RESUMEN

The epithelial-Na(+)-channel (alphabetagammaENaC) regulates kidney salt-transport and blood pressure. Each ENaC subunit contains a PY motif (PPxY) and its mutation in beta/gammaENaC causes Liddle syndrome, a hereditary hypertension. These (extended) PY motifs (PP(616)xY(618)xxL(621)) serve as binding sites for the ubiquitin ligase Nedd4-2, which decreases cell-surface expression of ENaC by unknown route(s). Using polarized kidney epithelia [Madin-Darby canine kidney I (MDCK-I)] cells stably expressing extracellularly myc-tagged wild type (WT) or PY-motif mutants of betaENaC (P616A, Y618A or L621A, with WT-alphagammaENaC), and live-imaging plus enzyme-linked immunosorbent assay (ELISA)-type assays to analyze routes/rates of ENaC internalization/recycling, we show here that cell-surface half-life of all PY mutants was fourfold longer than WT-ENaC (approximately 120 versus 30 minutes), reflecting primarily reduced channel internalization but also attenuated replenishment of cell-surface ENaC from a large subapical pool. The Y618A mutant revealed more severe internalization and replenishment defects than the other PY mutants. Internalized WT-ENaC was detected in sorting/recycling and late endosomes/lysosomes, while the Y618A mutant accumulated in the former. Nedd4-2 ubiquitinated ENaC at the apical membrane causing channel internalization and degradation. Cyclic AMP (cAMP) accelerated mobilization of subapical ENaC to the cell surface and long-term ENaC recycling, but only mobilization, not recycling, was inhibited in the PY mutants. These results suggest that the ENaC PY motifs (and Nedd4-2) primarily regulate channel internalization but also affect cAMP-dependent replenishment, providing important insight into the Liddle syndrome defects.

Asunto(s)

Endocitosis/fisiología , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Secuencias de Aminoácidos/genética , Animales , Línea Celular , Membrana Celular/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Endocitosis/efectos de los fármacos , Complejos de Clasificación Endosomal Requeridos para el Transporte , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Humanos , Hipertensión/genética , Cinética , Microscopía Confocal , Microvellosidades/metabolismo , Modelos Biológicos , Mutación , Ubiquitina-Proteína Ligasas Nedd4 , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Síndrome , Tionucleótidos/farmacología , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

In renal proximal brush borders the Na/Pi cotransporter NaPi-IIa is part of a heteromultimeric complex including the PDZ proteins PDZK1 and NHERF1, which interact with the C terminus of NaPi-IIa. In this study, a yeast two-hybrid screen against the N terminus of the Na/Pi cotransporter NaPi-IIa was performed. Thereby we identified visinin-like protein-3 (VILIP-3), a member of neuronal calcium sensors. In this study, expression and protein localization of VILIP-3 in the mouse kidney was performed by immunofluorescence and RT-PCR using laser-assisted microdissected nephron segments. VILIP-3 was found to be abundant in distal and collecting ducts where it partly colocalized with calbindin D28K. In addition VILIP-3 was observed in the brush borders of proximal tubular S1 and S3 segments of both superficial and deep nephrons.

Asunto(s)

Proteínas de Unión al Calcio/genética , Riñón/metabolismo , Proteínas del Tejido Nervioso/genética , Animales , Proteínas de Unión al Calcio/metabolismo , Expresión Génica , Inmunohistoquímica , Riñón/química , Masculino , Ratones , Ratones Endogámicos , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa , Simportadores/genética , Simportadores/metabolismo , Técnicas del Sistema de Dos Híbridos

