CGMP Inhibition of Na+/H+ Antiporter 3 (NHE3) Requires PDZ Domain Adapter NHERF2, a Broad Specificity Protein Kinase G-anchoring Protein

Boyoung Cha,Jae Ho Kim,Hans Hut,Boris M Hogema,Janani Nadarja,Mirza Zizak,Megan Cavet,Whaseon Lee-Kwon,Suzanne M Lohmann,Albert Smolenski,Chung Ming Tse,Chris Yun,Hugo R De Jonge,Mark Donowitz

doi:10.1074/jbc.m500505200

Abstract

Electroneutral NaCl absorption mediated by Na+/H+ exchanger 3 (NHE3) is important in intestinal and renal functions related to water/Na+ homeostasis. cGMP inhibits NHE3 in intact epithelia. However, unexpectedly it failed to inhibit NHE3 stably transfected in PS120 cells, even upon co-expression of cGMP-dependent protein kinase type II (cGKII). Additional co-expression of NHERF2, the tandem PDZ domain adapter protein involved in cAMP inhibition of NHE3, restored cGMP as well as cAMP inhibition, whereas NHERF1 solely restored cAMP inhibition. In vitro conditions were identified in which NHERF2 but not NHERF1 bound cGKII. The NHERF2 PDZ2 C terminus, which binds NHE3, also bound cGKII. A non-myristoylated mutant of cGKII did not support cGMP inhibition of NHE3. Although cGKI also bound NHERF2 in vitro, it did not evoke inhibition of NHE3 unless a myristoylation site was added. These results show that NHERF2, acting as a novel protein kinase G-anchoring protein, is required for cGMP inhibition of NHE3 and that cGKII must be bound both to the plasma membrane by its myristoyl anchor and to NHERF2 to inhibit NHE3.

Highlights

The rapid elevation of intestinal cAMP and cGMP levels by activation of adenylate cyclase and guanylate cyclase, respec
CGMP Inhibits Na؉/H؉ exchanger 3 (NHE3) in PS120 Cells, an Effect Requiring NHERF2 Plus cGMP-dependent protein kinase type II (cGKII)—Earlier studies have indicated that NHERF1 or NHERF2 is necessary for second messenger regulation of NHE3 [1, 2, 13, 16]
The amount of cGKII expression was estimated by Western analysis, and infection efficiency was estimated by determining the percent of infected cells displaying immunofluorescence when labeled with anti-cGKII antibody/Alexa 488-labeled goat anti-rabbit secondary antibody. cGKII was absent from PS120 cells and from cells infected with adenovirus empty vector, but was present in cells infected ϳ48 h earlier with adenovirus containing cGKII (Fig. 1A)

Summary

Introduction

The rapid elevation of intestinal cAMP and cGMP levels by activation of adenylate cyclase and guanylate cyclase, respec-. Some details of the mechanisms of acute regulation of intestinal NaCl absorption by cAMP are understood. Hormones such as vasoactive intestinal peptide or secretin and enterotoxins such as cholera toxin activate adenylate cyclase and increase cellular cAMP content. NHERF1/ NHERF2 each contain two homologous PDZ domains (PDZ1 and PDZ2) and an ERM (ezrin-radixin-moesin) binding domain, which anchors NHERF and its binding partners to the actin cytoskeleton via NHERF binding to ezrin Ezrin binds both NHERF1/NHERF2 and PKAII and acts as a low affinity cAMP kinase-anchoring protein (AKAP), positioning PKAII so it can phosphorylate NHE3, which is required for cAMP inhibition of NHE3 [1, 6, 7]. From the consequences of activating guanylate cyclase-C/ cGMP/cGKII from the apical surface, elevating cGMP from the serosal side through the angiotensin-nitric oxide/GC-S signaling pathway stimulated rather than inhibited intestinal NaCl absorption [11]. The cell type initially affected by the latter signaling pathway has not been defined

Methods

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Conclusion