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【摘要】 The product of gene RSC1A1, named RS1, participates in transcriptional and posttranscriptional regulation of the sodium- D -glucose cotransporter SGLT1. Using coexpression in oocytes of Xenopus laevis, posttranscriptional inhibition of human SGLT1 (hSGLT1) and some other transporters by human RS1 (hRS1) was demonstrated previously. In the present study, histidine-tagged hRS1 was expressed in oocytes or Sf9 cells and purified using nickel(II)-charged nitrilotriacetic acid-agarose. hRS1 protein was injected into oocytes expressing hSGLT1 or the human organic cation transporter hOCT2, and the effect on hSGLT1-mediated uptake of methyl- - D -[ 14 C]glucopyranoside ([ 14 C]AMG) or hOCT2-mediated uptake of [ 14 C]tetraethylammonium ([ 14 C]TEA) was measured. Within 30 min after the injection of hRS1 protein, hSGLT1-expressed AMG uptake or hOCT2-expressed TEA uptake was inhibited by 50%. Inhibition of AMG uptake was decreased when a dominant negative mutant of dynamin I was coexpressed and increased after stimulation of PKC. Inhibition remained unaltered when endocytosis was inhibited by chlorpromazine, imipramine, or filipin but was prevented when exocytosis was inhibited by botulinum toxin B or when the release of vesicles from the TGN and endosomes was inhibited by brefeldin A. Inhibition of hSGLT1-mediated AMG uptake and hOCT2-mediated TEA uptake by hRS1 protein were decreased at an enhanced intracellular AMG concentration. The data suggest that hRS1 protein exhibits glucose-dependent, short-term inhibition of hSGLT1 and hOCT2 by inhibiting the release of vesicles from the trans -Golgi network.
【关键词】 Na + D glucose cotransport SGLT RS glucosedependent regulation exocytotic pathway dynamin brefeldin A
GLUCOSE ABSORPTION IN THE SMALL intestine is mediated by two types of glucose transporters in the enterocytes: the sodium-dependent D -glucose cotransporter SGLT1 in the brush-border membrane and the sodium-independent glucose transporter GLUT2 in the basolateral membrane ( 12, 22 ). GLUT2 has been located to the brush border membrane as well ( 22 ). Glucose absorption in the small intestine is highly regulated and plays a pivotal role in the maintenance of blood glucose concentration ( 8, 31 ), glucose metabolism in the liver ( 9 ), and intestinal motility and secretion of intestinal glands ( 27, 36 ). The Na + - D -glucose cotransporter SGLT1 plays a key role in small intestinal glucose absorption as illustrated by defective mutants of SGLT1 in humans which lead to glucose galactose malabsorption that is characterized by severe diarrhea in newborn babies ( 54 ). The expression and activity of SGLT1 in the small intestine exhibit circadian periodicity and are increased following a carbohydrate-rich diet ( 8, 38 ). Regulation of SGLT1 can be mediated by adrenergic innervation, insulin, glucagon 37, glucagon-like peptide 2, cholecystokinin, and insulin-like growth factors ( 4, 17, 18, 41, 44, 45 ). It has been shown that SGLT1 can be regulated by changes in transcription ( 30, 42, 50 ), mRNA stability ( 29 ), amount of transporter within the plasma membrane ( 16 ), and transporter activity ( 49 ). However, the detailed mechanisms for these regulations, the cross talk between different regulatory mechanisms, and the physiological importance of individual regulatory pathways are not understood.
Previously, an intracellular regulatory protein termed RS1 (human gene RSC1A1 ) was identified that is involved in the transcriptional and the posttranscriptional regulation of SGLT1 ( 25, 28, 33, 37, 51, 52 ). RSC1A1 is an intronless single-copy gene located at human chromosome 1p36.1, which is specific for mammals. The RSC1A1 genes code for 67- to 68-kDa RS1 proteins in humans, pigs, rabbits, and mice that exhibit 57-74% amino acid identity. RS1 proteins contain consensus sequences for phosphorylation by PKC and casein kinase 2, and one ubiquitin-associated (UBA) domain. They have a broad tissue distribution, including renal proximal tubular cells, small intestinal epithelial cells, hepatocytes, and neurons ( 28, 35, 37, 51 ). In LLC-PK 1 cells, RS1 was located on the intracellular side of the plasma membrane, at vesicles below the plasma membrane, at the trans -Golgi network (TGN), and within the nucleus [47; see also the companion paper in this issue ( 26a )]. Nuclear location was dependent on the state of confluence, being mainly observed in the subconfluent LLC-PK 1 cells. Data were obtained suggesting that RS1 participates in transcriptional downregulation of SGLT1 ( 25 ) in subconfluent LLC-PK 1 cells. In addition, it was shown 1 ) that transcriptional upregulation of SGLT1 after reaching confluence was associated with a posttranscriptional downregulation of RS1 protein; 2 ) that the expression of SGLT1 was upregulated by reduction of RS1 via an antisense strategy; and, inversely, 3 ) that the expression of SGLT1 was largely decreased by overexpression of RS1 ( 25 ). In previous studies aiming to understand the posttranscriptional regulation of plasma membrane transporters by RS1, coexpression of RS1 and transporters was performed by injecting the respective cRNAs into oocytes of Xenopus laevis ( 25, 28, 33, 52 ). It has been demonstrated that the expression of human SGLT1 (hSGLT1) was inhibited when human RS1 (hRS1) was coexpressed. The inhibition of hSGLT1 expression by hRS1 was abolished when a dominant negative dynamin mutant was coexpressed or increased when PKC was stimulated in oocytes expressing hSGLT1 and hRS1 ( 52 ). Given that dynamin is required for endocytosis and for vesicle budding from intracellular compartments such as endosomes or the TGN ( 20, 48 ), we could not distinguish whether the observed effects of RS1 were due to stimulation of dynamin-dependent endocytosis or to inhibition of dynamin-dependent release of vesicles from intracellular compartments.
Considering the ubiquitous expression of RS1 and the fact that posttranscriptional inhibition by RS1 was not only observed for SGLT1 but also for the human organic cation transporter hOCT2 and the Na + - myo -inositol cotransporter, but not, however, for the human glucose transporter GLUT1 or the Na + /Cl - GABA cotransporter GAT1 ( 28, 51, 52 ), RS1 is supposed to be involved in the regulation of various plasma membrane transporters. Nevertheless, evidence was obtained that SGLT1 is a physiologically important target of RS1 because in RS1 knockout mice upregulation of SGLT1 and of glucose absorption in small intestine was observed and the mice developed an obese phenotype ( 33 ). Recently Jiang and co-workers ( 19 ) reported that RS1 is associated with the 28-kDa protein IRIP that is upregulated in the kidney after ischemia and reperfusion. Like RS1, IRIP inhibits the expression of a variety of plasma membrane transporters such as the organic cation transporter hOCT2, the organic anion transporter OAT1, and the sodium cotransporters for norepinephrine, dopamine, and serotonin. Since inhibition of OCT2 by RS1 and IRIP was not additive and inhibition of OCT2 by RS1 was prevented by coexpression of a dominant negative mutant of IRIP, RS1 and IRIP are supposed to be parts of a common regulatory pathway controlling transporter activities.
