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

Abstract

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...

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