Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, C131

Oral Communications

Megalin binds to NHERF1 and NHERF2 scaffold proteins

D. Hryciw1, C. Slattery2, K. Jenkin1, P. Poronnik3

1. School of Biomedical and Health Sciences, Victoria University, Melbourne, Victoria, Australia. 2. UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, Dublin, Ireland. 3. School of Medical Sciences, Royal Melbourne Institute of Technology, Bundoora, Victoria, Australia.


Albumin endocytosis in the renal proximal tubule is regulated by a number of transmembrane and accessory proteins including the scavenger receptor megalin. Previously we have demonstrated an essential role for the scaffold proteins NHERF1 and NHERF2 in albumin uptake. NHERF1 and NHERF2 are PDZ domain containing proteins that interact with specific sequences that form a PDZ binding domain (S/TXXΦ) in the C-terminus of proteins. Interestingly, megalin contains a functional PDZ binding domain (SDV), however the interaction with the scaffold proteins NHERF1 and NHERF2 has not been investigated. In this study we investigated if there is an interaction between megalin and NHERF1 and NHERF2, and then characterized the specific domains required for this interaction. Firstly, we investigated if the proteins co-localize in a proximal tubule cell model, the opossum kidney (OK) cell line. Confocal analysis of OK cells demonstrated that the distribution of megalin was predominantly apical with some cytosolic localization. Importantly, NHERF1 had a strong apical localization which overlapped with megalin. Further, as previously described (Hryciw et al, 2006) NHERF2 was predominantly cytosolic, and this protein co-localized with megalin in this region. This indicated that the proteins had an overlap in distribution in proximal tubule cells. Further, immunoprecipitation experiments were performed using anti-megalin, anti-NHERF1 and anti-NHERF2 antibodies that were incubated with rat kidney lysate. The immunoprecipitates were analysed by Western blot analysis using the anti-NHERF1 and anti-NHERF2 and anti-megalin antibodies, respectively. These studies clearly indicated that megalin bound to NHERF1 and NHERF2 in vivo. To determine which domains in NHERF1 and NHERF2 were required for this interaction, GST fusion proteins were generated as described previously (Hryciw et al. 2006; Lee et al 2007). These fusion proteins included the full length NHERF proteins as well as their 2 PDZ domains (PDZ1 and PDZ2) and C-terminal ezrin binding domain. Incubation with rat kidney lysate and analysis by Western blot analysis indicated that megalin bound to PDZ2 of NHERF1 and PDZ2 and the C-terminal ezrin binding domain of NHERF2. Finally, we used fusion proteins to determine if the C-terminus of megalin was the site of interaction with NHERF1 and NHERF2. GST-pull down experiments and rat kidney lysate supported this. Therefore, we have described for the first time an interaction between megalin and the scaffold proteins NHERF1 and NHERF2. As the NHERF proteins have been shown to be required for the formation of macromolecular complexes in other cell systems, as well as binding to NHE3 and ClC-5 that are essential transmembrane proteins required for endocytosis, further investigation should determine if the a complex is required for albumin endocytosis in proximal tubule cells.

Where applicable, experiments conform with Society ethical requirements