Since in the previously performed coexpression experiments long-term effects of RS1 on transporter expression have been described ( 19, 52 ), it could not be excluded that the observed effects were secondary to counterregulations of other proteins or to changes in cellular metabolism. To exclude these possibilities, we injected hRS1 protein into oocytes expressing hSGLT1 and measured the short-term effects on glucose transport. We observed that hSGLT1-mediated methyl- - D -[ 14 C]glucopyranoside (AMG) uptake in X. laevis oocytes was inhibited within minutes after injection of purified hRS1 protein. The inhibition of hSGLT1 after injection of hRS1 protein was abolished when a dominant negative dynamin mutant was coexpressed, and was increased when PKC was activated. It was blocked after inhibition of the exocytotic pathway but not altered by inhibitors of endocytosis. Importantly, the inhibitory effect of injected RS1 protein on the expression of glucose uptake by hSGLT1 was decreased when the intracellular concentration of the nonmetabolizable glucose analog AMG was enhanced.
MATERIALS AND METHODS
Biochemicals. [ 14 C]AMG (11.7 GBq/mmol) was obtained from Amersham Biosciences (Freiburg, Germany) and [ 14 C]TEA (2.0 GBq/mmol) from Biotrend (Cologne, Germany). The proteasome inhibitor MG-132, chlorpromazine, imipramine, filipin complex, and botulinum toxin B (BTXB) were supplied by Sigma (Taufkirchen, Germany). Brefeldin A (BFA) was obtained from Calbiochem (Schwalbach, Germany), nickel(II)-charged nitrilotriacetic acid-agarose (Ni 2+ -NTA-agarose) from Qiagen (Hilden, Germany), conjugate of protein G with horseradish peroxidase from Bio-Rad (Munich, Germany), and prestained molecular weight markers (BenchMark) from Life Technologies (Karlsruhe, Germany). The other chemicals and enzymes were purchased as described earlier ( 2, 21, 51 ).
Plasmids. DNA of rat wild-type dynamin I and dominant negative mutant of dynamin I (K44A) ( 32 ) were digested with Ssp I and Kpn I and cloned into the oocyte expression vector pRSSP ( 2 ). For expression of hRS1 in oocytes of X. laevis, a cDNA sequence coding for a COOH-terminal His 8 tag downstream of the last amino acid of hRS1 was introduced into the plasmid pBS2953 ( 28 ) using PCR and the primers 5'-ACTTCAGGAGTCTAGGTG-3' (forward, position 3373-3390 on the hRS1 sequence, accession number X82877 ) and 5'-CG TCTAGA TCA GTGGTGGTGGTGGTGGTGGTGGTG TGTAGGAACTACGATGTT-3' (reverse, the Xba I site is underlined, and the His 8 sequence is shown in italics). The PCR product was digested with Hin dII and Xba I and cloned into the pBS2953 replacing the Hin dII/ Xba I fragment of the original plasmid. The sequence of the modified hRS1 (hRS1H) was verified by DNA sequencing. For the expression in Sf9 cells, human RS1 was provided at its NH 2 terminus with an S-tag and thrombin cleavage site (originated from pET44a plasmid, Novagen, Schwalbach/Ts., Germany) and at its COOH terminus with another thrombin cleavage site and His 8 tag (originated from pET42b plasmid, Novagen) and cloned in several steps into the plasmid pSL1180 (Amersham Pharmacia Biotech). The sequence of the tagged hRS1 was verified by DNA sequencing and recloned using Pst I and Xba I restriction enzymes into the vector pVL1392 (BD Biosciences, Erembodegem, Belgium). The resulting vector (S-tag-hRS1-His/pVL1392) was transfected together with linearized baculovirus BaculoGold DNA (BD Biosciences) into Sf9 insect cells. Selection of plaques for the recombinant virus was performed according to the manufacturer?s recommendations. The recombinant virus was grown to a titer of 4-6 x 10 7 pfu/ml and used to transfect insect cells for hRS1 expression.
In vitro synthesis of cRNA. For injection into X. laevis oocytes, m7G(5')G-capped cRNA was prepared, purified, and stored as described earlier ( 51, 52 ). To prepare sense cRNA from hRS1 ( 28 ), hRS1H, hSGLT1 ( 13 ), hOCT2 ( 10 ), wild-type dynamin (DyWt), and a dominant negative mutant of dynamin I from rat (DyMu) ( 32 ), the respective purified plasmids were linearized with Eco RI (hSGLT1), Not I (hOCT2), Mlu I (DyWt, DyMu), Xba I (hRS1), or Sac I (hRS1H). cRNA was synthesized using T3 polymerase (hSGLT1), SP6 polymerase (DyWt, DyMu), or T7 polymerase (hRS1, hRS1H, hOCT2) as described earlier ( 51, 52 ). cRNAs were prepared employing an "mMESSAGE mMACHINE" kit (Ambion, Austin, TX) using ammonium acetate precipitation. cRNA concentrations were estimated from ethidium bromide-stained agarose gels using a polynucleotide marker as a standard ( 11 ).
Expression of transporters, RS1 and dynamin in oocytes of X. laevis. Stage V-VI oocytes were obtained from X. laevis by partial ovariectomy, selected, and injected with cRNAs as described earlier ( 51 ). For uptake measurements, 50 nl/oocyte of water-solved hSGLT1 cRNA (2.5 ng) or hOCT2 cRNA (2.5 ng ) were injected alone or together with either hRS1 cRNA (7.5 ng), hRS1H cRNA (7.5 ng), dynamin I wild-type cRNA (10 ng), and/or dynamin I mutant cRNA (10 ng). Water injected or noninjected oocytes served as controls. To allow translation and targeting of the expressed proteins, the injected oocytes were incubated for 2-3 days at 16°C in Ori buffer (5 mM MOPS, 100 mM NaCl, 3 mM KCl, 2 mM CaCl 2, and 1 mM MgCl 2, adjusted to pH 7.4 using NaOH) containing 50 mg/l gentamicin. For isolation of hRS1H protein, 100 oocytes/experiment were injected with 15 ng of hRS1H cRNA/oocyte, and the oocytes were incubated at 16°C for 2 days in Ori buffer, and for another day in Ori buffer containing 5 µM proteasome inhibitor MG-132. Control proteins were prepared from noninjected oocytes incubated in the same way.
