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Ezrin promotes functional expression and parathyroid hormone-mediated regulation of the sodium-phosphate cotransporter 2a in LLC-PK1 cells

Department of Medicine, Harvard Medical School, and Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts

Submitted 15 June 2007 ; accepted in final form 7 January 2008

The sodium-phosphate cotransporter 2a (NPT2a) is the principal phosphate transporter expressed in the brush border of renal proximal tubules and is downregulated by parathyroid hormone (PTH) through an endocytic mechanism. Apical membrane expression of NPT2a is dependent on interactions with the sodium-hydrogen exchanger regulatory factor 1 (NHERF-1). An LLC-PK1 renal cell line stably expressing the PTH receptor (PTH1R) and NHERF-1, termed B28-N1, fails to functionally express NPT2a. In B28-N1 cells, NHERF-1 and NPT2a are inappropriately localized to the cytoplasm. Ezrin, in the activated state, is capable at linking NHERF-1-assembled complexes to the actin cytoskeleton. Early-passage LLC-PK1 cells stably transfected with either empty vector or wild-type ezrin express a comparable level of the active, T567 phosphorylated form of ezrin and are capable of functionally expressing NPT2a. Colocalization of the PTH1R, NPT2a, and ezrin exists and is prominently associated with actin-containing microvilli in apical domains of these cells. Upon PTH treatment, the PTH1R, NPT2a, NHERF-1, and ezrin colocalize to endocytic vesicles and NPT2a-dependent phosphate uptake is markedly inhibited. LLC-PK1 cells expressing the constitutively active ezrin (T567D) display enhanced NPT2a functional expression and PTH-mediated regulation of phosphate. Expression of a dominant-negative ezrin, consisting of the NH₂-terminal half of the protein, markedly disrupts NPT2a-dependent phosphate uptake. PTH does not appear to alter ezrin phosphorylation at T567. Instead, PTH perhaps initiates NPT2a endocytosis by inducing reorganization of the actin-containing microvilli in a process that is blocked by the actin-stabilizing compound jasplakinolide.

proximal tubule; hormonal regulation