Chakravarthi Chintalapati

University of Würzburg

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Hermann Koepsell

University of Würzburg

Valentin Gorboulev

University of Würzburg

Thorsten Keller

University of Würzburg

Jürgen Gröll

University of Würzburg

Maike Veyhl-Wichmann

University of Würzburg

Ilona Zilkowski

Bavarian Polymer Institute

Marina Leyerer

University of Würzburg

Yvonne Reinders

Leibniz Institute for Analytical Sciences - ISAS

Alina Filatova

University of Giessen

Dietmar Geiger

University of Würzburg

Cooperative Institutions

University of Würzburg

Universitätsklinikum Würzburg

Leibniz Association

Institute for Research in Biomedicine

Heidelberg University

Centro de Investigación Biomédica en Red

German Cancer Research Center

Düsseldorf University Hospital

Heinrich Heine University Düsseldorf

Goethe University Frankfurt

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Protein RS1 (RSC1A1) Downregulates the Exocytotic Pathway of Glucose Transporter SGLT1 at Low Intracellular Glucose via Inhibition of Ornithine Decarboxylase

Molecular Pharmacology (2016)

Chakravarthi Chintalapati Thorsten Keller Thomas D. Mueller Valentin Gorboulev Nadine Schäfer

Na+-d-glucose cotransporter 1 (SGLT1) is rate-limiting for glucose absorption in the small intestine. Shortly after intake of glucose-rich food, SGLT1 abundance in the luminal membrane of the small intestine is increased. This upregulation occurs via glucose-induced acceleration of the release of SGLT1-containing vesicles from the trans-Golgi network (TGN), which is regulated by a domain of protein RS1 (RSC1A1) named RS1-Reg. Dependent on phosphorylation, RS1-Reg blocks release of vesicles containing SGLT1 or concentrative nucleoside transporter 1. The hypothesis has been raised that RS1-Reg binds to different receptor proteins at the TGN, which trigger release of vesicles with different transporters. To identify the presumed receptor proteins, two-hybrid screening was performed. Interaction with ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme of polyamine synthesis, was observed and verified by immunoprecipitation. Binding of RS1-Reg mutants to ODC1 was characterized using surface plasmon resonance. Inhibition of ODC1 activity by RS1-Reg mutants and the ODC1 inhibitor difluoromethylornithine (DFMO) was measured in the absence and presence of glucose. In addition, short-term effects of DFMO, RS1-Reg mutants, the ODC1 product putrescine, and/or glucose on SGLT1 expressed in oocytes of Xenopus laevis were investigated. High-affinity binding of RS1-Reg to ODC1 was demonstrated, and evidence for a glucose binding site in ODC1 was provided. Binding of RS1-Reg to ODC1 inhibits the enzymatic activity at low intracellular glucose, which is blunted at high intracellular glucose. The data suggest that generation of putrescine by ODC1 at the TGN stimulates release of SGLT1-containing vesicles. This indicates a biomedically important role of ODC1 in regulation of glucose homeostasis.

10.1124/mol.116.104521

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Citations (9)

Phosphorylation of RS1 (RSC1A1) Steers Inhibition of Different Exocytotic Pathways for Glucose Transporter SGLT1 and Nucleoside Transporter CNT1, and an RS1-Derived Peptide Inhibits Glucose Absorption

Molecular Pharmacology (2015)

Maike Veyhl-Wichmann Alexandra Friedrich Alexandra Vernaleken Smriti Singh Helmut Kipp

Cellular uptake adapts rapidly to physiologic demands by changing transporter abundance in the plasma membrane. The human gene RSC1A1 codes for a 67-kDa protein named RS1 that has been shown to induce downregulation of the sodium-D-glucose cotransporter 1 (SGLT1) and of the concentrative nucleoside transporter 1 (CNT1) in the plasma membrane by blocking exocytosis at the Golgi. Injecting RS1 fragments into Xenopus laevis oocytes expressing SGLT1 or CNT1 and measuring the expressed uptake of α-methylglucoside or uridine 1 hour later, we identified a RS1 domain (RS1-Reg) containing multiple predicted phosphorylation sites that is responsible for this post-translational downregulation of SGLT1 and CNT1. Dependent on phosphorylation, RS1-Reg blocks the release of SGLT1-containing vesicles from the Golgi in a glucose-dependent manner or glucose-independent release of CNT1-containing vesicles. We showed that upregulation of SGLT1 in the small intestine after glucose ingestion is promoted by glucose-dependent disinhibition of the RS1-Reg–blocked exocytotic pathway of SGLT1 between meals. Mimicking phosphorylation of RS1-Reg, we obtained a RS1-Reg variant that downregulates SGLT1 in the brush-border membrane at high luminal glucose concentration. Because RS1 mediates short-term regulation of various transporters, we propose that the RS1-Reg–navigated transporter release from Golgi represents a basic regulatory mechanism of general importance, which implies the existence of receptor proteins that recognize different phosphorylated forms of RS1-Reg and of complex transporter-specific sorting in the trans-Golgi. RS1-Reg–derived peptides that downregulate SGLT1 at high intracellular glucose concentrations may be used for downregulation of glucose absorption in small intestine, which has been proposed as strategy for treatment of type 2 diabetes.