Preparation of cytosol containing hRS1H protein from oocytes. To prepare cytosolic proteins from oocytes, 100 oocytes expressing hRS1H or 100 noninjected oocytes were homogenized in 2 ml K-Ori buffer (5 mM MOPS, 103 mM KCl, 2 mM CaCl 2, and 1 mM MgCl 2, adjusted to pH 7.4 using KOH) containing protease inhibitors [1 mM 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride, 0.8 µM aprotinin, 50 µM bestatin, 15 µM N -(trans-epoxysuccinyl)- L -leucine-4-guanidinobutylamide, 20 µM leupeptin, and 10 µM pepstatin A]. The homogenates were centrifuged for 10 min (4°C) at 5,000 g, and the intermediate phase was collected to remove egg yolk (top layer) and large debris (sediment). Centrifugation and collection of the intermediate phase were repeated four times. To remove metabolites, the cytosolic extract was concentrated three times to 20 µl on Centricon YM-30 filters from Millipore (Bedford, MA), diluted 50-fold with K-Ori buffer and finally concentrated to 10 µl.
For affinity purification of hRS1H protein on Ni 2+ -NTA-agarose, the cytosolic extract was prepared from oocytes as described above using 20 mM Tris·HCl, pH 8.0, that contained 500 mM NaCl and 5 mM imidazole (binding buffer). Samples were frozen in liquid nitrogen and stored at -80°C.
Expression of hRS1H protein in Sf9 insect cells. Sf9 cells were cultured at 27°C in Grace?s insect medium containing 10% (vol/vol) fetal calf serum and 50 mg/l of gentamicin. A suspension of 2 x 10 8 Sf9 cells (Invitrogen, Karlsruhe, Germany) was transfected with a recombinant baculovirus (MOI = 1) ( 23 ) and incubated at 27°C in 75-cm 2 flasks. After 3 days the cells were harvested by scraping them off the surface of the flasks and collected by a 10-min centrifugation at 300 g. Cells were washed twice by suspending in 25 ml 1.9 mM NaH 2 PO 4, 8.1 mM Na 2 HPO 4, and 154 mM NaCl, pH 7.4 (PBS) followed by centrifugation at 300 g. Cells from five 14.5-cm-diameter culture plates were suspended in 2 ml of binding buffer containing protease inhibitors, sonicated for 3 min at 400 W (4°C), diluted to 20 ml with binding buffer, centrifuged for 10 min at 10,000 g, and the supernatant was collected (cytosolic proteins).
Purification of hRS1H protein on Ni 2+ -NTA. For purification of hRS1H from oocytes, 0.2 ml of Ni 2+ -NTA-agarose equilibrated with binding buffer was added to 1 ml of cytosolic proteins. For purification of hRS1H from Sf9 cells, 1 ml of Ni 2+ -NTA-agarose was added to 20 ml of the cytosolic proteins prepared from Sf9 cells. The suspensions were shaken for 2 h at 4°C and placed into columns. Columns were washed with five volumes of 20 mM Tris·HCl and 500 mM NaCl, pH 8, containing 20 mM imidazole. For elution of protein, columns were washed with 20 mM Tris·HCl and 500 mM NaCl, pH 8, containing 50 mM imidazole or 100 mM imidazole. Fractions (100 or 500 µl) were collected during purification of hRS1H from oocytes and Sf9 cells, respectively, dialyzed against K-Ori buffer, concentrated using Centricon YM-30 filters, and stored at -80°C.
SDS-PAGE and Western blotting. Protein concentration was determined according to Bradford using bovine serum albumin as a standard ( 1 ). For SDS-PAGE, protein samples were pretreated for 30 min at 37°C in 60 mM Tris·HCl, pH 6.8, 100 mM dithiothreitol, 2% (wt/vol) SDS, and 7% (vol/vol) glycerol. Electrophoresis and Western blotting were performed as described ( 21, 25 ). Proteins separated by SDS-PAGE were transferred by electroblotting to polyvinylidene difluoride membrane and stained for hRS1 protein using an earlier described antibody that had been raised against porcine RS1 expressed in Escherichia coli (pRS1-ab) ( 47 ). For antibody reaction, the blot was incubated for 2 h at room temperature with pRS1-ab diluted 1:20,000 in PBS containing 2% (wt/vol) bovine serum albumin and 0.1% Tween 20. After a washing, the blot was incubated for 2 h at room temperature with a conjugate of protein G and horseradish peroxidase from Bio-Rad diluted 1:20,000 in PBS. The immunoreaction was visualized by enhanced chemiluminescence (ECL system; Amersham Biosciences Europe). Prestained molecular weight markers (BenchMark, Life Technologies) were used to determine apparent molecular masses.
Injection of proteins or biochemicals into oocytes. Two to three days after cRNAs of hSGLT1, hOCT2, hSGLT1 plus dynamin wild-type, or hSGLT1 plus dynamin mutant had been injected into oocytes, 50 nl of K-Ori buffer/oocyte were injected containing between 50 and 500 ng of proteins, 2.5 pmol of MG-132 (inhibitor of the proteasome), 2 ng of BTXB (inhibitor of exocytosis), 2.7 pmol of BFA (inhibitor of vesicle release from the trans -Golgi network), and/or 1 pmol of sn -1,2-dioctanoyl-glycerol (DOG; activator of PKC). After injection of proteins and/or inhibitors, oocytes were routinely incubated for 30 min at 21°C in Ori buffer (see Figs. 2, 4 - 10 ) before uptake of [ 14 C]AMG or [ 14 C]TEA was measured. In one type of experiment, oocytes were incubated for various time periods after hRS1H protein and MG-132 had been injected (see Fig. 3 ).
Fig. 1. Expression of human RS1 (hRS1) with His-tag (hRS1H) in oocytes and insect cells and affinity chromatographic purification with nickel(II)-charged nitrilotriacetic acid (Ni 2+ -NTA)-agarose. hRS1H was expressed in Xenopus laevis oocytes ( A ) or Sf9 cells ( B ). Cytosolic proteins from hRS1H-expressing oocytes ( A, lanes 1 and 5 ), noninjected control oocytes ( A, lanes 2 and 6 ), or from Sf9 cells expressing hRS1H ( B, lanes 1 and 3 ) were prepared. Cytosolic proteins were incubated with Ni 2+ -NTA-agarose beads, washed with buffers containing 20 mM imidazole, and eluted with 50 or 100 mM imidazole. Proteins were separated by SDS-PAGE and stained by Coomassie brilliant blue or analyzed by Western blotting using a specific antibody against pRS1. Lanes 3 and 7 in A show 1 of the elution fractions from beads that were incubated with cytosol of hRS1H-expressing oocytes (hRS1H enr.), and lanes 4 and 8 show 1 of the elution fractions from beads incubated with cytosol of noninjected control oocytes (110-kDa prot.). In B, elution fractions from beads that were incubated with cytosol of hRS1H-expressing insect cells ( lanes 2 and 4 ) are shown. Per lane, 40 µg ( A, lanes 1-4; B, lanes 1 and 3 ) or 3 µg of protein ( A, lanes 5-8; B, lanes 2 and 4 ) were applied. The data show minor enrichment of hRS1H expressed in oocytes and purification of hRS1H expressed in insect cells.
