TY  - THES
A1  - Fetting, Doreen [verh: Korb]
T1  - Novel Cav1.2 and PMCA4b interacting PDZ domain containing proteins
T1  - Neue PDZ-Domain Protein-Interaktionspartner von Cav1.2 und PMCA4b
N2  - The voltage –gated calcium channel, Cav1.2, and the plasma membrane calcium ATPase, PMCA4b, play important roles in excitable and non-excitable cells. The central function of Cav1.2 is to regulate the calcium entry into cells upon depolarization, while PMCA4b is responsible for calcium extrusion and has an influence on cellular calcium homeostasis. Both proteins control fundamental functions in the heart and brain, but the specific functions and the precise mechanisms are still investigated. In order to identify new interaction partners that may regulate the activities of the Cav1.2 and the PMCA4b, we used three independent assays and co-localization studies. The assays, which were used are PDZ domain arrays (testing 124 different PDZ domains), GST pull-downs, and conventional immunoprecipitation assays. In the PDZ arrays, strongest interactions with Cav1.2 and PMCA4b were found for the PDZ domains of MAST-205, MAGI-1, MAGI-2, MAGI-3, and ZO-1. Additionally, we established interactions between Cav1.2 and the PDZ domains of NHERF1/2, Mint-2, and CASK. PMCA4b was observed to interact with Mint-2, and its interactions with Chapsyn-110 and CASK were confirmed. Furthermore, we validated interaction of Cav1.2 and PMCA4b with NHERF1, CASK, MAST-205 and MAGI-3 via immunoprecipitation. We also demonstrated direct interaction of the C-terminus of Cav1.2 and the PDZ domain of nNOS. We assumed that nNOS overexpression would reduce Ca2+ influx through Cav1.2. To address this question, we measured Ca2+ currents in stably transfected HEK 293 cells expressing the Cav1.2 (α1b and β2a subunit of the smooth muscle L-type calcium channel) and nNOS. It has been shown that NO modulates ion channel activity by nitrosylation of sulfhydryl groups on the channel protein. So we propose that the interaction between the C-terminus of Cav1.2 and the PDZ domain of nNOS inhibits the currents by an S-nitrosylation of the channel protein. All these interactions connect both proteins to signaling networks involved in signal transmission, cell adhesion, and apoptosis, which may help provide new hints about the physiological functions of Cav1.2 and PMCA4b in intra- and intercellular signaling.
N2  - Der spannungsabhängige Calcium-Kanal, Cav1.2, und die Plasmamembran Calcium ATPase, PMCA4b, spielen eine wichtige Rolle in erregbaren und nicht-erregbaren Zellen. Der Cav1.2 Kanal reguliert den Calciumeintritt in die Zelle nach einer Depolarisation, während die PMCA4b für den Calciumausstrom und für die Calcium-Homöostase verantwortlich ist. Beide Proteine haben einen grossen Einfluss auf die Funktionen von Herz und Gehirn, aber die genauen Aufgaben und spezifischen Mechanismen, sind noch nicht geklärt. In dieser Arbeit benutzten wir drei unabhängige Assays und Kolokalisationen, um Interaktionspartner von Cav1.2 und PMCA4b zu identifizieren, welche möglicherweise die Aktivitäten von Cav1.2 und PMCA4b regulieren. Die Assays, die wir benutzten waren PDZ Domain Arrays (getestet wurden 124 unterschiedliche PDZ Domänen), GST Pull Downs und konventionelle Immunopräzipitationen. Die Ergebnisse des PDZ Arrays zeigten, dass die PDZ Liganden Cav1.2 und PMCA4b stark mit den PDZ Domänen von MAST-205, MAGI-1, MAGI-2, MAGI-3 und ZO-1 interagierten. Zusätzlich, konnten wir Interaktionen zwischen Cav1.2 und den PDZ Domänen von NHERF1/2, Mint-2 und CASK nachweisen. Es wurde beobachtet, dass PMCA4b mit dem PDZ Protein Mint-2 ein starkes Signal auf der Membran zeigte. Andere Interaktionen von PMCA4b und PDZ Proteinen, konnten durch unseren PDZ Domain Array bestätigt werden (z.B. Chapsyn-110 und CASK). Weiterhin untersuchten wir die Interaktionspartner (NHERF1, CASK, MAST-205 und MAGI-3) von Cav1.2 und PMCA4b durch Immunopräzipitationen genauer. Ein sehr interessantes PDZ Protein, welches wir durch alle drei unabhängigen Assays bestätigen konnten, war nNOS. Schuh et al. konnte schon 2001 zeigen, dass die PDZ Domäne von nNOS mit der PMCA4b interagiert. In der vorliegenden Arbeit konnten wir eine direkte Interaktion des C-terminus von Cav1.2 und dem PDZ Protein nNOS nachweisen. Wir fomulierten eine Hypothese, die lautete, dass eine nNOS Überexpression den Calcium-Einstrom durch den Cav1.2 Kanal reduziert. Um diese Hypothese zu bestätigen wurden Calcium-Ströme in stabil transfizierten HEK 293 Zellen gemessen. Diese HEK 293 Zellen waren stabil transfiziert mit der α1b und β2a Untereinheit des L-type Calcium Kanals und mit nNOS. Es konnte in anderen Studien gezeigt werden, dass NO die Ionenkanal-Aktivität durch Nitrosylierung von Sulfhydryl-Gruppen an den Kanal-Proteinen moduliert. Wir denken, dass die Interaktion zwischen dem C-terminus von Cav1.2 und dem PDZ Protein nNOS, die Calcium-Ströme durch eine S-Nitrosylierung von Cav1.2 inhibiert. Durch all diese Interaktionen wird klar, dass Cav1.2 und PMCA4b eine wichtige Rolle spielen im signalen Netzwerk, in der zellulären Erregung, in Zelladhäsion und Apoptose. Und das wiederum gibt Aufschluss über die physiologischen Funktionen von Cav1.2 und PMCA4b in intra- und interzellulären Signalen.
KW  - Calciumkanal
KW  - Calcium-ATPasen
KW  - Interaktion
KW  - Proteine
KW  - Protein-Interaktionspartner
KW  - CaV1.2 und PMCA4b
KW  - PDZ-Domain
KW  - interacting PDZ domain
KW  - Cav1.2 and PMCA4b
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-66440
ER  - 
TY  - JOUR
A1  - Wiegering, Armin
A1  - Korb, Doreen
A1  - Thalheimer, Andreas
A1  - Kämmerer, Ulrike
A1  - Allmanritter, Jan
A1  - Matthes, Niels
A1  - Linnebacher, Michael
A1  - Schlegel, Nicolas
A1  - Klein, Ingo
A1  - Ergün, Süleyman
A1  - Germer, Christoph-Thomas
A1  - Otto, Christoph
T1  - E7080 (Lenvatinib), a Multi-Targeted Tyrosine Kinase Inhibitor, Demonstrates Antitumor Activities Against Colorectal Cancer Xenografts
N2  - Clinical prognosis of metastasized colorectal carcinoma (CRC) is still not at desired levels and novel drugs are needed. Here, we focused on the multi-tyrosine kinase inhibitor E7080 (Lenvatinib) and assessed its therapeutic efficacy against human CRC cell lines in vitro and human CRC xenografts in vivo. The effect of E7080 on cell viability was examined on 10 humanCRCcell lines and humanendothelial cells (HUVEC). The inhibitory effect of E7080 on VEGF-induced angiogenesis was studied in an ex vivo mouse aortic ring angiogenesis assay. In addition, the efficacy of E7080 against xenografts derived fromCRC cell lines and CRC patient resection specimenswithmutated KRASwas investigated in vivo. Arelatively low cytotoxic effect of E7080 on CRC cell viabilitywas observed in vitro. Endothelial cells (HUVEC)weremore susceptible to the incubation with E7080. This is in line with the observation that E7080 demonstrated an anti-angiogenic effect in a three-dimensional ex vivo mouse aortic ring angiogenesis assay. E7080 effectively disrupted CRC cell-mediated VEGF-stimulated growth of HUVEC in vitro. Daily in vivo treatment with E7080 (5 mg/kg) significantly delayed the growth of KRAS mutated CRC xenografts with decreased density of tumor-associated vessel formations and without tumor regression. This observation is in line with results that E7080 did not significantly reduce the number of Ki67-positive cells in CRC xenografts. The results suggest antiangiogenic activity of E7080 at a dosage thatwas well tolerated by nudemice. E7080 may provide therapeutic benefits in the treatment of CRC with mutated KRAS.
KW  - Chirurgie
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111165
ER  - 
TY  - JOUR
A1  - Korb, Doreen
A1  - Tng, Priscilla Y.
A1  - Milenkovic, Vladimir M.
A1  - Reichhart, Nadine
A1  - Strauss, Olaf
A1  - Ritter, Oliver
A1  - Fischer, Tobias
A1  - Benz, Peter M.
A1  - Schuh, Kai
T1  - Identification of PDZ domain containing proteins interacting with \(Ca_v1.2\) and PMCA4b
JF  - ISRN Cell Biology
N2  - PDZ (PSD-95/Disc large/Zonula occludens-1) protein interaction domains bind to cytoplasmic protein C-termini of transmembrane proteins. In order to identify new interaction partners of the voltage-gated L-type \(Ca^{2+}\) channel Cav1.2 and the plasma membrane \(Ca^{2+}\) ATPase 4b (PMCA4b), we used PDZ domain arrays probing for 124 PDZ domains. We confirmed this byGST pulldowns and immunoprecipitations. In PDZ arrays, strongest interactionswith \(Ca_v1.2\) and PMCA4b were found for the PDZ domains of SAP-102, MAST-205, MAGI-1, MAGI-2, MAGI-3, and ZO-1. We observed binding of the \(Ca_v1.2\) C-terminus to PDZ domains of NHERF1/2, Mint-2, and CASK. PMCA4b was observed to interact with Mint-2 and its known interactions with Chapsyn-110 and CASK were confirmed. Furthermore, we validated interaction of \(Ca_v1.2\) and PMCA4b with NHERF1/2, CASK,MAST-205 and MAGI-3 viaimmunoprecipitation. We also verified the interaction of \(Ca_v1.2\) and nNOS and hypothesized that nNOS overexpression might reduce \(Ca^{2+}\) influx through \(Ca_v1.2\). To address this, we measured \(Ca^{2+}\) currents in HEK 293 cells co-expressing \(Ca_v1.2\) and nNOS and observed reduced voltage-dependent \(Ca_v1.2\) activation. Taken together, we conclude that \(Ca_v1.2\) and PMCA4b bind promiscuously to various PDZ domains, and that our data provides the basis for further investigation of the physiological consequences of these interactions.
KW  - Cell
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130585
IS  - Article ID 265182
ER  -