GABARAP deficiency modulates expression of NaPi-IIa in renal brush-border membranes

1 Institute of Physiology and Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland; 2 Department of Internal Medicine and 3 Charles and Jane Pak Center of Mineral Metabolism, University of Texas Southwestern Medical Center, Dallas, Texas; and 4 Department of Neurochemistry, Max Planck Institute for Brain Research, Frankfurt am Main, Germany Submitted 14 August 2008 ; accepted in final form 13 February 2009 Renal reabsorption of inorganic phosphate (P i ) is mainly mediated by the Na + -dependent P i -cotransporter NaPi-IIa that is expressed in the brush-border membrane (BBM) of renal proximal tubules. Regulation and apical expression of NaPi-IIa are known to depend on a network of interacting proteins. Most of the interacting partners identified so far associate with the COOH-terminal PDZ-binding motif (TRL) of NaPi-IIa. In this study GABA A receptor-associated protein (GABARAP) was identified as a novel interacting partner of NaPi-IIa applying a membrane yeast-two-hybrid system (MYTH 2.0) to screen a mouse kidney library with the TRL-truncated cotransporter as bait. GABARAP mRNA and protein are present in renal tubules, and the interaction of NaPi-IIa and GABARAP was confirmed by using glutathione S -transferase pulldowns from BBM and coimmunoprecipitations from transfected HEK293 cells. Amino acids 36–68 of GABARAP were identified as the determinant for the described interaction. The in vivo effects of this interaction were studied in a murine model. GABARAP –/– mice have reduced urinary excretion of P i , higher Na + -dependent 32 P i uptake in BBM vesicles, and increased expression of NaPi-IIa in renal BBM compared with GABARAP +/+ mice. The expression of Na + /H + exchanger regulatory factor (NHERF)1, an important scaffold for the apical expression of NaPi-IIa, is also increased in GABARAP –/– mice. The absence of GABARAP does not interfere with the regulation of the cotransporter by either parathyroid hormone or acute changes of dietary P i content. epithelial transport; renal proximal tubules; phosphate homeostasis Address for reprint requests and other correspondence: N. Hernando, Institute of Physiology and Zurich Center for Integrative Human Physiology (ZIHP), Univ. of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland (e-mail: hernando{at}physiol.uzh.ch )