A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.

The Na+/H+ exchanger regulatory factor (NHERF) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of Na+/H+ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the beta2 receptor. Mutagenesis studies of the beta2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the beta2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling

The Role of the NHERF-1 and NHERF-2 Adapter Proteins in Intestinal Ion Transport Regulation

The chloride channel CFTR (cystic fibrosis transmembrane conductance regulator) and the sodium/proton exchanger NHE3 are key proteins involved in transepithelial ion and water transport in several epithelial tissues, including the intestine. In this thesis we mainly focus on the role of the NHERF (NHE3 Regulatory Factor) proteins as regulators of CFTR and NHE3 activity. In chapter 2 we show that cAMP- and cGMP-dependent activation of CFTR was moderately reduced in the duodenum and jejunum (but not in ileum) of NHERF-1 deficient mice. This reduced activation of CFTR in may be explained by the ~30% reduced local abundance of CFTR protein in the jejunal crypts of the NHERF-1 deficient mice. As shown in chapter 3, the basal NHE3 activity was decreased in the jejunum and colon of NHERF-1 null mice. In addition, NHERF-1 deficient mice displayed a reduced sodium absorption in the jejunum. These observations may be explained by the reduced NHE3 protein level in the brush border membrane fraction of jejunum and colon of these mice. The cAMP- and/or cGMP dependent inhibition of NHE3 was not appreciably affected in NHERF-1 deficient mice. As described in chapter 4, NHERF-1 and/or NHERF-2 gene knockdown in polarized epithelial cells indicated that NHERF-2 is required for cGKII-dependent regulation of NHE3, but not of CFTR. An overview of the proteome of the jejunal brush border of the mouse is presented in chapter 5. Finally, chapter 6 shows that the glial fibrillary acidic protein (GFAP) is a specific binding partner for cGKII. cGKII was able to phosphorylate GFAP in vitro and expression of active cGKII in cultured glioblastoma cells increased GFAP abundance. We propose that cGKII-mediated phosphorylation of GFAP results in a more stable configuration and a prolonged half-life of this protein

Az apikális anion transzporterek/csatornák szerepe a pancreas duktális sejtek szekréciójában = The role of apical anion transporters/channels in secretion by pancreatic duct cells

Kutatásaink során összességében fontos élettani és kórélettani információkat szereztünk a cisztás fibrózis transzmembrán konduktancia regulátor (CFTR), az SCL26A6 (PAT-1 – putatív anion transzporter 1) és a Na+/H+ cserélő regulátor faktor 1 (NHERF-1) hasnyálmirigy duktális epitél sejtek bikarbonát és folyadék szekréciójában betöltött szerepéről egér pankreászban. A CFTR, PAT-1 és NHERF-1 csökkent expressziója a pankreász vezetéksejtek bikarbonát- és folyadék szekréciójának a csökkenéséhez vezet. A NHERF-1 központi szerepet játszik a CFTR apikális membránba való kijutásában, és biztosítja a CFTR apikális membránban való lokalizációját. Kimutattuk azt is, hogy a NHERF-1 génkiütött egerekben az akut hasnyálmirigygyulladás (főleg az acinus sejtek károsodása) két modellben (ceruleines és taurokólsavas) is súlyosabb volt a vad-típusú állatokhoz képest. Az in vivo adatok alátámasztják, hogy a pankreász duktális sejteknek fontos szerepük van az akut pankreatitisz patogenezisében. Végső soron eredményeink utat nyithatnak egy új terápiás lehetőség felé (duktális szekréció fokozása) a pankeász gyulladásos megbetegedéseinek kezelésében, amik eddig főleg az acinus sejtekre fókuszáltak. | Overall, the project provided important information on the roles of cystic fibrosis transmembrane conductance regulator (CFTR), putative anion transporter 1 (PAT-1) and Na+/H+ exchanger regulatory factor-1 (NHERF-1) in the pancreatic ductal bicarbonate and fluid secretion in mice. NHERF-1 plays a critical role in regulating the apical trafficking/retention of CFTR in mouse pancreatic duct cells. The decrease in CFTR, PAT-1 and NHERF-1 expression resulted in greatly reduced pancreatic ductal secretion. Moreover, NHERF-1 expression also influenced the development of acute necrotizing pancreatitis. In fact, acute pancreatitis severity (especially acinar necrosis) was higher in NHERF-1 knock-out vs. wild-type mice. Importantly, we provide in vivo data that suggest the involvement of pancreatic ducts in the pathogenesis of acute pancreatitis. The results obtained from this study may eventually open up new therapeutic possibilities (targeting ductal secretion) in the treatment of pancreatic inflammation which have mainly focused on acinar cells

Differential association of the Na+/H+ exchanger regulatory factor (NHERF) family of adaptor proteins with the raft- and the non-raft brush border membrane fractions of NHE3

Background/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes ('lipid rafts') were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3 - mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs

The CD34-Related Molecule Podocalyxin Is a Potent Inducer of Microvillus Formation

BACKGROUND: Podocalyxin is a CD34-related transmembrane protein involved in hematopoietic cell homing, kidney morphogenesis, breast cancer progression, and epithelial cell polarization. Although this sialomucin has been shown to block cell adhesion, the mechanisms involved remain enigmatic. It has, however, been postulated that the adaptor proteins NHERF-1 and 2 could regulate apical targeting of Podocalyxin by linking it to the actin cytoskeleton. PRINCIPAL FINDINGS: Here, in contrast, we find that full-length Podocalyxin acts to recruit NHERF-1 to the apical domain. Moreover, we show that ectopic expression of Podocalyxin in epithelial cells leads to microvillus formation along an expanded apical domain that extends laterally to the junctional complexes. Removal of the C-terminal PDZ-binding domain of Podocalyxin abolishes NHERF-1 recruitment but, surprisingly, has no effect on the formation of microvilli. Instead, we find that the extracellular domain and transmembrane region of Podocalyxin are sufficient to direct recruitment of filamentous actin and ezrin to the plasma membrane and induce microvillus formation. CONCLUSIONS/SIGNIFICANCE: Our data suggest that this single molecule can modulate NHERF localization and, independently, act as a key orchestrator of apical cell morphology, thereby lending mechanistic insights into its multiple roles as a polarity regulator, tumor progression marker, and anti-adhesin

Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)–binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions