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Diese Arbeit bedient sich der Immunfluoreszenzmikroskopie, um die intrazelluläre Lokalisation des mit der Plasmamembran assoziierten Regulatorproteins RS1 und eines seiner Zielproteine, des Natrium-D-Glucose-Kotransporters SGLT1, in Zellkulturmodellen des Nierenepithels (LLC-PK1- und HEK293-Zellen) zu untersuchen. Zwei polyklonale Antikörper gegen das RS1-Protein des Schweins (pRS1) wurden dafür erzeugt. In Untersuchungen am konfokalen Laser-Scanning-Mikroskop fand sich pRS1 an der Plasmamembran, im Zellkern, intrazellulär an Vesikeln sowie an einem perinukleären Kompartiment. Die Lokalisation des Proteins im Kern von LLC-PK1-Zellen nahm mit zunehmender Differenzierung der Zellen ab, pRS1 wurde in differenzierten Zellen lediglich im perinukleären Kompartiment gefunden. Dieses wurde in Kolokalisationsstudien als trans-Golgi-Netzwerk (TGN) identifiziert und dort eine Kolokalisation von pRS1 mit Clathrin und Dynamin nachgewiesen. Durch Behandlung der Zellen mit Brefeldin A wurde der Verlust von pRS1 vom TGN induziert. SGLT1 wurde überwiegend in Endosomen nachgewiesen, die entlang von Microtubuli organisiert waren. Auch im trans-Golgi-Netzwerk wurde die Anwesenheit von SGLT1 gezeigt. pSGLT1 kolokalisierte dort mit Dynamin aber nicht mit Clathrin. Es wurde demonstriert, dass experimentelle Hemmung der Proteasoms die Menge an pRS1 drastisch erhöht und gegenläufig die des Natrium-D-Glucose-Kotransporter (pSGLT1) abnimmt. Die gewonnenen Daten wurden in einem hypothetischen Modell zusammengefasst, das die gezeigten Ergebnisse mit früher gewonnenen funktionellen Experimente zu einem schlüssigen Konzept zusammenführt.
The RS1 protein (gene RSC1A1) participates in regulation of Na+-D-glucose cotransporter SGLT1 and some other solute carriers. In subconfluent LLC-PK1 cells, RS1 inhibits release of SGLT1 from the trans-Golgi network and transcription of SGLT1. In subconfluent cells, RS1 is localized in the nucleus and the cytoplasm whereas confluent cells contain predominantly cytoplasmic RS1. In the present study, the mechanism and regulation of confluence-dependent nuclear location of RS1 was investigated. Confluence dependent nuclear location of RS1 was shown to be regulated by the cell cycle. A nuclear shuttling signal (NS) in pRS1 was identified that ensures confluence-dependent distribution of pRS1 and comprises nuclear localization signal (NLS) and nuclear export signal (NES). The NLS and NES of RS1 mediate translocation into and out of the nucleus via importin ß1 and CRM1, respectively, and the nuclear/cytoplasmic distribution of the RS1 protein is determined by the nuclear export activity. The adjacent protein kinase C (PKC) phosphorylation site at serine 370 of pRS1 was shown to control nuclear localization driven by NS and is necessary for the differential localization of RS1 in quiescent versus proliferating cells. Basing on the data of site-directed mutagenesis, PKC activation experiments and mass spectrometry analysis of RS1 phosphorylation, the following model of the regulation of RS1 nuclear location in LLC-PK1 cells was proposed. In subconfluent cells, RS1 is actively imported into the nucleus whereas nuclear export of RS1 is not active leading to accumulation of RS1 in the nucleus. After confluence, phosphorylation of serine 370 of pRS1 by PKC takes place leading to enhancement of RS1 nuclear export and predominantly cytoplasmic distribution of the protein in the confluent cells. The confluence-dependent regulation of RS1 localization may control SGLT1 expression during regeneration of enterocytes in small intestine and during regeneration of renal tubular cells after hypoxemic stress. Moreover, the gene expression profiling of mouse embryonic fibroblasts with RS1-/- genotype suggests that transcriptional regulation by RS1 might be important for the cell cycle and cell division. Since RS1 localization depends on the cell cycle, RS1 might play a role in the regulation of the solute carriers during specific phases of the cell cycle.
The Na+-D-glucose cotransporter in small intestine is regulated in response to food composition. Short term regulation of SGLT1 occurs post-transcriptionally in response to changes in luminal glucose. Adaptation to dietary carbohydrate involves long term regulation at the transcriptional level. The intracellular protein RS1 (gene RSC1A1) is involved in transcriptional and post-transcriptional regulation of SGLT1. RS1 contains an N-terminal domain with many putative phosphorylation sites. By Expressing SGLT1 in oocytes of Xenopus laevis it was previously demonstrated that the post-transcriptional down-regulation of SGLT1 by RS1 was dependent on the intracellular glucose concentration and activated by protein kinase C (PKC). The role of RS1 for short term regulation of SGLT1 in mouse small intestine in response to glucose and PKC was investigated comparing effects in RS1-/- mice and wildtype mice. Effects on SGLT1 activity were determined by measuring phlorizin inhibited uptake of α-methylglucoside (AMG). The involvement of RS1 in glucose dependent short term regulation could not be elucidated for technical reasons. However, evidence for RS1 independent short-term downregulation of SGLT1 after stimulation of PKC could be provided. It was shown that this downregulation includes decrease in the amount and/or in turnover of SGLT1 in the brush-border membrane as well as an increase of substrate affinity for AMG transport. Trying to elucidate the role of RS1 in long term regulation of SGLT1 in small intestine in response to glucose and fat content of the diet, wildtype and RS1-/- mice were kept for 2 months on a normo-caloric standard diet with high glucose and low fat content (ND), on a hyper-caloric glucose-galactose reduced diet with high fat content (GGRD) or on a hyper-caloric diet with a high fat and high glucose content (HFHGD). Thereafter the animals were starved overnight and SGLT1 mediated AMG uptake was measured. Independent of diet AMG uptake in ileum was smaller compared to duodenum and jejunum. In jejunum of wildtype and RS1-/- mice kept on the fat rich diets (GGRD and HFHGH) transport activity of SGLT1 was lower compared to mice kept on ND with low fat content. This result suggests an RS1 independent downregulation due to fat content of diet. Different to RS1-/- mice, the duodenum of wildtype mice showed transport activity of SGLT1 smaller in mice kept on glucose galactose reduced diet (GGRD) compared to the glucose galactose rich diets (ND and HFHGG). These data indicate that RS1 is involved in glucose dependent long term regulation in duodenum.