; 95: 2143-2149Crossref PubMed Scopus (299) Google Scholar, 20.Yun C.H. Lamprecht G. Forster D.V. Sidor A. NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border Na+/H+ exchanger NHE3 and the cytoskeletal protein ezrin.J Biol Chem. 1998; 273: 25856-25863Crossref PubMed Scopus (244) Google Scholar, 21.Murthy A. Gonzalez-Agosti C. Cordero E. et al.NHE-RF, a regulatory cofactor for Na+-H+ exchange, is a common interactor for merlin and ERM (MERM) proteins.J Biol Chem. 1998; 273: 1273-1276Crossref PubMed Scopus (214) Google Scholar They are expressed in the apical/subapical domain of murine proximal tubules, respectively.16.Gisler S.M. Stagljar I. Traebert M. et al.Interaction of the type IIa Na/Pi cotransporter with PDZ proteins.J Biol Chem. 2001; 276: 9206-9213Crossref PubMed Scopus (198) Google Scholar,22.Wade J.B. Liu J. Coleman R.A. et al.Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse.Am J Physiol Cell Physiol. 2003; 285: C1494-C1503Crossref PubMed Scopus (97) Google Scholar NaPi-IIa binds to the first PDZ domain on both proteins.16.Gisler S.M. Stagljar I. Traebert M. et al.Interaction of the type IIa Na/Pi cotransporter with PDZ proteins.J Biol Chem. 2001; 276: 9206-9213Crossref PubMed Scopus (198) Google Scholar Renal proximal cells (opossum kidney cells) transfected with dominant-negative NHERF1 constructs23.Hernando N. Deliot N. Gisler S.M. et al.PDZ-domain interactions and apical expression of type IIa Na/Pi cotransporters.Proc Natl Acad Sci USA. 2002; 99: 11957-11962Crossref PubMed Scopus (151) Google Scholar and young NHERF1-/- animals24.Shenolikar S. Voltz J.W. Minkoff C.M. et al.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 USA. 2002; 99: 11470-11475Crossref PubMed Scopus (269) Google Scholar show a reduced amount of NaPi-IIa at the BBM. In animals, this reduction associates with urinary loss of Pi, a phenotype that reverts with age.25.Weinman E.J. Mohanlal V. Stoycheff N. et al.Longitudinal study of urinary excretion of phosphate, calcium, and uric acid in mutant NHERF-1 null mice.Am J Physiol Renal Physiol. 2006; 290: F838-F843Crossref PubMed Scopus (32) Google Scholar These findings suggest that NHERF1 contributes to stabilize NaPi-IIa at the BBM. This stabilization depends on the Merlin-Ezin-Radixin-Moesin-binding domain,23.Hernando N. Deliot N. Gisler S.M. et al.PDZ-domain interactions and apical expression of type IIa Na/Pi cotransporters.Proc Natl Acad Sci USA. 2002; 99: 11957-11962Crossref PubMed Scopus (151) Google Scholar which mediates binding to the actin-associated protein Ezrin. In contrast to the effect on NaPi-IIa, deficiency in NHERF1 does not affect the expression of NHE3.24.Shenolikar S. Voltz J.W. Minkoff C.M. et al.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 USA. 2002; 99: 11470-11475Crossref PubMed Scopus (269) Google Scholar PDZK1 and PDZK2 are related proteins, also expressed in murine proximal tubules, each containing four PDZ domains. In both cases, NaPi-IIa binds to the third PDZ domain.16.Gisler S.M. Stagljar I. Traebert M. et al.Interaction of the type IIa Na/Pi cotransporter with PDZ proteins.J Biol Chem. 2001; 276: 9206-9213Crossref PubMed Scopus (198) Google Scholar,26.Kocher O. Comella N. Gilchrist A. et al.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. 