Downregulation of the Na + -D-glucose Cotransporter SGLT1 by Protein RS1 (RSC1A1) is Dependent on Dynamin and Protein Kinase C

Abstract

We have previously shown that the regulatory protein RS1, cloned from pig, rabbit and human (RSC1A1), is localized intracellularly and inhibits the transcription of the Na(+)- D-glucose cotransporter SGLT1 in LLC-PK(1) cells. We also reported that transport activities of human SGLT1 (hSGLT1) and human organic cation transporter hOCT2 expressed in Xenopus oocytes were decreased upon co-expression of human RS1 (hRS1). The present paper indicates that the glucose transporter GLUT1 and the peptide transporter PEPT1 are not influenced by hRS1. Voltage-clamp experiments in oocytes expressing hSGLT1 demonstrated that hRS1 reduced the maximal substrate-induced currents but did not change substrate activation, membrane potential dependence, Na(+) dependence or substrate selectivity of hSGLT1. Co-expression experiments with a dominant-negative dynamin mutant showed that the posttranslational inhibition of hSGLT1 by hRS1 was dependent on the function of dynamin. Finally, we observed that hRS1 changed the short-term effect of protein kinase C (PKC) on hSGLT1. Whereas the PKC activators phorbol-12-myristate-13-acetate (PMA) and sn-1,2-dioctanoyl glycerol (DOG) increased alpha-methyl glucose (AMG) uptake expressed by hSGLT1 alone as described earlier, PMA and DOG decreased AMG uptake mediated by hSGLT1 when hRS1 was co-expressed. Taken together, these data indicate that hRS1 modulates dynamin-dependent trafficking of intracellular vesicles containing hSGLT1 in Xenopus oocytes, and modulates PKC-dependent short-term regulation of this transporter.

Tripeptides of RS1 (RSC1A1) Inhibit a Monosaccharide-dependent Exocytotic Pathway of Na+-d-Glucose Cotransporter SGLT1 with High Affinity

“…The observed changes in distribution correlated with functional data showing that RS1 down-regulates the transcrip-* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB tion of SGLT1 in subconfluent LLC-PK 1 cells (25). In Xenopus oocytes, expression of SGLT1 was inhibited when human RS1 was coexpressed (23,24,27) or when purified RS1 protein was injected into SGLT1 expressing oocytes shortly before uptake measurements (21). This indicated that hSGLT1 was inhibited by hRS1 on the posttranscriptional level.…”

“…Expressed by hSGLT1-Previously we described that uptake of AMG into oocytes of X. laevis expressing hSGLT1 was decreased when hRS1 was coexpressed or when purified hRS1 protein was injected into hSGLT1 expressing oocytes (21,24,27). We showed that hRS1 protein is a coat protein of the TGN and that it blocks the release of SGLT1 containing vesicles (21,30).…”

“…To prepare sense cRNA from hRS1 (24), fragments of hRS1, hSGLT1 (34), or hPepT1 (37), the respective purified plasmids were linearized with EcoRI (hSGLT1), NotI (hPepT1), or MluI (hRS1 and hRS1 fragments). cRNA was synthesized using T3 polymerase (hSGLT1), SP6 polymerase (hRS1, fragments of hRS1), or T7 polymerase (hPepT1) as described earlier (27). cRNAs were prepared employing the "mMESSAGE mMACHINE" kit (Ambion) using sodium acetate precipitation.…”

“…Previously, we identified the intracellular protein RS1 (human gene RSC1A1) that participates in the transcriptional and post-transcriptional regulation of SGLT1 (21,(23)(24)(25)(26)(27)(28). RSC1A1 is an intronless single copy gene that was only detected in mammals and codes for 67-68-kDa proteins in human, pig, rabbit, and mouse that exhibit about 70% amino acid identity.…”

The Large Extracellular Loop of Organic Cation Transporter 1 Influences Substrate Affinity and Is Pivotal for Oligomerization

“…The regions of interest were selected in the CFP (donor) or YFP (acceptor) image (to avoid bleaching-associated bias) and transmitted to the N FRET image by the ImageJ Multi Measure Tool. As a negative control, we employed the CFP-labeled form of rOct1 (C-Oct1) with a membrane-bound form of YFP (25) In Vitro Synthesis of cRNA-For injection into X. laevis oocytes, m7G(5Ј)G-capped cRNA was prepared, purified, and stored as described previously (27,28). To prepare sense cRNA from rOct1 (4), rOct1 mutants, or rOct1 tandem protein, the respective purified plasmids were linearized with MluI.…”