PDZ-domain interactions and apical expression of type IIa Na/P _i cotransporters

Abstract

Type IIa Na͞Pi cotransporters are expressed in renal proximal brush border and are the major determinants of inorganic phosphate (Pi) reabsorption. Their carboxyl-terminal tail contains information for apical expression, and interacts by means of its three terminal amino acids with several PSD95͞DglA͞ZO-1-like domain (PDZ)-containing proteins. Two of these proteins, NaPi-Cap1 and Na͞H exchanger-regulatory factor 1 (NHERF1), colocalize with the cotransporter in the proximal brush border. We used opossum kidney cells to test the hypothesis of a potential role of PDZ-interactions on the apical expression of the cotransporter. We found that opossum kidney cells contain NaPi-Cap1 and NHERF1 mRNAs. For NHERF1, an apical location of the protein could be documented; this location probably reflects interaction with the cytoskeleton by means of the MERM-binding domain. Overexpression of PDZ domains involved in interaction with the cotransporter (PDZ-1͞ NHERF1 and PDZ-3͞NaPi-Cap1) had a dominant-negative effect, disturbing the apical expression of the cotransporter without affecting the actin cytoskeleton or the basolateral expression of Na͞K-ATPase. These data suggest an involvement of PDZ-interactions on the apical expression of type IIa cotransporters.opossum kidney cells ͉ Na͞H exchanger-regulatory factor 1 ͉ proximal tubules P roximal tubular reabsorption of inorganic phosphate (P i ) plays a key role in P i metabolism (1, 2). Up to 80% of the renal reabsorption of P i is mediated by the brush border membrane (BBM)-associated type IIa Na͞P i -cotransporters (NaPi IIa; refs. 3 and 4; for review, see ref.2). According to their key physiological role, these cotransporters are up-regulated by factors that stimulate renal reabsorption of P i (ref. 5; for review see ref.2), whereas they are down-regulated by phosphaturic factors (refs. 6 and 7; for review, see ref. 2). Their expression is also affected in pathological states associated with P i wasting, such as X-linked hypophosphatemic rickets: a primary defect on the PHEX gene leads by a yet-unknown mechanism to a reduced expression of NaPi IIa (8, 9). Many of the proximal tubular characteristics in terms of P i handling are retained in a cell line derived from opossum kidney (OK) cells. These OK cells contain an endogenous NaPi IIa cotransporter (NaPi4) apically located and regulated by the same hormones and factors as NaPi IIa cotransporters in proximal tubules (6,10,11).NaPi IIa cotransporters are predicted to contain eight transmembrane domains with intracellular N-and C-terminal tails (12). The C-terminal tail contains two signals involved in apical expression: a terminal PSD95͞DglA͞ZO-1-like domain (PDZ)-binding motif (TRL) and an internal determinant (13). The C-terminal tail interacts, by means of TRL residues, with several PDZ-containing proteins, among them NaPi-Cap1 and Na͞H exchanger-regulatory factor 1 (NHERF1; ref. 14). Similar to NaPi IIa, both proteins are located on the BBM of proximal tubules (14, 15). NaPi-Cap1 is a protein of about 500 residues that co...

Role of PDZK1 Protein in Apical Membrane Expression of Renal Sodium-coupled Phosphate Transporters

“…S7). Both transporters showed robust interaction with PDZK1 in accordance with previous studies that used diverse biochemical techniques (12)(13)(14)(15).…”

“…For example, it is well known that NaPi-2a is integrated in a series of macromolecular complexes whose architecture is based on PDZ (PSD-95, discs-large, and ZO-1) protein interactions (12)(13)(14)(15)(16)(17)(18)(19). NaPi-2a participates in this complex by means of a class I PDZ-binding site located at its C terminus, comprising its last three amino acids (TRL).…”

The Role of the C Terminus and Na+/H+ Exchanger Regulatory Factor in the Functional Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Nonpolarized Cells and Epithelia

“…The D1 and D2 domains bind with nanomolar affinity to the PDZbinding motifs of CFTR ( 1477 DTRL) and to the C terminus of other transport proteins, signaling molecules, and receptors (18,(22)(23)(24)(25)(26). The ERM-binding domain tethers the complex via ezrin to cytoskeletal elements in a phosphorylation-dependent manner and to the catalytic and regulatory subunit of PKA (19,(27)(28)(29).…”

Disorders of Renal Tubular Phosphate Transport

“…Furthermore, NaPi-Cap1 and NHERF-1 may be involved in the interaction of type IIa protein with the cytoskeleton (S. Gisler, S. Pribanic, H. Murer, J. Biber; submitted manuscript). Experiments in OK cells have suggested a role for NHERF-1 and NaPi-Cap1 in apical positioning of the type IIa Na/P i cotransporter (48), and a similar conclusion was reached from studies in mice homozygous for the disrupted NHERF-1 gene (49). Thus, these type IIa interacting proteins might build a local regulatory complex and provide regulatory specificity.…”