Identification of Sites Required for Down-regulation of Na+/H+ Exchanger NHE3 Activity by cAMP-dependent Protein Kinase

Na؉ /H ؉ exchanger 3 (NHE3) plays a pivotal role in transepithelial Na ؉ and HCO 3 ؊ absorption across a wide range of epithelia in the digestive and renal-genitourinary systems. Accumulating evidence suggests that PDZ-based adaptor proteins play an important role in regulating the trafficking and activity of NHE3. A search for NHE3-binding modular proteins using yeast two-hybrid assays led us to the PDZ-based adaptor Shank2. The interaction between Shank2 and NHE3 was further confirmed by immunoprecipitation and surface plasmon resonance studies. When expressed in PS120/ NHE3 cells, Shank2 increased the membrane expression and basal activity of NHE3 and attenuated the cAMP-dependent inhibition of NHE3 activity. Furthermore, knock-down of native Shank2 expression in Caco-2 epithelial cells by RNA interference decreased NHE3 protein expression as well as activity but amplified the inhibitory effect of cAMP on NHE3. These results indicate that Shank2 is a novel NHE3 interacting protein that is involved in the fine regulation of transepithelial salt and water transport through affecting NHE3 expression and activity.Maintenance of intracellular and systemic pH, Na ϩ concentration, and fluid volume is essential for maintaining the physiological status in cells and whole organisms (1, 2). First demonstrated almost 30 years ago (3), members of the mammalian Na ϩ /H ϩ exchanger (NHE) 2 family participate in the regulation of these parameters at both cellular and systemic levels. To date, nine NHE family members have been identified in mammalian cells with unique tissue distribution and functional properties (2). As a better characterized isoform, NHE3 is primarily found in the apical membrane of epithelial cells of the renal and gastrointestinal tracts, where it mediates transepithelial absorption of Na ϩ and HCO 3 Ϫ (2, 4). Lack of NHE3 activity impairs acid-base balance and extracellular fluid volume homeostasis (5).NHE3 is known to be regulated by a large variety of hormones, such as ␣-and ␤-adrenergic agonists, dopamine, parathyroid hormone, and angiotensin II via multiple signaling systems (6, 7), but the exact underlying mechanisms are still only partially understood. Nevertheless, it has been known for many years that acute regulation of NHE3 activity is linked to protein phosphorylation, as in the case of inhibition by cAMPdependent protein kinase A (PKA) (7,8). Subsequently, it has been demonstrated that adaptor proteins with PDZ (PSD-95/discs large/ ZO-1) domains play an important role in the cAMP-dependent inhibition of NHE3 in a number of systems (7, 9, 10). For example, EBP50 (also known as NHERF1) and E3KARP (also known as NHERF2, SIP-1, or TKA-1) were found to be necessary modular proteins that participated in the cAMP-dependent PKA phosphorylation of NHE3 by forming a multiprotein signaling complex (11,12).In recent years, there has been a growing interest in PDZ domains and modular proteins having PDZ domains. Best studied in the post-synaptic density (PSD) region of neurons, PDZ domain proteins have em...

Section: Discussionmentioning

confidence: 99%

Shank2 Associates with and Regulates Na+/H+ Exchanger 3

Han

Kim

et al. 2006

“…For example, it is possible that the association of NHE3 with megalin causes inactivation of transport activity. However, it is known that phosphorylation of NHE3 by protein kinase A causes inhibition of NHE3 activity (36,37). It is possible that phosphorylation of NHE3 causes both its inactivation and its association with megalin.…”

Section: Fig 10 Namentioning

confidence: 99%

Active (9.6 S) and Inactive (21 S) Oligomers of NHE3 in Microdomains of the Renal Brush Border

Biemesderfer

DeGray

Aronson

2001

We have previously shown that Na ؉ -H ؉ exchanger isoform NHE3 exists as both 9.6 and 21 S (megalin-associated) oligomers in the renal brush border (1). To characterize the oligomeric forms of the renal brush border Na ؉ -H ؉ exchanger in more detail, we performed membrane fractionation studies. We found that similar amounts of NHE3 were present in microvilli and a nonmicrovillar membrane domain of high density (dense vesicles). Horseradish peroxidase-labeled endosomes were not prevalent in the dense membrane fraction. However, megalin, which localizes primarily to the intermicrovillar microdomain of the brush border, was enriched in the dense vesicles, implicating this microdomain as the likely source of these membranes. Immunolocalization of NHE3 confirmed that a major fraction of the transporter colocalized with megalin in the intermicrovillar region of the brush border. Immunoprecipitation studies demonstrated that in microvilli the majority of NHE3 was not bound to megalin, while in the dense vesicles most of the NHE3 coprecipitated with megalin. Moreover, sucrose velocity gradient centrifugation experiments revealed that most NHE3 in microvilli sedimented with an S value of 9.6, while the S value of NHE3 in dense vesicles was 21. Finally, we examined the functional state of NHE3 in both membrane fractions. As assayed by changes in acridine orange fluorescence, imposing an outwardly directed Na ؉ gradient caused generation of an inside acid pH gradient in the microvilli, indicating Na ؉ -H ؉ exchange activity, but not in the dense vesicles. Taken together, these data demonstrate that renal brush border NHE3 exists in two oligomeric states: a 9.6 S active form present in microvilli and a 21 S, megalin-associated, inactive form in the intermicrovillar microdomain of the apical plasma membrane. Thus, regulation of renal brush border Na ؉ -H ؉ exchange activity may be mediated by shifting the distribution between these forms of NHE3.In the kidney, the activity of the Na ϩ -H ϩ exchanger isoform NHE3, located on the apical microvillar membrane of the proximal tubule, plays a major role in mediating transepithelial bicarbonate and NaCl reabsorption (2-4). Numerous physiologic studies of brush border Na ϩ -H ϩ exchange have shown that this activity is regulated by such hormones as angiotensin II (5) and parathyroid hormone (6) as well as by systemic alterations in acid-base balance (2, 3). Several laboratories have presented evidence suggesting that NHE3 is regulated by posttranslational mechanisms that may include membrane trafficking between an intracellular compartment and the plasma membrane (7-9). Such models predict that NHE3 must be localized in a nonmicrovillar membrane compartment, which functions as a store of transporter, as well as on the microvillar membrane where NHE3 is active.We have recently reported an association between NHE3 and the putative scavenger receptor megalin in renal brush border membranes (1). Moreover, we found that renal brush border NHE3 exists in two states with distinct sediment...

“…We recently have shown that A 1 R-mediated inactivation of NHE3 is dependent on protein kinase C (PKC) (7,9). Phosphorylation of NHE3 is an early event in regulation of NHE3 by protein kinases (36,37,55,56). Elimination of phosphoserines on NHE3 may disrupt CPA regulation of NHE3 activity.…”

Section: Identification Of Structural Elements Required For Downregulmentioning

confidence: 99%

Acute Regulation of Na/H Exchanger NHE3 by Adenosine A1 Receptors Is Mediated by Calcineurin Homologous Protein

Sole

Cerull

Babich

et al. 2004