Fig. 2. Short-term inhibition of human SGLT1 (hSGLT1)-expressed methyl- - D -glucopyranoside (AMG) uptake by hRS1 protein. Oocytes were injected with 2.5 ng of hSGLT1 cRNA, with 2.5 ng hSGLT1 cRNA plus 7.5 ng hRS1 cRNA, or with 2.5 ng hSGLT1 cRNA plus 7.5 ng hRS1H cRNA, and incubated for 3 days. Then, uptake of [ 14 C]AMG for 15 min was measured. Alternatively, oocytes expressing hSGLT1 were injected with 50 nl of K-Ori buffer containing cytosolic proteins from control oocytes (ooc. control cyt.); cytosolic proteins from oocytes expressing hRS1H (ooc. hRS1H cyt.); hRS1H from hRS1H-expressing oocytes that was enriched by affinity chromatography on a Ni 2+ -NTA-agarose column (ooc. hRS1 enr.); 110-kDa protein from control oocytes that was copurified during affinity chromatography on a Ni 2+ -NTA-agarose column (ooc. 110 kDa prot.); and hRS1H protein that was expressed in Sf9 cells and purified on a Ni 2+ -NTA-agarose column (Sf9 hRS1H pur.). Thirty minutes after injection of proteins, uptake of 50 µM [ 14 C]AMG was measured. The data show that injection of hRS1H protein inhibits hSGLT1-expressed AMG uptake within 30 min to the same degree as observed after coexpression of hRS1 cRNA or hRS1H cRNA. Mean values that differ significantly from AMG uptake values obtained after expression of SGLT1 without coexpression of hRS1 and without injection of RS1 protein are indicated (** P < 0.01).
Fig. 3. Proteasomal degradation of hRS1H protein. Oocytes were injected with 2.5 ng of hSGLT1 cRNA and incubated for 3 days in Ori buffer (circles). hRS1H protein that had been enriched from hRS1H-expressing oocytes, was injected into oocytes. In some oocytes, 2.5 pmol of MG-132/oocyte were injected together with hRS1H (squares). Oocytes were incubated for various periods of time, and uptake of 50 µM [ 14 C]AMG for 15 min was measured. The data show that the activity of injected hRS1H protein was decreased after 60 or 90 min and that this inactivation of hRS1H was prevented when the proteasomal inhibitor MG-132 was present. Mean values of AMG uptake in oocytes injected with enriched hRS1H protein that were significantly different in the absence or presence of MG-132 are indicated (** P < 0.01, *** P < 0.001).
Fig. 4. Dynamin dependence of the inhibition of hSGLT1-expressed AMG uptake by hRS1H protein. Oocytes were injected with 2.5 ng of hSGLT1 cRNA, 2.5 ng of hSGLT1 cRNA plus 10 ng of dynamin wild-type cRNA (DyWt), or with 2.5 ng of hSGLT1-cRNA plus 10 ng dynamin I dominant negative mutant cRNA (DyMu) and incubated 3 days for expression. hRS1H protein enriched from oocytes injected with hRS1H (hRS1H) was injected, oocytes were incubated for 30 min, and uptake of 50 µM [ 14 C]AMG was measured. In control 1, no injection into the hSGLT1-expressing oocytes was performed whereas in control 2, K-Ori buffer with 110-kDa protein isolated from control oocytes was injected. The data suggest that the inhibition of SGLT1 by injected hRS1H protein is dynamin dependent. Significant (* P < 0.05. *** P < 0.001) and some nonsignificant (n.s.) differences between mean values of AMG uptake are indicated.
Fig. 10. Effect of different intracellular AMG concentrations on the inibition of hOCT2-expressed uptake of 10 µM [ 14 C]TEA by hRS1H protein. Oocytes were injected with 2.5 ng of hOCT2 cRNA and incubated 3 days in Ori buffer. Fifty nanoliters of K-Ori buffer, of K-Ori buffer containing 2 mM AMG, or of K-Ori buffer containing 80 mM AMG was injected without or with enriched hRS1H. After 30-min incubation in Ori buffer, uptake of 10 µM [ 14 C]TEA was measured. The data show that hRS1H protein inhibits hOCT2 in addition to hSGLT1 and that the inhibition of hOCT2 by hRS1H protein is modulated by (an) intracellular glucose binding site(s). Significant (* P < 0.05, ** P < 0.01, *** P < 0.001) and n.s. differences between uptake rates are indicated.
To investigate whether intracellular AMG influences the inhibition of hSGLT1 by hRS1H protein (see Figs. 8 and 9 ), oocytes expressing hSGLT1 were incubated for 15 min with various concentrations of AMG. Then, partially purified hRS1H protein or control protein was injected and the oocytes were incubated for 30 min in the presence of the respective concentrations of AMG. Thereafter, uptake of radioactively labeled AMG was measured at the indicated concentrations of AMG. To determine whether intracellular AMG influences the inhibition of hOCT2-mediated TEA uptake (see Fig. 10 ), hOCT2-expressing oocytes were injected with 50 nl K-Ori buffer containing partially purified hRS1H protein or control protein plus different concentrations of AMG. After a 30-min incubation, uptake of 50 µM [ 14 C]TEA was measured.
Fig. 8. Inhibition of hSGLT1-expressed AMG uptake by hRS1H protein measured in the presence of different AMG concentrations. Oocytes were injected with 2.5 ng of hSGLT1 cRNA/oocyte and incubated for 3 days in Ori buffer. Thereafter, they were incubated for 15 min with Ori buffer containing the indicated concentrations of nonradioactive AMG. Then, K-Ori buffer ( ) or K-Ori buffer containing hRS1H protein that was enriched from oocytes expressing hRS1H ( ) were injected. After 30-min incubation in Ori buffer containing the respective concentrations of nonradioactive AMG, [ 14 C]AMG was added and uptake of [ 14 C]AMG was measured ( top panel). The data indicate that the inhibition of hSGLT1-expressed AMG uptake by hRS1H protein ( bottom panel) is dependent on the AMG concentration during hRS1H injection and/or on the AMG concentration during the uptake measurement.
Fig. 9. Effect of different intracellular AMG concentrations on the inhibition of hSGLT1-expressed uptake of 50 µM [ 14 C]AMG by hRS1H protein. Oocytes were injected with 2.5 ng of hSGLT1 cRNA and kept for 3 days in Ori buffer. They were preincubated for 15 min in Ori buffer containing the indicated concentrations of AMG, washed, and K-Ori buffer or K-Ori buffer containing hRS1H protein were injected as in Fig. 8. After 30-min incubation in Ori buffer containing the same AMG concentrations as during the preincubation period, uptake of 50 µM [ 14 C]AMG during 15 min was measured. The data show that the inhibition of hSGLT1 by hRS1H protein is dependent on the intracellular concentration of AMG. Significant differences between uptake rates (** P < 0.01, *** P < 0.001) and a n.s. difference are indicated.
