1,166 research outputs found

    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.

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    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

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    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

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    Kutat√°saink sor√°n √∂sszess√©g√©ben fontos √©lettani √©s k√≥r√©lettani inform√°ci√≥kat szerezt√ľnk a ciszt√°s fibr√≥zis transzmembr√°n konduktancia regul√°tor (CFTR), az SCL26A6 (PAT-1 ‚Äď putat√≠v anion transzporter 1) √©s a Na+/H+ cser√©lŇĎ regul√°tor faktor 1 (NHERF-1) hasny√°lmirigy dukt√°lis epit√©l sejtek bikarbon√°t √©s folyad√©k szekr√©ci√≥j√°ban bet√∂lt√∂tt szerep√©rŇĎl eg√©r pankre√°szban. A CFTR, PAT-1 √©s NHERF-1 cs√∂kkent expresszi√≥ja a pankre√°sz vezet√©ksejtek bikarbon√°t- √©s folyad√©k szekr√©ci√≥j√°nak a cs√∂kken√©s√©hez vezet. A NHERF-1 k√∂zponti szerepet j√°tszik a CFTR apik√°lis membr√°nba val√≥ kijut√°s√°ban, √©s biztos√≠tja a CFTR apik√°lis membr√°nban val√≥ lokaliz√°ci√≥j√°t. Kimutattuk azt is, hogy a NHERF-1 g√©nki√ľt√∂tt egerekben az akut hasny√°lmirigygyullad√°s (fŇĎleg az acinus sejtek k√°rosod√°sa) k√©t modellben (ceruleines √©s taurok√≥lsavas) is s√ļlyosabb volt a vad-t√≠pus√ļ √°llatokhoz k√©pest. Az in vivo adatok al√°t√°masztj√°k, hogy a pankre√°sz dukt√°lis sejteknek fontos szerep√ľk van az akut pankreatitisz patogenezis√©ben. V√©gsŇĎ soron eredm√©nyeink utat nyithatnak egy √ļj ter√°pi√°s lehetŇĎs√©g fel√© (dukt√°lis szekr√©ci√≥ fokoz√°sa) a panke√°sz gyullad√°sos megbeteged√©seinek kezel√©s√©ben, amik eddig fŇĎleg az acinus sejtekre f√≥kusz√°ltak. | 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

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    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

    Regulation of cystic fibrosis transmembrane conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction

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

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    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

    Suppression of breast cancer cell growth by Na(+)/H(+ )exchanger regulatory factor 1 (NHERF1)

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    INTRODUCTION: Na(+)/H(+ )exchanger regulatory factor 1 (NHERF1, also known as EBP50 or NHERF) is a putative tumour suppressor gene in human breast cancer. Located at 17q25.1, NHERF1 is frequently targeted during breast tumourigenesis. Loss of heterozygosity (LOH) at the NHERF1 locus is found in more than 50% of breast tumours. In addition, NHERF1 is mutated in a subset of primary breast tumours and breast cancer cell lines. LOH at the NHERF1 locus is strongly associated with aggressive features of breast tumours, implicating NHERF1 as a haploinsufficiency tumour suppressor gene. However, the putative NHERF1 tumour suppressor activity has not been functionally verified. METHODS: To confirm the NHERF1 tumour suppressor activity suggested by our genetic analyses, we used retrovirus-transduced short hairpin RNA (shRNA) to knock down NHERF1 expression in breast cancer cell lines MCF7 and T47D. These cells were then assessed for cell growth in vitro and in vivo. The control and NHERF1 knockdown cells were also serum-starved and re-fed to compare their cell cycle progression as measured by fluorescence-activated cell sorting analyses. RESULTS: We found that downregulation of the endogenous NHERF1 in T47D or MCF7 cells resulted in enhanced cell proliferation in both anchorage-dependent and -independent conditions compared with that of the vector control cells. NHERF1 knockdown T47D cells implanted at mammary fat pads of athymic mice formed larger tumours than did control cells. We found that serum-starved NHERF1 knockdown cells had a faster G(1)-to-S transition after serum re-stimulation than the control cells. Immunoblotting showed that the accelerated cell cycle progression in NHERF1 knockdown cells was accompanied by increased expression of cyclin E and elevated Rb phosphorylation level. CONCLUSION: Our findings suggested that the normal NHERF1 function in mammary epithelial cells involves blockage of cell cycle progression. Our study affirmed the tumour suppressor activity of NHERF1 in breast which may be related to its regulatory effect on cell cycle. It warrants future investigation of this novel tumour suppressor pathway in human breast cancer which may turn up therapeutic opportunities
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