1999; 79: 1161-1170PubMed Google Scholar Opossum kidney cells transfected with a dominant-negative PDZK1 construct show reduced levels of NaPi-IIa at the BBM.23.Hernando N. Deliot N. Gisler S.M. et al.PDZ-domain interactions and apical expression of type IIa Na/Pi cotransporters.Proc Natl Acad Sci USA. 2002; 99: 11957-11962Crossref PubMed Scopus (151) Google Scholar Although NaPi-IIa expression is unaffected in normally fed PDZK1-/- mice, its abundance decreases when the animals are fed a high Pi-diet.27.Capuano P. Bacic D. Stange G. et al.Expression and regulation of the renal Na/phosphate cotransporter NaPi-IIa in a mouse model deficient for the PDZ protein PDZK1.Pflugers Arch. 2005; 449: 392-402Crossref PubMed Scopus (63) Google Scholar Thus, in extreme dietary conditions PDZK1 may contribute to stabilize NaPi-IIa at the BBM. Shank2E is an epithelial-specific isoform of Shank2. The three members of the Shank family share a similar domain structure consisting of six N-terminal ankyrin repeats followed by an SH3 domain, a PDZ domain, and a proline-rich region.28.Boeckers T.M. Bockmann J. Kreutz M.R. Gundelfinger E.D. ProSAP/Shank proteins – a family of higher order organizing molecules of the postsynaptic density with an emerging role in human neurological disease.J Neurochem. 2002; 81: 903-910Crossref PubMed Scopus (245) Google Scholar In rats, Shank2E is expressed at the BBM of proximal tubules and, as for the other PDZ proteins, association with NaPi-IIa requires the C-terminal TRL motif of the cotransporter.17.McWilliams R.R. Breusegem S.Y. Brodsky K.F. et al.Shank2E binds NaPi cotransporter at the apical membrane of proximal tubule cells.Am J Physiol Cell Physiol. 2005; 289: C1042-C1051Crossref PubMed Scopus (27) Google Scholar Shank2 can bind dynamin,29.Okamoto P.M. Gamby C. Wells D. et al.Dynamin isoform-specific interaction with the shank/ProSAP scaffolding proteins of the postsynaptic density and actin cytoskeleton.J Biol Chem. 2001; 276: 48458-48465Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar a guanosine triphosphatase that mediates fission of endocytic vesicles.30.De Camilli P. Takei K. McPherson P.S. The function of dynamin in endocytosis.Curr Opin Neurobiol. 1995; 5: 559-565Crossref PubMed Scopus (132) Google Scholar Thus, Shank2E may connect NaPi-IIa with the endocytic machinery. In the proximal tubule, PTH binds to apical and basolateral receptors. Stimulation of either receptor leads to an increase in urinary excretion of Pi as consequence of the reduction of NaPi-IIa in the BBM.31.Traebert M. Volkl H. Biber J. et al.Luminal and contraluminal action of 1–34 and 3–34 PTH peptides on renal type IIa Na-Pi cotransporter.Am J Physiol Renal Physiol. 2000; 278: F792-F798PubMed Google Scholar Apical application of PTH to isolated proximal tubules activates preferentially the phospholipase C/protein kinase C (PKC) pathway, whereas basolateral application leads to activation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling.31.Traebert M. Volkl H. Biber J. et al.Luminal and contraluminal action of 1–34 and 3–34 PTH peptides on renal type IIa Na-Pi cotransporter.Am J Physiol Renal Physiol. 2000; 278: F792-F798PubMed Google Scholar The molecular explanation for this dual response may relay on the presence (apical) or absence (basolateral) of NHERF. Thus, it has been shown that NHERF associates with both the PTH receptor and the phospholipase Cβ1.32.Mahon M.J. Donowitz M. Yun C.C. Segre G.V. Na+/H+ exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling.Nature. 2002; 417: 858-861Crossref PubMed Scopus (259) Google Scholar The consequence of this intermolecular association is the preferential activation of phospholipase C upon binding of PTH to apical receptors. In accordance with this mechanism, both 1–34 PTH (a fragment that activates PKA and PKC) and 3–34 PTH (a fragment that activates only PKC) fail to activate phospholipase C in kidney slices from NHERF1-/- mice.33.Capuano P. Bacic D. Roos M. et al.Defective coupling of apical PTH-receptors to phospholipase C prevents internalization of the Na+/phosphate cotransporter NaPi-IIa in NHERF1 deficient mice.Am J Physiol-Renal. 