44 research outputs found

    A Novel PDZ Protein Regulates the Activity of Guanylyl Cyclase C, the Heat-stable Enterotoxin Receptor

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    Secretory diarrhea is the leading cause of infectious diarrhea in humans. Secretory diarrhea may be caused by binding of heat-stable enterotoxins to the intestinal receptor guanylyl cyclase C (GCC). Activation of GCC catalyzes the formation of cGMP, initiating a signaling cascade that opens the cystic fibrosis transmembrane conductance regulator chloride channel at the apical cell surface. To identify proteins that regulate the trafficking or function of GCC, we used the unique COOH terminus of GCC as the "bait" to screen a human intestinal yeast two-hybrid library. We identified a novel protein, IKEPP (intestinal and kidney-enriched PDZ protein) that associates with the COOH terminus of GCC in biochemical assays and by co-immunoprecipitation. IKEPP is expressed in the intestinal epithelium, where it is preferentially accumulated at the apical surface. The GCC-IKEPP interaction is not required for the efficient targeting of GCC to the apical cell surface. Rather, the association with IKEPP significantly inhibits heat-stable enterotoxin-mediated activation of GCC. Our findings are the first to identify a regulatory protein that associates with GCC to modulate the catalytic activity of the enzyme and provides new insights in mechanisms that regulate GCC activity in response to bacterial toxin

    Elevated Intracellular Calcium Stimulates NHE3 Activity by an IKEPP (NHERF4) Dependent Mechanism

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    The ileal brush border (BB) contains four evolutionarily related multi-PDZ domain proteins including NHERF1, NHERF2, PDZK1 (NHERF3) and IKEPP (NHERF4). Why multiple related PDZ proteins are in a similar location in the same cell is unknown. However, some specificity in regulation of NHE3 activity has been identified. For example, elevated intracellular Ca2+ ([Ca2+]i) inhibition of NHE3 is reconstituted by NHERF2 but not NHERF1, and involves the formation of large NHE3 complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether the four PDZ domain containing protein IKEPP reconstitutes elevated [Ca2+]i regulation of NHE3. In vitro, IKEPP bound to the F2 region (aa 590-667) of NHE3 in overlay assays, which is the same region where NHERF1 and NHERF2 bind. PS120 cells lack endogenous NHE3 and IKEPP. Treatment of PS120/NHE3/IKEPP cells (stably transfected with NHE3 and IKEPP) with the Ca2+ ionophore, 4-Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (0.5őľM), stimulated NHE3 Vmax activity by ‚ąľ40%. This was associated with an increase in plasma membrane expression of NHE3 by a similar amount. NHE3 activity and surface expression were unaffected by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 in PS120/NHE3 cells lacking IKEPP. Based on sucrose density gradient centrifugation, IKEPP was also shown to exist in large complexes, some of which overlap in size with NHE3, and the size of both NHE3 and IKEPP complexes decreased in parallel after [Ca2+]i elevation. FRET experiments on fixed cells demonstrated that IKEPP and NHE3 directly associated at an intracellular site. Elevating [Ca2+]i decreased this intracellular NHE3 and IKEPP association. In summary: (1) In the presence of IKEPP, elevated [Ca2+]i stimulates NHE3 activity. This was associated with increased expression of NHE3 in the plasma membrane as well as a shift to smaller sizes of NHE3 and IKEPP containing complexes. (2) IKEPP directly binds NHE3 at its F2 C-terminal domain and directly associates with NHE3 in vivo (FRET). (3) Elevated [Ca2+]i decreased the association of IKEPP and NHE3 in an intracellular compartment. Based on which NHERF family member is expressed in PS120 cells, elevated [Ca2+]i stimulates (IKEPP), inhibits (NHERF2) or does not affect (NHERF1) NHE3 activity. This demonstrates that regulation of NHE3 depends on the nature of the NHERF family member associating with NHE3 and the accompanying NHE3 complexes