RESUMEN

BACKGROUND: In proximal tubular cells, PDZK1 (NaPi-Cap1) has been implicated in apical expression of the Na+-dependent phosphate cotransporter (NaPi-IIa) via interaction with its C-terminus. PDZK1 represents a multidomain protein consisting of four PDZ domains and thus is believed to have a broader specificity besides NaPi-IIa. METHODS: We subjected single PDZ domains derived from PDZK1 either to yeast two-hybrid screens or yeast trap assays. Different pull-down assays and blot overlays were applied to corroborate the PDZK1-mediated interactions in vitro. Co-localization of interacting proteins with PDZK1 in proximal tubular cells was assessed by immunohistochemistry. RESULTS: In the yeast screens, the most abundant candidate protein to interact with PDZK1 was the membrane-associated protein of 17 kD (MAP17). Besides MAP17, C-terminal parts of following transporters were also identified: NaPi-IIa, solute carrier SLC17A1 (NaPi-I), Na+/H+ exchanger (NHE-3), organic cation transporter (OCTN1), chloride-formate exchanger (CFEX), and urate-anion exchanger (URAT1). In addition, other regulatory factors were found among the clones, such as a protein kinase A (PKA)-anchoring protein (D-AKAP2) and N+/H+ exchanger regulator factor (NHERF-1). All interactions of itemized proteins with PDZK1 were affirmed by in vitro techniques. Apart from PDZK1, strong in vitro interactions of NHERF-1 were also observed with the solute transporters (excluding MAP17) and D-AKAP2. All identified proteins were immunolocalized in proximal tubular cells, wherein all membrane proteins co-localized with PDZK1 in brush borders. CONCLUSION: We hypothesize that PDZK1 and NHERF-1 establish an extended network beneath the apical membrane to which membrane proteins and regulatory components are anchored.

Asunto(s)

Túbulos Renales Proximales/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Animales , Glicosilfosfatidilinositoles/metabolismo , Inmunohistoquímica , Túbulos Renales Proximales/citología , Proteínas de la Membrana/genética , Ratones , Microvellosidades/metabolismo , Unión Proteica/fisiología , Intercambiadores de Sodio-Hidrógeno , Técnicas del Sistema de Dos Híbridos , Levaduras

RESUMEN

BACKGROUND: PDZK1, a multiple PDZ protein, was recently found to interact with the type IIa Na/Pi cotransporter (NaPi-IIa) in renal proximal tubular cells. In a preceding study, yeast two-hybrid screens using single PDZ domains of PDZK1 as baits were performed. Among the identified proteins, a C-terminal fragment of the dual-specific A-kinase anchoring protein 2 (D-AKAP2) was obtained by screening PDZ domain 4. METHODS: After its molecular cloning by means of RACE, the renal expression of D-AKAP2 was analyzed by real-time polymerase chain reaction (PCR) and immunohistochemistry. Protein interactions were characterized by overlays, pull-downs, and immunoprecipitations from transfected opossum kidney (OK) cells. RESULTS: Based on 5'-RACE and PDZK1 overlays of mouse kidney cortex separated by two-dimensional electrophoresis, it was suggested that the renal isoform of D-AKAP2 in mouse comprises 372 amino acids and exists as a protein of >40 kD. Immunohistochemistry and real-time PCR localized D-AKAP2 only to the subapical pole of proximal tubular cells in mouse kidney. In pull-down experiments, D-AKAP2 tightly bound PDZK1 as well as N+/H+ exchanger regulator factor (NHERF-1), but the latter with an apparent fourfold lower affinity. Similarly, His-tagged D-AKAP2 specifically retained PDZK1 from mouse kidney cortex homogenate. In addition, myc-tagged D-AKAP2 and HA-tagged PDZK1 co-immunoprecipitated from transfected OK cells. CONCLUSION: We conclude that D-AKAP2 anchors protein kinase A (PKA) to PDZK1 and to a lesser extent to NHERF-1. Since PDZK1 and NHERF-1 both sequester NaPi-IIa to the apical membrane, D-AKAP2 may play an important role in the parathyroid hormone (PTH)-mediated regulation of NaPi-IIa by compartmentalization of PKA.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Túbulos Renales Proximales/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Anclaje a la Quinasa A , Animales , Células Cultivadas , Clonación Molecular , Túbulos Renales Proximales/citología , Proteínas de la Membrana/genética , Ratones , Oocitos , Unión Proteica/fisiología , Xenopus laevis