Incubation of oocytes with membrane-permeant inhibitors. To block endocytosis, oocytes were incubated with 40 µM chlorpromazine, 40 µM imipramine, or 1 µg/ml filipin complex ( 40, 53 ). The incubation with these inhibitors was started 1 h before the injection of proteins, and the inhibitors remained present during the 30-min incubation period after protein injection and during the transport measurement. To stimulate PKC, oocytes were incubated for 2 min with 1 µM PMA before the transport measurements were started.
Uptake measurements. Uptake of [ 14 C]AMG mediated by hSGLT1 was measured as described earlier ( 51, 52 ). Briefly, oocytes expressing hSGLT1, noninjected control oocytes, or water-injected control oocytes were incubated for 15 min at room temperature in Ori buffer containing 50 µM [ 14 C]AMG or the indicated AMG concentrations without or with 200 µM phlorizin. Thereafter, the oocytes were washed four times in ice-cold Ori buffer and single oocytes were solubilized in 5% (wt/vol) SDS and analyzed for radioactivity by scintillation counting. In the presence of phlorizin, tracer uptake in oocytes expressing hSGLT1 was similar to noninjected or water-injected oocytes and <5% of uptake in the absence of phlorizin. Transport of [ 14 C]TEA in oocytes expressing the organic cation transporter hOCT2 was measured as described ( 52 ). Oocytes were incubated for 15 min with 10 µM [ 14 C]TEA in the absence or presence of 100 µM cyanine863. TEA uptake in the presence of cyanine863 was identical to TEA uptake in noninjected or water-injected oocytes.
Calculations and statistics. The uptake rates shown in the figures represent arithmetic means ± SE from 8-10 oocytes expressing SGLT1 or hOCT2 that were corrected for the uptake rates measured in noninjected control oocytes. In control oocytes without expression of SGLT1 or hOCT2, uptake of 50 µM [ 14 C]AMG and of 10 µM [ 14 C]TEA was smaller than 1 pmol·oocyte -1 ·15 min -1. In the presence of 200 µM phlorizin, uptake of [ 14 95%. Representative experiments of two to four experiments are shown that were performed with different batches of oocytes. One-way ANOVA with a post hoc Tukey comparison was used to test for the significance of a difference between mean values.
RESULTS
Short-term inhibition of hSGLT1-expressed transport of AMG after injection of RS1 protein. Previously, we observed that AMG uptake into oocytes of X. laevis coinjected with cRNA of hSGLT1 and hRS1 was decreased compared with control oocytes expressing hSGLT1 alone ( 28, 52 ). The decrease in AMG uptake was due to the reduction of SGLT1 protein within the plasma membrane of the oocytes ( 47 ). To distinguish whether hRS1 exhibits a long-term effect on the expression of hSGLT1 or a short-term effect on the turnover of the transporter, we expressed hSGLT1 by injecting SGLT1 cRNA into oocytes and incubating them for 2 or 3 days, and subsequently injected hRS1 protein with a His-tag at the COOH terminus (hRS1H). After 30 min, we measured the uptake of 50 µM [ 14 C]AMG over a time period of 15 min. For hRS1H protein injection, three types of preparations were used: 1 ) cytosolic proteins from X. laevis oocytes expressing hRS1H (hRS1H cyt. in Figs. 1 and 2 ), 2 ) hRS1H expressed in oocytes that was enriched on Ni 2+ -NTA-agarose but contained an abundant endogenous 110-kDa oocyte protein (called enriched hRS1H protein or hRS1H enr. in Figs. 1 and 2 ), and 3 ) hRS1H expressed in Sf9 insect cells and purified on Ni 2+ -NTA-agarose (hRS1H pur. in Figs. 1 and 2 ). Identical results were obtained with the three preparations. In Fig. 1, the three preparations are characterized by SDS-PAGE and Western blotting using a previously described antibody against porcine RS1 that cross-reacts with human RS1 ( 47 ). Cytosolic proteins isolated from control oocytes and hRS1H-expressing oocytes (hRS1H cyt.) are characterized in Fig. 1 A, lanes 1, 2, 5, and 6. Note that the cytosol of the oocytes contains an abundant endogenous 110-kDa protein comigrating with hRS1 that is not recognized by the antibody against RS1 (pRS1-ab). By affinity chromatography on Ni 2+ -NTA-agarose, some cytosolic proteins were removed; however, the abundant 110-kDa protein was copurified ( Fig. 1 A, lanes 3, 4, 7, and 8 ). After Ni 2+ -NTA-agarose chromatography of cytosolic proteins from hRS1H expressing Sf9 insect cells, the hRS1 protein running at an apparent molecular mass of 100 kDa was purified ( Fig. 1 B ). From insect cells expressing hRS1H, no significant copurification of endogenous proteins was observed. Contamination of purified hRS1H from insect cells with an endogenous protein migrating to the same position as hRS1H was excluded since no band running at 90-110 kDa was observed when the organic cation transporter rOCT1 expressed in Sf9 cells was purified on Ni 2+ -NTA-agarose (see Fig. 4 in Ref. 21 ).
As shown in Fig. 2, the uptake of 50 µM [ 14 C]AMG mediated by hSGLT1 was inhibited by 50-70% when hRS1 was coexpressed as described previously ( 52 ). The same effect was observed after coexpression of His-tagged hRS1 ( Fig. 2, hSGLT1+hRS1H). When cytosolic proteins prepared from noninjected control oocytes ( Fig. 2, ooc. control cyt.) were injected into oocytes expressing hSGLT1, hSGLT1-induced uptake of 50 µM [ 14 C]AMG measured 30 min after injection was not altered. In contrast, hSGLT1-mediated uptake of 50 µM [ 14 C]AMG was inhibited by 50% when cytosolic proteins from oocytes expressing hRS1H ( Fig. 2, ooc. hRS1H cyt.) were injected. A similar inhibition was observed when enriched hRS1H protein expressed in oocytes ( Fig. 2, ooc. hRS1H enr.) or purified hRS1H protein expressed in insect cells ( Fig. 2, Sf9 hRS1H pur.) was injected. In contrast, injection of the endogenous 110-kDa protein from noninjected control oocytes that was copurified with hRS1H on Ni 2+ -NTA-agarose ( Fig. 2, ooc. 110 kDa-prot.) had no effect on hSGLT1-mediated AMG uptake. The data indicate that hRS1 protein inhibits SGLT1-induced AMG uptake within 30 min.