    Expressionsstudie von Transport- und Adapterproteinen sowie hormonellen Rezeptoren in der Darmwand von Wildtyp- und NOD2-knockout-Mäusen

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    In der vorliegenden Arbeit wurde die basale Expression von Transportproteinen, Adapterproteinen und hormonellen Rezeptoren in der Darmwand von WT- und NOD2-KO-Mäusen untersucht. Die Transportproteine, die in dieser Arbeit untersucht wurden, sind DRA, PAT1, NHE3, CFTR und ANO1. Zu den untersuchten Adapterproteinen gehören NHERF1, NHERF2, PDZK1, IKEPP und SNX27. Des Weiteren wurde die Expression der hormonellen Rezeptoren GLP2- und IGF1-Rezeptor bestimmt, welche postoperativ nach einer ausgedehnten Darmresektion eine Adaptation der Darmschleimhaut vermitteln können

    Dr. Jekyll and Mr. Hyde: MAP17’s up-regulation, a crosspoint in cancer and inflammatory diseases

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    Inflammation is a common defensive response that is activated after different harmful stimuli. This chronic, or pathological, inflammation is also one of the causes of neoplastic transformation and cancer development. MAP17 is a small protein localized to membranes with a restricted pattern of expression in adult tissues. However, its expression is common in destabilized cells, as it is overexpressed both in inflammatory diseases and in cancer. MAP17 is overexpressed in most, if not all, carcinomas and in many tumors of mesenchymal origin, and correlates with higher grade and poorly differentiated tumors. This overexpression drives deep changes in cell homeostasis including increased oxidative stress, deregulation of signaling pathways and increased growth rates. Importantly, MAP17 is associated in tumors with inflammatory cells infiltration, not only in cancer but in various inflammatory diseases such as Barret‚Äôs esophagus, lupus, Crohn‚Äôs, psoriasis and COPD. Furthermore, MAP17 also modifies the expression of genes connected to inflammation, showing a clear induction of the inflammatory profile. Since MAP17 appears highly correlated with the infiltration of inflammatory cells in cancer, is MAP17 overexpression an important cellular event connecting tumorigenesis and inflammation?Espa√Īa Ministerio de Econom√≠a y Competitividad Fis: PI15/00045Espa√Īa Consejeria de Ciencia e Innovacion CTS-1848Espa√Īa Consejeria de Salud de la Junta de Andaluc√≠a PI-0096-201

    Az ABCA1 membránfehérje funkciójának és fehérje-kölcsönhatásainak vizsgálata = Investigation of the function and protein interactions of the ABCA1 membrane protein

