At the brush borders of renal proximal tubules the bulk of filtered solutes is reabsorbed. Among the many solute transporters localized in this cellular domain the Na-phosphate cotransporter NaPi-lla is of particular interest because its abundance in the brush borders is regulated according to the body needs. In order to elucidate the mechanisms which are involved in the specific endocytosis of NaPi-lla we searched for proteins which interact with NaPi-lla by the yeast two-hybrid method. A screen against the C-terminus of NaPi-lla resulted in several clones coding for the PDZ protein PDKZ1. As PDZK1 encompasses four PDZ domains in tandem each single PDZ domain was used as a bait for further yeast two-hybrid screens. Besides NaPi-lla, the following transporters were identified to interact with PDZK1: the chloride-formate exchanger CFEX, the urate-anion exchanger URAT1 and the organic cation transporter OCTN1. In addition, the PK-A anchoring protein D-AKAP2 was identified to interact with PDZK1.
All interactions were confirmed by different in-vitro binding assays such as pull downs and gel overlay techniques. In-vitro binding studies were performed also with NHERF1 (regulatory factor for the Na/H-exchanger NHE-3) in order to establish an eventual specifity of the PDZ-transporter intreactions. Interestingly, all above mentioned proteins also interacted with NHERF1.
By immunofluorescence, colocalization of PDKZ1 (and NHERF1) with mentioned solute transporters was observed in the brush borders of proximal cells. Similarly, D-AKAP2 was localized in the brush borders but also in the subapical compartment.
Based on these findings we suggest that in the microvilli of proximal tubular cells, PDZK1 and NHERF1 form a network of PDZ domains to which some of the regulated and non-regulated membrane transporters are bound and to which regulatory components such as PK-A are anchored. We propose that interactions of the different transporters to this network is important for apical sorting and/or positioning. In the case of the hormonal regulation of NaPi-lla it is assumed that its interaction with PDZK1 and NHERF1 is of dynamic nature and specifically regulated.