Parathyroid hormone treatment induces dissociation of type IIa Na+-Pi cotransporter-Na+/H+ exchanger regulatory factor-1 complexes

Institutes of 1 Physiology and 2 Anatomy, Zurich University, Zurich, Switzerland; and 3 Genzyme Corporation, Framingham, Massachusetts Submitted 15 September 2004 ; accepted in final form 14 February 2005 The type IIa Na + -P i cotransporter (NaP i -IIa) and the Na + /H + exchanger regulatory factor-1 (NHERF1) colocalize in the apical membrane of proximal tubular cells. Both proteins interact in vitro. Herein the interaction between NaP i -IIa and NHERF1 is further documented on the basis of coimmunoprecipitation and co-pull-down assays. NaP i -IIa is endocytosed and degraded in lysosomes upon parathyroid hormone (PTH) treatment. To investigate the effect of PTH on the NaP i -IIa-NHERF1 association, we first compared the localization of both proteins after PTH treatment. In mouse proximal tubules and OK cells, NaP i -IIa was removed from the apical membrane after hormonal treatment; however, NHERF1 remained at the membrane. Moreover, PTH treatment led to degradation of NaP i -IIa without changes in the amount of NHERF1. The effect of PTH on the NaP i -IIa-NHERF1 interaction was further studied using coimmunoprecipitation. PTH treatment reduced the amount of NaP i -IIa coimmunoprecipitated with NHERF antibodies. PTH-induced internalization of NaP i -IIa requires PKA and PKC; therefore, we next analyzed whether PTH induces changes in the phosphorylation state of either partner. NHERF1 was constitutively phosphorylated. Moreover, in mouse kidney slices, PTH induced an increase in NHERF1 phosphorylation; independent activation of PKA or PKC also resulted in increased phosphorylation of NHERF1 in kidney slices. However, NaP i -IIa was not phosphorylated either basally or after exposure to PTH. Our study supports an interaction between NHERF1 and NaP i -IIa on the basis of their brush-border membrane colocalization and in vitro coimmunoprecipitation/co-pull-down assays. Furthermore, PTH weakens this interaction as evidenced by different in situ and in vivo behavior. The PTH effect takes place in the presence of increased phosphorylation of NHERF1. proximal tubule; opossum kidney cells; phosphorylation; endocytosis Address for reprint requests and other correspondence: N. Hernando, Physiologisches Institut, Universität Zürich-Irchel, Wintherthurerstrasse 190, CH-8057 Zurich, Switzerland (e-mail: hernando{at}physiol.unizh.ch )