To determine how long hRS1H protein is active after injection into the oocytes expressing hSGLT1, we expressed hSGLT1 by cRNA injection and after a 3-day incubation, injected enriched hRS1H protein from oocytes. After incubation of the oocytes for various periods of time, we measured the uptake of 50 µM [ 14 C]AMG for 15 min. Figure 3 shows that the inhibitory effect of hRS1H protein was decreased after 60-min incubation and was undetectable if oocytes were incubated for 90 min. To test whether hRS1H protein is degraded by the proteasome, oocytes were injected with 2.5 pmol of the proteasome inhibitor MG-132 together with enriched hRS1H protein. MG-132 did not change SGLT1-mediated AMG uptake; however, in the presence of MG-132 the inhibition of hSGLT1-induced AMG uptake by injected hRS1H protein remained identical when the oocytes were incubated 15-90 min after the protein injection ( Fig. 3 ). The data suggest that hRS1H is degraded by the proteasome.
hRS1 protein inhibits a dynamin- and PKC-dependent exocytotic pathway of hSGLT1. Previous coexpression experiments using hSGLT1 and hRS1 without and with additional expression of a dominant negative dynamin I mutant showed that functionally active dynamin is required for inhibition of hSGLT1 by hRS1 ( 52 ). Since dynamin is involved in scission of vesicles during endocytosis and exocytosis ( 15, 20, 26 ), it remained unclear whether hRS1 downregulates hSGLT1 by stimulating endocytosis or inhibiting exocytosis. In Fig. 4, we compared the inhibition of AMG uptake after injection of hRS1H protein into oocytes that expressed either hSGLT1, hSGLT1 plus dynamin I wild-type (DyWt), or hSGLT1 plus dominant negative dynamin I mutant (DyMu). After injection of hRS1H protein, similar results were obtained as after coexpression of hRS1 cRNA ( 52 ). The inhibition of hSGLT1 mediated AMG uptake by injected hRS1 protein was significantly reduced when DyMu was coexpressed ( Fig. 4 ).
To identify whether the short-term inhibition of hSGLT1 by hRS1 protein is due to an increase in dynamin-dependent endocytosis, we investigated the inhibition of SGLT1-expressed AMG uptake by hRS1H protein when clathrin-dependent endocytosis was blocked by removing clathrin-coated pits from the plasma membrane with chlorpromazine or imipramine ( 53 ) or by inhibiting caveolae-mediated endocytosis with filipin ( 40 ). Oocytes were preincubated for 1 h with 40 µM chlorpromazine, 40 µM imipramine, or 1 µg/ml filipin. Effectivity of the inhibitors under the employed experimental conditions is indicated by the observations that hSGLT1-expressed AMG uptake 1 ) was 25% stimulated by chlorpromazine (see Fig. 5 A; significant stimulation was observed in all 3 performed experiments) and imipramine (see Fig. 5 A; significant stimulation was observed in 1 of 3 experiments); and 2 ) 50% stimulated by filipin (see Fig. 5 B; significant stimulation was observed in all 3 performed experiments). In the presence of chlorpromazine, imipramine, or filipin, hRS1H protein inhibited AMG uptake to the same level as in the absence of these compounds ( Fig. 5 ). The data contradict the hypothesis that posttranscriptional regulation of hSGLT1 by hRS1 is due to an increase in clathrin- or caveolae-mediated endocytosis of hSGLT1.
Fig. 5. Inhibition of hSGLT1-expressed AMG uptake by hRS1H protein in the presence of inhibitors of endocytosis. Oocytes were injected with 2.5 ng of hSGLT1 cRNA, incubated for 3 days in Ori buffer, injected with enriched hRS1H protein (hRS1H), incubated for 30 min in Ori buffer, and uptake of 50 µM [ 14 C]AMG was measured. In some experiments, 40 µM chlorpromazine ( A ), 40 µM imipramine ( A ), or 1 µg/ml filipin complex ( B ) was added to incubation media and transport buffers 1 h before hRS1H protein was injected. The data indicate that hRS1H protein inhibits hSGLT1 when endocytosis is blocked. Significant (* P < 0.05, *** P < 0.001) and some n.s. differences between mean values are indicated.
If hRS1 protein inhibits vesicle release during the exocytotic pathway or during recycling of hSGLT1, no effect of hRS1H protein on hSGLT1-mediated AMG uptake should be expected when fusion of hSGLT1-containing vesicles with the plasma membrane is blocked. To test this prediction, we blocked exocytosis in oocytes expressing hSGLT1 by injecting 2 ng BTXB/oocyte 30 min before AMG uptake was measured ( Fig. 6 A ). BTXB cleaves synaptobrevin located at intracellular vesicles ( 46 ). In our experiments, BTXB inhibited hSGLT1-mediated AMG uptake by 40% within 30 min. This indicates that BTXB was active in the oocytes and that hSGLT1 exhibits a rapid turnover in the plasma membrane during which synaptobrevin-dependent exocytosis is a critical step. At variance, it has been previously described that botulinum toxin C1 that inactivates syntaxin 1A does not effect the amount of SGLT1 in the plasma membrane ( 34 ). Moreover, Fig. 6 A shows also that BTXB inhibited SGLT1-mediated AMG uptake by about the same degree as hRS1H protein. When hRS1H protein was injected in the presence of BTXB, no additional inhibition of SGLT1-induced AMG uptake was observed. The data suggest that hRS1 protein inhibits either recycling and/or the exocytotic pathway of SGLT1.
Fig. 6. hRS1H protein does not inhibit hSGLT1 expressed AMG uptake when the exocytotic pathway is blocked. For expression of hSGLT1, oocytes were injected with 2.5 ng of hSGLT1 cRNA, incubated for 3 days, and inhibition of AMG uptake by hRS1H protein was measured in the presence of botulinum toxin B (BTXB) or brefeldin A (BFA). A : BTXB (2 ng/oocyte), hRS1H protein that was enriched from hRS1H-expressing oocytes, or BTXB plus hRS1H protein were injected, oocytes were incubated for 30 min and uptake of 50 µM [ 14 C]AMG was measured. B : in other oocytes expressing hSGLT1, BFA (2.7 pmol/oocyte) and/or enriched hRS1H protein was injected, oocytes were incubated for 30 min in Ori buffer, and uptake of 50 µM [ 14 C]AMG was measured. Significant differences between mean values of oocytes without and with hRS1H, BTXB, and BFA (*** P < 0.001) and n.s. effects of hRS1H in the presence of BTXB or BFA are indicated.
To further investigate how RS1 inhibits SGLT1 posttranscriptionally, we investigated whether BFA influences posttranscriptional inhibition of hSGLT1-mediated AMG uptake by hRS1 ( 24 ). BFA inhibits guanosine nucleotide exchange factors that activate ADP-ribosylation factors, which regulate the assembly of coat complexes at the TGN and endosomes involved in protein sorting and release of vesicles ( 3, 6, 14 ). When 2.7 pmol BFA/oocyte were injected into oocytes expressing hSGLT1 and oocytes were incubated for 30 min, the uptake of 50 µM [ 14 C]AMG was decreased by 50% ( Fig. 6 B ). When enriched hRS1H protein was injected into hSGLT1-expressing oocytes, expressed AMG uptake was decreased to a similar degree (see Figs. 2, 4, 5, 6, and 7 ). No significant further decrease in hSGLT1-mediated AMG uptake by hRS1H protein was observed when 2.7 pmol BFA/oocyte were injected together with hRS1H ( Fig. 6 B ). The data indicate that hRS1 inhibits the dynamin-dependent release of vesicles derived from the TGN or endosomes.
