The type IIa Na ؉ -dependent inorganic phosphate (Na/ P i ) cotransporter is localized in the apical membrane of proximal tubular cells and is regulated by an endocytotic pathway. Because molecular processes such as apical sorting, internalization, or subsequent degradation might be assisted by associated proteins, a yeast twohybrid screen against the C-terminal, cytosolic tail of type IIa cotransporter was designed. Most of the potential proteins found belonged to proteins with multiple PDZ modules and were either identical/related to PDZK1 or identical to NHERF-1. Yeast trap truncation assays confined the peptide-protein association to the C-terminal amino acid residues TRL of type IIa cotransporter and to single PDZ domains of each identified protein, respectively. The specificity of these interactions were confirmed in yeast by testing other apical localized transmembraneous proteins. Moreover, the type IIa protein was recovered in vitro by glutathione S-transferase-fused PDZ proteins from isolated renal brush border membranes or from type IIa-expressing oocytes. Further, these PDZ proteins are immunohistochemically detected either in the microvilli or in the subapical compartment of proximal tubular cells. Our results suggest that the type IIa Na/P i cotransporter interacts with various PDZ proteins that might be responsible for the apical sorting, parathyroid hormone controlled endocytosis or the lysosomal sorting of internalized type IIa cotransporter.In kidney, reabsorption of filtered inorganic phosphate (P i ) takes place along the proximal tubules and is controlled by a variety of hormones (e.g. parathyroid hormone, PTH) 1 and other factors (e.g. dietary intake of P i ) (1, 2). Three structurally unrelated sodium-dependent phosphate (Na/P i ) cotransporter families have been identified (1, 3). By immunohistochemistry, it was apparent that members of the type I and the type IIa Na/P i cotransporters are located in the apical membrane of proximal tubular cells (4, 5). Targeted inactivation of the type IIa Na/P i cotransporter gene (npt2) provided strong evidence that ϳ70% of Na-dependent P i transport across the brush border membrane is mediated by the type IIa Na/P i cotransporter (6). Furthermore, the type IIa cotransporter represents the major target for the many factors described to regulate proximal tubular P i reabsorption (2). Additionally, reduced proximal P i -reabsorption, as observed in X-linked hypophosphatemia, is due to a decreased abundance of the type IIa Na/P i cotransporter (7).According to the current mechanistic view, inhibition of proximal tubular Pi-reabsorption, such as by PTH or by a diet of high P i content (acutely given), is achieved by a removal of type IIa cotransporters (2) from the apical membrane. Results obtained from in vivo (rats) and in vitro (OK cells) studies indicated that internalized type IIa Na/P i cotransporters are subjected to degradation in the lysosomes (8, 9). Besides Na/P i cotransport, the activity of the brush border Na/H exchanger, NHE-3, is regulated ...

We hypothesize that PDZK1 and NHERF-1 establish an extended network beneath the apical membrane to which membrane proteins and regulatory components are anchored.

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...