Nucleoside transporter

10.1124/mol.115.101162

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Differential regulation of G-protein coupled bile acid receptor (Gpbar-1) by Sp1/KLF5 family transcription factors

Zeitschrift für Gastroenterologie (2015)

Chakravarthi Chintalapati Christina Wöhler Christian Ehlting Juergen Bode D Häussinger

TGR5 (Gpbar-1) is a G-protein coupled bile acid receptor, which is highly expressed in cholangiocytes, liver sinusoidal endothelial cells, liver macrophages and CD14 positive monocytes of peripheral blood. TGR5-/- mice show impaired liver regeneration after partial hepatectomy and the increased inflammation in liver disease models, thus emphasizing a protective role of TGR5 in liver. While TGR5 functions are being studied extensively, little is known about the transcriptional regulation of this receptor. Aim: To investigate the mechanism and transcriptional regulation of TGR5. Methods: The potential TGR5 promoter (-154/-79) was cloned into a pGL3 Luciferase expression vector. Luciferase gene expression was used to evaluate the effect of Sp1 and KLF5 on TGR5 expression by co-transfection. Binding of the two transcription factors to the promoter was verified by Chromatin Immunoprecipitation (ChIP). Immunoprecipitation was used to confirm the interaction of HDAC1 and 2 with Sp1. Transfection of KLF5 siRNA was used to verify the TGR5 transcriptional regulation. Results: KLF5 increased the luciferase gene expression in a concentration dependent manner. The effect was lost when the binding site for KLF5 was mutated. High expression of SP1 mediated a downregulation of TGR5 promoter luciferase activity, which was absent when mutated Sp1 with a defective DNA binding domain was co-transfected. ChIP analysis confirmed the binding of both transcription factors to the predicted TGR5 promoter region. Sp1 forms an inhibitory complex with HDAC1 and 2 at the site of binding there by downregulating the transcription of TGR5. Inhibition of casein kinase 2 mediated phosphorylation of HDAC proteins by apigenin resulted in a decreased complex formation with Sp1 there by loss of Sp1 mediated downregulation. TGR5 is differentially regulated by Sp1 in different carcinoma cell lines (colon carcinoma and cholangiocyte carcinoma). KLF5 siRNA decreased the mRNA expression of TGR5 in colon and cholangiocyte carcinoma cell lines. Conclusion: This study demonstrates that the transcription factor Sp1 downregulates whereas KLF5 upregulates the gene expression of TGR5 in vitro. Sp1 decreases the transcription of TGR5 by binding to the promoter region and recruiting HDAC1 and 2 to forming an inhibitory complex thereby leading to the deacetylation of histones. Phosphorylation of HDAC proteins by casein kinase 2 is important in the formation of inhibitory complex. Differential regulation of TGR5 by Sp1 was confirmed in different carcinoma cell lines.

Chromatin immunoprecipitation

Farnesoid X receptor

Sp1 transcription factor

10.1055/s-0035-1567990

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Citations (1)

Novel Shuttling Domain in a Regulator (RSC1A1) of Transporter SGLT1 Steers Cell Cycle‐Dependent Nuclear Location

Traffic (2009)

Alina Filatova Marina Leyerer Valentin Gorboulev Chakravarthi Chintalapati Yvonne Reinders

The gene product of RSC1A1, RS1, participates in the regulation of the Na(+)-D-glucose cotransporter SGLT1. RS1 inhibits release of SGLT1 from the trans Golgi network. In subconfluent LLC-PK(1) cells, RS1 migrates into the nucleus and modulates transcription of SGLT1, whereas most confluent cells do not contain RS1 in the nuclei. We showed that confluence-dependent nuclear location of RS1 is because of different phases of the cell cycle and identified a RS1 nuclear shuttling domain (RNS) with an associated protein kinase C (PKC) phosphorylation site (RNS-PKC) that mediates cell cycle-dependent nuclear location. RNS-PKC contains a novel non-conventional nuclear localization signal interacting with importin beta1, a nuclear export signal mediating export via protein CRM1 and a Ca(2+)-dependent calmodulin binding site. PKC and calmodulin compete for binding to RNS-PKC. Mutagenesis experiments and analyses of the phosphorylation status suggest the following sequences of events. Subconfluent cells without and with synchronization to the G2/M phase contain non-phosphorylated RNS-PKC that mediates nuclear import of RS1 but not its export. During confluence or synchronization of subconfluent cells to the G2/M phase, phosphorylation of RNS-PKC mediates rapid nuclear export of RS1.

Nuclear export signal

Importin

10.1111/j.1600-0854.2009.00982.x

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Citations (15)