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    A kutat√°s c√©lja az ABCA1 membr√°nfeh√©rje mŇĪk√∂d√©s√©nek √©s feh√©rje-k√∂lcs√∂nhat√°sainak jellemz√©se volt. √öj modellrendszereket alak√≠tottunk ki Sf9 rovarsejt-bakulov√≠rus √©s retrovir√°lis expresszi√≥s rendszerek seg√≠ts√©g√©vel, r√©szletesen vizsg√°ltuk a vad-t√≠pus√ļ √©s mut√°ns ABCA1 feh√©rj√©k sejten bel√ľli lokaliz√°ci√≥j√°t, mŇĪk√∂d√©s√©t √©s PDZ feh√©rj√©kkel val√≥ k√∂lcs√∂nhat√°s√°t. Bizony√≠tottuk, hogy az ABCA1 feh√©rje mind az ApoA1-f√ľggŇĎ koleszterin ki√°raml√°s, mind a Ca2+-aktiv√°lt sejtfelsz√≠ni foszfatidilszerin expoz√≠ci√≥ folyamat√°ban fontos szerepet j√°tszik. ElsŇĎk√©nt mutattunk ki √∂sszef√ľgg√©st egy v√©rz√©kenys√©gi betegs√©g √©s az ABCA1 mŇĪk√∂d√©se k√∂z√∂tt. Elemezt√ľk az ABCA1 mut√°ci√≥nak hat√°s√°t a betegs√©gre jellemzŇĎ hib√°s foszfatidilszerin expoz√≠ci√≥ban. Vizsg√°ltuk a lipidanyagcser√©re hat√≥ vegy√ľletek hat√°s√°t az ABCA1-hez k√∂thetŇĎ funkci√≥kra, azonos√≠tottunk k√©t √ļj g√°tl√≥ vegy√ľletet. Meg√°llap√≠tottuk, hogy egy speci√°lis PDZ feh√©rje a vizsg√°lt ABC feh√©rj√©k k√∂z√ľl egyed√ľl az ABCA1 feh√©rj√©vel l√©p k√∂lcs√∂nhat√°sba, m√°s ABC transzporterekhez k√∂tŇĎ egy√©b PDZ feh√©rj√©k nem k√∂tŇĎdtek az ABCA1-hez. Kimutattuk polariz√°lt sejtekben az ABCA1, a b2-syntrophin √©s az utrophin bazolater√°lis ko-lokaliz√°ci√≥j√°t. A kidolgozott m√©r√©si m√≥dszereket m√°s ABC transzporterek mŇĪk√∂d√©s√©nek vizsg√°lat√°ra is eredm√©nyesen alkalmaztuk. Megkezdt√ľk a foszfolipid-transzport√©rt felelŇĎs ABC feh√©rj√©k azonos√≠t√°s√°t trombocit√°kban. | The aims of this project were the functional characterization of the ABCA1 protein and identification of its potential interactions with intracellular proteins. We installed new assay systems to analyse the function, subcellular localization and protein interactions of the wild-type and mutant ABCA1 versions, by using two expression systems: the baculovirus-Sf9 insect cell system and retrovirus based expression system for mammalian cells. We proved that ABCA1 plays a key role both in cellular ApoAI-mediated cholesterol removal pathway, and in the exofacial translocation of phosphatidylserine. Our results provided the first link between a defect in a transbilayer phospholipid transport pathway, that of ABCA1, and the bleeding phenotype. We analysed the effects of various mutations of ABCA1 on the Ca2+-stimulated PS exposition. We screened the influence of potential inhibitors on the ABCA1-dependent processes and identified new inhibitors of the PS exposition. We demonstrated that among the examined ABC transporters only ABCA1 binds b2-syntrophin. A diverse group of PDZ proteins that interacts with other ABC proteins does not bind to ABCA1. We showed basolateral colocalization of ABCA1 protein with b2-syntrophin and utrophin. The assays for ABCA1 characterization were applied for studying other ABC proteins successfully. We started the identification of ABC proteins involved in phospholipid translocation in platelets

    PDZ Protein Regulation of ő≤-arrestin Recruitment and GPCR Trafficking

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    ő≤-arrestins are versatile adaptor proteins that play a vital role in regulation of G protein coupled receptor (GPCR) trafficking and signalling properties. PDZ proteins have previously been shown to modulate ő≤-arrestin2 recruitment and receptor internalization for many GPCRs including Corticotropin-Releasing Factor Receptor 1 (CRFR1), a receptor whose antagonists have been shown to demonstrate both anxiolytic- and antidepressant-like effects. Further characterization of the interplay between ő≤-arrestins and PDZ proteins may aid in determining a potential mechanism for PDZ protein regulation of GPCR trafficking. Our findings suggest that PDZ proteins PSD-95, MAGI1, and PDZK1 complex with ő≤-arrestin2 by interacting via the PDZ domain. Using a proteomic approach, mutational analyses were used to reveal that the ő≤-arrestin2 A175F mutant impairs interaction with PSD-95. Additionally, this mutant form of ő≤-arrestin2 shows decreased CRF-stimulated recruitment to CRFR1. Thus, investigating how ő≤-arrestins and PDZ proteins interact could provide further insight into GPCR trafficking properties and the development of novel therapeutics

    NHERF2 is crucial in ERM phosphorylation in pulmonary endothelial cells

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    NHERF2 is crucial in ERM phosphorylation in pulmonary endothelial cells

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