RESUMEN

An essential role in phosphate homeostasis is played by Na/Pi cotransporter IIa that is localized in the brush borders of renal proximal tubular cells. Recent studies identified several PDZ proteins interacting with the COOH-terminal tail of NaPi-IIa, such as PDZK1 and NHERF-1. Here, by using yeast two-hybrid screen of mouse kidney cDNA library, we attempted to find proteins interacting with the NH2-terminal part of NaPi-IIa. We identified MAP17, a 17-kDa membrane protein that has been described to be associated with various human carcinomas, but it is also expressed in normal kidneys. Results obtained by various in vitro analyses suggested that MAP17 interacts with the fourth domain of PDZK1 but not with other PDZ proteins localized in proximal tubular brush borders. As revealed by immunofluorescence, MAP17 was abundant in S1 but almost absent in S3 segments. No alterations of the apical abundance of MAP17 were observed after maneuvers undertaken to change the content of NaPi-IIa (parathyroid hormone treatment, different phosphate diets). In agreement, no change in the amount of MAP17 mRNA was observed. Results obtained from transfection studies using opossum kidney cells indicated that the apical localization of MAP17 is independent of PDZK1 but that MAP17 is required for apical localization of PDZK1. In summary, we conclude that MAP17 1) interacts with PDZK1 only, 2) associates with the NH2 terminus of NaPi-IIa within the PDZK1/NaPi-IIa/MAP17 complex, and 3) acts as an apical anchoring site for PDZK1.

Asunto(s)

Túbulos Renales Proximales/metabolismo , Proteínas de la Membrana/fisiología , Simportadores/fisiología , Animales , Línea Celular , Túbulos Renales Proximales/citología , Masculino , Ratones , Ratones Endogámicos , Proteínas de Neoplasias , Zarigüeyas , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa

RESUMEN

Inorganic phosphate (P(i)) reabsorption in the renal proximal tubule occurs mostly via the Na(+)/P(i) cotransporter type IIa (NaP(i)-IIa) located in the brush-border membrane (BBM) and is regulated, among other factors, by dietary P(i) intake and parathyroid hormone (PTH). The PTH-induced inhibition of P(i) reabsorption is mediated by endocytosis of Na/P(i)-IIa from the BBM and subsequent lysosomal degradation. Megalin is involved in receptor-mediated endocytosis of proteins from the urine in the renal proximal tubule. The recently identified receptor-associated protein (RAP) is a novel type of chaperone responsible for the intracellular transport of endocytotic receptors such as megalin. Gene disruption of RAP leads to a decrease of megalin in the BBM and to a disturbed proximal tubular endocytotic machinery. Here we investigated whether the distribution of NaP(i)-IIa and/or its regulation by dietary P(i) intake and PTH is affected in the proximal tubules of RAP-deficient mice as a model for megalin loss. In RAP-deficient mice megalin expression was strongly reduced and restricted to a subapical localization. NaP(i)-IIa protein distribution and abundance in the kidney was not altered. The localization and abundance of the NaP(i)-IIa interacting proteins MAP17, PDZK-1, D-AKAP2, and NHE-RF1 were also normal. Other transport proteins expressed in the BBM such as the Na(+)/H(+) exchanger NHE-3 and the Na(+)/sulphate cotransporter NaSi were normally expressed. In whole animals and in isolated fresh kidney slices the PTH-induced internalization of NaP(i)-IIa was strongly delayed in RAP-deficient mice. PTH receptor expression in the proximal tubule was not affected by the RAP knock-out. cAMP, cGMP or PKC activators induced internalization which was delayed in RAP-deficient mice. In contrast, both wildtype and RAP-deficient mice were able to adapt to high-, normal, and low-P(i) diets appropriately as indicated by urinary P(i) excretion and NaP(i)-IIa protein abundance.

Asunto(s)

Proteína Asociada a Proteínas Relacionadas con Receptor de LDL/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Hormona Paratiroidea/farmacología , Simportadores/genética , Animales , Regulación hacia Abajo/efectos de los fármacos , Endocitosis/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvellosidades/metabolismo , Fósforo Dietético/farmacología , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa , Simportadores/metabolismo