Fig. 7. Inhibition of hSGLT1-expressed AMG uptake by hRS1H protein is increased by stimulation of PKC. Oocytes were injected with 2.5 ng of hSGLT1 cRNA, incubated for 3 days, injected with 50 nl K-Ori buffer per oocyte without or with enriched hRS1H protein, incubated for 30 min, and uptake of 50 µM [ 14 C]AMG was measured. In part of the experiments PKC was stimulated by incubating the oocytes for 2 min with 1 µM PMA or by injecting 1 pmol of DOG per oocyte together with the K-Ori buffer. Significant differences are indicated (** P < 0.01, *** P < 0.001).
We also investigated whether the short-term inhibition of hSGLT1-mediated AMG uptake by hRS1H protein is increased when PKC is activated. Previously, we observed that stimulation of PKC resulted in an increase or decrease in hSGLT1-mediated AMG uptake when hSGLT1 was expressed alone or together with hRS1, respectively ( 52 ). Figure 7 shows that hSGLT1-expressed uptake of 50 µM AMG was significantly increased when PKC was activated by DOG or PMA as described earlier ( 52 ). Activation of PKC by DOG and PMA increased the inhibition of AMG uptake observed after injection of hRS1H protein. The data suggest that the hRS1-induced inhibition of dynamin-dependent release of SGLT1-containing vesicles from the TGN is regulated by PKC.
Inhibition of transport activity of hSGLT1 and the organic cation transporter hOCT2 by hRS1 protein is dependent on intracellular AMG. Oocytes expressing hSGLT1 were injected with K-Ori buffer or with K-Ori buffer containing hRS1H protein that was partially purified from oocytes. After 30 min, the oocytes were superfused with Ori buffer, clamped to -50 mV, and superfused with Ori buffer containing 1 or 3 mM AMG. The AMG-induced inward currents were measured as described ( 39 ). In several experiments, we were not able to detect a significant difference between AMG-induced inward currents in SGLT1-expressing oocytes without or with injection of hRS1H protein (data not shown). This finding is at variance with previous experiments in which SGLT1-expressed inward currents induced by 1, 3, or 10 mM AMG were inhibited after coexpression of hRS1 ( 52 ). It suggests that coexpression of hRS1 with hSGLT1 leads to more pronounced changes compared with short-term effects observed after injection of hRS1 protein.
To test whether the inhibition of hSGLT1-induced AMG uptake by hRS1H protein is dependent on the concentration of glucose, we preincubated hSGLT1-expressing oocytes for 15 min with various concentrations of the nonmetabolized glucose analog AMG, injected a part of the oocytes with K-Ori buffer and another part with K-Ori buffer containing enriched hRS1H protein, incubated the oocytes for 30 min in the presence of the respective concentrations of AMG, and measured the uptake of [ 14 C]AMG during a 15-min incubation at the respective AMG concentrations. Injection of hRS1H protein inhibited SGLT1-expressed [ 14 C]AMG uptake at AMG concentrations between 10 and 300 µM. In the presence of 500 µM AMG, the inhibition by hRS1H was much smaller, and no significant inhibition was observed at 2 mM AMG ( Fig. 8 ). The data show that the inhibition of SGLT1-mediated AMG uptake by hRS1H protein is dependent on the AMG concentration during preincubation and/or during the uptake measurements.
To differentiate whether hRS1H protein changes the glucose dependence of AMG uptake or whether the downregulation of hSGLT1 by hRS1H is dependent on the concentration of AMG, we incubated hSGLT1-expressing oocytes for 15 min in Ori buffer, Ori buffer containing 50 µM nonradioactive AMG, or Ori buffer containing 2 mM AMG. Then, we injected the oocytes with K-Ori or K-Ori containing enriched hRS1H protein and incubated them for 30 min in Ori buffer containing the same concentrations of nonradioactive AMG as during the preincubation period. From parallel performed uptake measurements with [ 14 C]AMG, intracellular concentrations of 50 µM and 0.5 mM AMG were estimated after incubation with 50 µM and 2 mM AMG, respectively (assuming an intracellular volume of 1 µl/oocyte). Thirty minutes after injection of hRS1H protein, we washed the oocytes with Ori buffer and measured uptake of 50 µM [ 14 C]AMG using an incubation period of 15 min. Figure 9 shows that hRS1H protein inhibited SGLT1-mediated uptake of 50 µM [ 14 C]AMG when the oocytes were preincubated without AMG or with 50 µM AMG; however, no significant inhibition was observed when the oocytes were preincubated with 2 mM AMG. The data show that high AMG concentrations prevent the inhibition of hSGLT1-expressed AMG uptake by hRS1 protein.
To distinguish whether the AMG dependence of inhibition of hSGLT1 by hRS1 protein is based on the interaction of AMG with hSGLT1 or requires an AMG binding site on a different protein, we investigated whether transport activity of the human organic cation transporter hOCT2 is inhibited by hRS1H protein and whether this inhibition of hOCT2 is influenced by AMG. Previously, we observed that coexpression of hRS1 with the organic cation transporter hOCT2 induced a significant decrease in the uptake of [ 14 C]TEA expressed by this transporter ( 52 ). In contrast, we were not able to inhibit hOCT2-expressed TEA uptake in oocytes by injection of hRS1H protein in three separate experiments (data not shown). Since the inhibition of hSGLT1 by hRS1H protein was always measured in the presence of intracellular AMG (because AMG enters the oocytes during the uptake measurements), we wondered whether a low intracellular concentration of AMG is required for the inhibitory effect of hRS1H protein on hSGLT1 or other transporters. We injected 2.5 ng of hOCT2 cRNA into oocytes, incubated the oocytes for 3 days, and injected 50 nl of K-Ori buffer containing different concentrations of AMG (0, 2, or 80 mM) or 50 nl of K-Ori buffer containing hRS1H protein partially purified from oocytes plus different concentrations of AMG (0, 2, or 80 mM). Assuming an internal oocyte volume of 1 µl, internal concentrations of 0.1 and 4.0 mM AMG were estimated for injection of 2 and 80 mM AMG, respectively. After a 30-min incubation in Ori buffer, uptake of 10 µM [ 14 C]TEA during 15 min was measured ( Fig. 10 ). Significant inhibition of hOCT2-mediated TEA uptake by hRS1H protein was only observed when 50 nl of 2 mM AMG were injected. The data indicate that intermediate intracellular concentrations of glucose are required for the downregulation of hSGLT1 and hOCT2 by hRS1H protein. These glucose effects appear to be mediated by intracellular glucose binding sites that are independent of the glucose binding site of hSGLT1.
