NHERF2 is necessary for basal activity, second messenger inhibition, and LPA stimulation of NHE3 in mouse distal ileum

Abstract

ϩ exchanger (NHE) regulatory factor 2 (NHERF2) is necessary for multiple aspects of acute regulation of NHE3 in intact mouse small intestine, distal ileal NHE3 activity was determined using two-photon microscopy/SNARF-4F in a NHERF2-null mouse model. The NHERF2-null mouse ileum had shorter villi, deeper crypts, and decreased epithelial cell number. Basal rates of NHE3 activity were reduced in NHERF2-null mice, which was associated with a reduced percentage of NHE3 in the apical domain and an increase in intracellular NHE3 amount but no change in total level of NHE3 protein. cAMP, cGMP, and elevated Ca 2ϩ due to apical exposure to UTP all inhibited NHE3 activity in wild-type mouse ileum but not in NHERF2-null mice, while inhibition by hyperosmolarity occurred normally. The cAMP-increased phosphorylation of NHE3 at aa 552; levels of PKAII␣ and cGMP-dependent protein kinase II (cGKII); and elevation of Ca 2ϩ were similar in wild-type and NHERF2-null mouse ileum. Luminal lysophosphatidic acid (LPA) stimulated NHE3 in wild-type but not in NHERF2-null ileum. In conclusion, 1) there are subtle structural abnormalities in the small intestine of NHERF2-null mouse which include fewer villus epithelial cells; 2) the decreased basal NHE3 activity and reduced brush border NHE3 amount in NHERF2-null mice show that NHERF2 is necessary for normal basal trafficking or retention of NHE3 in the apical domain; 3) hyperosmolar inhibition of NHE3 occurs similarly in wild-type and NHERF2-null ileum, demonstrating that some inhibitory mechanisms of NHE3 are not NHERF2 dependent; 4) cAMP inhibition of NHE3 is NHERF2 dependent at a step downstream of cAMP/PKAII phosphorylation of NHE3 at aa 552; 5) cGMP-and UTP-induced inhibition of NHE3 are NHERF2 dependent at steps beyond cGKII and the UTP-induced increase of intracellular Ca 2ϩ ; and 6) LPA stimulation of NHE3 is also NHERF2 dependent. Ca 2ϩ ; adenosine 3=,5=-cyclic monophosphate; guanosine 3=,5=-cyclic monophosphate; trafficking; Na ϩ /H ϩ exchanger regulation; Na ϩ /H ϩ exchanger regulatory factor; lysophosphatidic acid THE BRUSH BORDER sodium/hydrogen exchanger, NHE3, belongs to the SLC9A gene family of proteins and is predominantly expressed in the apical surface of small and large intestine and proximal tubules of the kidney where it accounts for the majority of intestinal and renal Na ϩ absorption (39). NHE3 activity is highly regulated. Much of this regulation involves large signaling complexes which NHE3 forms with multiple binding partners, some of which bind to the intracellular COOH-terminal ϳ377 aa of NHE3 (12, 14, 15). The Na ϩ /H ϩ exchanger regulatory factor (NHERF) family of multi-PDZ domain containing proteins (NHERFs 1-4) are scaffolds that bind to the middle of the intracellular COOH terminus of NHE3 and form some of the signaling complexes that regulate NHE3 (12-14, 36, 38). The NHERF proteins are present in the apical domain of intestinal and renal proximal tubule Na-absorptive cells and bind multiple proteins in addition to NHE3. These scaffold proteins contain t...

Role of epithelial ion transports in inflammatory bowel disease

“…One study found NHERF2 Ϫ/Ϫ mice to have lower ileal NHE3 activity due to impaired trafficking or retention of NHE3 in the apical membrane (99), which is possibly related to NHERF2 association with lipid-raft NHE3 (136). In fact, lysophosphatidic acid (LPA, released in inflammatory settings to repair wounded tissue) stimulates NHE3 activity and expression through NHERF2-dependent mechanisms in ileum (99) and kidney brush border (27, 84). In contrast, Chen et al…”

“…Conflicting results aside, it is clear that NHERF2 is important for NHE3 regulation in IT, including cGMP-and Ca 2ϩ -dependent NHE3 inhibition (99). In turn, PDZK1 is required for cAMP-and Ca 2ϩ -dependent NHE3 inhibition (30), and its deletion in rodents results in decreased colonic and jejunal electroneutral sodium absorption (30, 57).…”

Traditional and emerging roles for the SLC9 Na+/H+ exchangers

“…Recently significant insights into how the NHERFs and ezrin affect NHE3 activity, the mechanisms underlying angiotensin II mediated activation of the exchanger, the role of SGK signalling and NHE3 lipid interactions on activity have been made [1,149,30,8,9,184,112,68,117,64].…”

Regulation of G Protein-Coupled Receptor Function by Na+/H+ Exchange Regulatory Factors

“…(Broere et al, 2007;Cunningham et al, 2008). Recent results indicate morphological changes of the intestine of NHERF2-null mice with shorter villi, deeper crypts, and decreased epithelial cell number Murtazina et al, 2011). NHERF2-NHERF EFFECTS ON GPCR FUNCTION 893 null mice display augmented forskolin-stimulated intestinal HCO 3 Ϫ secretion and are refractory to the inhibitory action of lysophosphatidic acid on forskolin-stimulated HCO 3 Ϫ secretion ).…”