2006Crossref Scopus (70) Google Scholar Despite the heterogeneity of their initial steps, apical, and basolateral PTH receptors use common downstream effectors. Mitogen-activated protein kinase-kinase 1/2 inhibitors partially or fully prevent the effect of both cascades, suggesting that the PKC and PKA pathways coactivate extracellular signal-regulated protein kinase 1/2.34.Bacic D. Schulz N. Biber J. et al.Involvement of the MAPK-kinase pathway in the PTH-mediated regulation of the proximal tubule type IIa Na+/Pi cotransporter in mouse kidney.Pflugers Arch. 2003; 446: 52-60Crossref PubMed Scopus (24) Google Scholar Interestingly, NHERF1 plays a very different role in the regulation of NHE3, where acts as a scaffold for PKA via association with the cAMP-kinase associated protein Ezrin.35.Weinman E.J. Cunningham R. Shenolikar S. NHERF and regulation of the renal sodium-hydrogen exchanger NHE3.Pflugers Arch. 2005; 450: 137-144Crossref PubMed Scopus (34) Google Scholar Then, PKA phosphorylates (and inhibits) NHE3 without initial changes in the expression of NHE3 in the BBM.36.Collazo R. Fan L. Hu M.C. et al.Acute regulation of Na+/H+ exchanger NHE3 by parathyroid hormone via NHE3 phosphorylation and dynamin-dependent endocytosis.J Biol Chem. 2000; 275: 31601-31608Crossref PubMed Scopus (112) Google Scholar cAMP-induced inhibition of NHE3 can be reproduced with cAMP analogs that activate exchange protein directly activated by cAMP (EPAC1), whereas NaPi-IIa is downregulated by PKA- but not by EPAC1-activating analogs.37.Honegger K.J. Capuano P. Winter C. et al.Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC).Proc Natl Acad Sci USA. 2006; 103: 803-808Crossref PubMed Scopus (76) Google Scholar Binding of PTH leads to the axial movement of NaPi-IIa along the microvilli and finally to its endocytosis from the microvillar clefts.38.Traebert M. Roth J. Biber J. et al.Internalization of proximal tubular type II Na-Pi cotransporter by PTH: immunogold electron microscopy.Am J Physiol Renal Physiol. 2000; 278: F148-F154PubMed Google Scholar,39.Yang L.E. Maunsbach A.B. Leong P.K. McDonough A.A. Differential traffic of proximal tubule Na+ transporters during hypertension or PTH: NHE3 to base of microvilli vs. NaPi2 to endosomes.Am J Physiol Renal Physiol. 2004; 287: F896-F906Crossref PubMed Scopus (74) Google Scholar NaPi-IIa colocalizes with insulin upon PTH administration, suggesting its internalization via receptor-mediated endocytosis.40.Bacic D. Lehir M. Biber J. et al.The renal Na+/phosphate cotransporter NaPi-IIa is internalized via the receptor-mediated endocytic route in response to parathyroid hormone.Kidney Int. 2006; 69: 495-503Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar This is further supported by the finding that NaPi-IIa endocytosis is prevented in mice with kidney-specific megalin deficiency and in receptor-associate-protein-deficient mice.41.Bacic D. Capuano P. Gisler S.M. et al.Impaired PTH-induced endocytotic down-regulation of the renal type IIa Na+/Pi-cotransporter in RAP-deficient mice with reduced megalin expression.Pflugers Arch. 2003; 446: 475-484Crossref PubMed Scopus (35) Google Scholar The immunostainings shown in Figure 3 illustrate the route followed by NaPi-IIa in response to PTH.40.Bacic D. Lehir M. Biber J. et al.The renal Na+/phosphate cotransporter NaPi-IIa is internalized via the receptor-mediated endocytic route in response to parathyroid hormone.Kidney Int. 2006; 69: 495-503Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar Endocytosis takes place via clathrin-coated pits and it is detected shortly upon PTH administration. Later on, NaPi-IIa is observed in clathrin-coated pits and in endosomes (early endosome-associated protein 1 (EEA1) positive). Finally, the cotransporter is targeted to late endosomes/lysosomes (lgp120 positive). Endocytosis associates with microtubule rearrangement, owing to the formation of apical to basolateral oriented bundles.42.Lotscher M. Scarpetta Y. Levi M. et al.Rapid downregulation of rat renal Na/Pi cotransporter in response to parathyroid hormone involves microtubule rearrangement.J Clin Invest. 1999; 104: 483-494Crossref PubMed Scopus (101) Google Scholar Prevention of microtubular rearrangement or microtubular depolymerization causes the delay of intracellular depletion of NaPi-IIa (i.e. lysosomal degradation), although it does not affect its downregulation (i.e. endocytosis). Clathrin-mediated internalization of many proteins depends on discrete intracellular sequences, among them tyrosine (Y)- and dileucine (LL)-based motifs. These motifs link the protein to be endocytosed to the adaptor protein AP2 which in turn binds to clathrin (for review, see Robinson43.Robinson M.S. Adaptable adaptors for coated vesicles.Trends Cell Biol. 2004; 14: 167-174Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). AP2 is a heterotetramer consisting of α, β2, μ2, and σ2 subunits. Y-based motifs bind to the μ subunit whereas LL-based motifs interact with the β subunit. NaPi-IIa contains several putative Y- or LL-motifs (GY402FAM, Y509RWF, LL101, LL374, and LI590) and two diacidic sequences (EE81 and EE616) that can control lysosomal targeting. Mutations of these motifs did not affect the PTH-induced retrieval of NaPi-IIa.44.Hernando N. Forgo J. Biber J. Murer H. PTH-induced downregulation of the type IIa Na/Pi-cotransporter is independent of known endocytic motifs.J Am Soc Nephrol. 2000; 11: 1961-1968PubMed Google Scholar Instead, a dibasic sequence (KR) within the last intracellular loop (Figure 4a) is required for PTH sensitivity.45.Karim-Jimenez Z. Hernando N. Biber J. et al.A dibasic motif involved in parathyroid hormone-induced down-regulation of the type IIa NaPi cotransporter.Proc Natl Acad Sci USA. 2000; 97: 12896-12901Crossref PubMed Scopus (45) Google Scholar These two positively charged residues are replaced by uncharged residues (NI) in the PTH-insensitive NaPi-IIb isoform. Swapping the specific residues inverts the PTH sensitivity of the protein. The KR-containing loop, but not a mutant with the KR sequence replaced by NI, interacts with PEX19.46.Ito M. Iidawa S. Izuka M. et al.Interaction of a farnesylated protein with renal type IIa Na/Pi co-transporter in response to parathyroid hormone and dietary phosphate.Biochem J. 2004; 377: 607-616Crossref PubMed Google Scholar In opossum kidney cells, NaPi-IIa endocytosis is accelerated upon transfection of PEX19, suggesting a role of this protein in the internalization of the cotransporter.46.Ito M. Iidawa S. Izuka M. et al.Interaction of a farnesylated protein with renal type IIa Na/Pi co-transporter in response to parathyroid hormone and dietary phosphate.Biochem J. 2004; 377: 607-616Crossref PubMed Google Scholar NHERF1 and PDZK1 remain attached to the BBM upon PTH administration; Deliot et al.,47.Deliot N. Hernando N. Horst-Liu Z. et al.Parathyroid hormone treatment induces dissociation of type IIa Na+-Pi cotransporter-Na+/H+ exchanger regulatory factor-1 complexes.Am J Physiol Cell Physiol. 2005; 289: C159-C167Crossref PubMed Scopus (86) Google Scholar in preparation. This suggests the disassembly of protein complexes before internalization of NaPi-IIa. In opossum kidney cells, the amount of NaPi-IIa that coimmunoprecipitates with NHERF1 is reduced upon PTH treatment.47.Deliot N. Hernando N. Horst-Liu Z. et al.Parathyroid hormone treatment induces dissociation of type IIa Na+-Pi cotransporter-Na+/H+ exchanger regulatory factor-1 complexes.Am J Physiol Cell Physiol. 2005; 289: C159-C167Crossref PubMed Scopus (86) Google Scholar Thus, PTH may negatively regulate the association between NaPi-IIa and NHERF1/PDZK1. PDZ-based interactions can be regulated by phosphorylation of either the PDZ-binding motif or the corresponding PDZ-domain. Studies using cell culture models have demonstrated that NHERF1 is constitutively phosphorylated, and the residues responsible for constitutive and regulated
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