DISCUSSION
In this report, we present evidence that RS1 participates in short-term regulation of SGLT1 by inhibiting the exocytotic pathway. The accompanying paper by Kroiss et al. ( 26a ) describes the subcellular localization of RS1 showing that RS1 is located at the intracellular side of the plasma membrane, coats the TGN, and can migrate into the nucleus of nonconfluent LLC-PK 1 cells. Previous data indicated that RS1 is a transcriptional and posttranscriptional inhibitor of the Na + - D - glucose cotransporter SGLT1 and some other plasma membrane transporters ( 28, 33, 37, 51 ). Evidence has been published that RS1 is critically involved in long-term regulation of glucose absorption in small intestine ( 33 ) and plays an important role in differentiation dependent downregulation of SGLT1 in LLC-PK 1 cells which are derived from porcine kidney ( 25 ). It was observed that the concentration of RS1 in LLC-PK 1 cells decreased drastically when the cells became confluent and that this decrease in RS1 concentration was correlated with an increase in SGLT1 expression ( 25 ). Since RS1 is located in the nucleus of subconfluent in contrast to confluent LLC-PK 1 cells, RS1 is considered to downregulate SGLT1 as well as some other plasma membrane transporters in nondifferentiated cells ( 25 ). Long-term regulation of transporters by RS1 was supported by the recent finding that a protein called IRIP participates in the same regulatory pathway as RS1 ( 19 ). IRIP is upregulated during dedifferentiation of renal cells after ischemia and reperfusion, binds to RS1, inhibits the expression of the same transporters as RS1, and the dominant negative mutant of IRIP blocks the inhibition of hOCT2-mediated TEA uptake by RS1 ( 19 ).
In the present report, we show that the injection of hRS1 protein in oocytes decreases the transport activity of hSGLT1 within minutes. The hRS1-induced decrease in transport activity is most probably due to a decrease in hSGLT1 protein in the plasma membrane because it was shown to be dependent on the presence of functionally active dynamin, which is involved in the release of vesicles from membranes during endocytosis and exocytosis ( 15, 20, 26 ). The effect of hRS1 protein was not reduced by inhibitors of endocytosis but was abolished by BTXB, which inactivates synaptobrevin and thereby inhibits the fusion of vesicles with the plasma membrane ( 46 ) or by BFA, which disturbs the release of vesicles from the TGN and endosomes ( 3, 6, 14 ). Together with the immunohistochemical data described in the accompanying paper by Kroiss and co-workers ( 26a ), our data indicate that RS1 inhibits the exocytotic pathway of hSGLT1 at the TGN. Comparing the subcellular locations of RS1, SGLT1, dynamin and TGN marker protein TGN46, RS1 was localized to the entire TGN whereas SGLT1 was localized to parts of the TGN where it colocalized with dynamin I. A few minutes after treatment of LLC-PK 1 cells with BFA, SGLT1 was accumulated within tubulovesicular structures that extended from the Golgi complex, whereas RS1 protein was dissociated from the TGN. These data indicate that RS1 is a part of coat complexes of the TGN that may be involved in the sorting of SGLT1 or in the release of SGLT1-containing vesicles from the TGN ( 3, 6, 14 ). Since RS1 contains a UBA binding domain at the COOH terminus that binds tetraubiquitin (Müller T and Koepsell H, unpublished observations), and a recent report suggested that ubiquitination of SGLT1 plays an important role in the turnover of SGLT1 ( 5 ), we speculate that RS1 interacts with ubiquitinated transporters at the TGN.
After injection of hRS1 protein into oocytes expressing hSGLT1, we observed a significant inhibition of AMG uptake as early as 10 min after the incubation was started (see Fig. 3 ). Considering the unknown lag period required for distribution of injected hRS1 protein throughout the cytosol and the 15-min incubation period used for uptake measurements, the time between interaction of hRS1 with the TGN and the downregulation of hSGLT1 at the plasma membrane may be only a few minutes and cannot be longer than 25 min. Previously, we described that the stimulation of PKC increased AMG uptake in X. laevis oocytes expressing hSGLT1 within several minutes whereas it decreased AMG uptake in oocytes expressing hSGLT1 plus hRS1 ( 52 ). This suggested an involvement of hRS1 in PKC-dependent short-term regulations of hSGLT1. The observation of the present paper that PKC stimulated the short-term inhibition of hSGLT1-expressed AMG uptake by hRS1 protein supports this interpretation. It indicates that PKC modulates the hRS1-dependent regulation of the exocytotic pathway of hSGLT1. The involvement of PKC suggests that hRS1 may regulate hSGLT1 abundance in the plasma membrane in response to extracellular stimuli such as solutes from small intestinal or renal proximal tubular lumen (see below), hormones, or neurotransmitters.
In vivo, various short-term regulations have been described in which PKC and hRS1 may be involved. For example, regulation of SGLT1 may be mediated by adrenergic innervation, glucagon 37, glucagon-like peptide 2, or cholecystokinin ( 4, 17, 18, 44, 45 ). Concerning the potential extracellular stimuli for regulation, it was an important observation that the inhibition of SGLT1-expressed AMG uptake by injected RS1 protein was dependent on intracellular AMG concentration, which, in turn, is linked to extracellular AMG concentration. Inhibition of hSGLT1-expressed AMG uptake and of hOCT2-expressed TEA uptake by hRS1 protein was observed when the intracellular concentrations of AMG were between 50 and 200 µM, whereas no significant inhibition was observed when the intracellular concentration of AMG was in the millimolar range. Measuring the effect of hRS1 protein on hOCT2-expressed TEA uptake, the effect of hRS1 could not be observed in the absence of AMG; however, in the presence of intracellular AMG, hRS1 inhibited TEA uptake by hOCT2. This indicates that a defined intracellular glucose concentration is required for the inhibition of plasma membrane transporters by hRS1 and suggests that RS1 participates in posttranscriptional glucose-dependent regulation. The AMG dependence of the RS1-mediated inhibition of TEA uptake by hOCT2 indicates that the effect of AMG on RS1 is independent of the glucose binding site at SGLT1 and requires an additional intracellular glucose binding site(s). These may have different selectivity compared with SGLT1. Since AMG cannot be metabolized, and it is difficult to explain the biphasic effect of intracellular AMG on hOCT2 by an inhibitory effect of AMG on glucose metabolism, our data suggest that the posttranscriptional inhibition of SGLT1 and other plasma membrane transporters by hRS1 is activated and/or inhibited by an intracellular glucose binding protein. It is a challenge for future experiments to identify this protein, to determine its selectivity for monosaccharides, and to elucidate the physiological role of glucose-dependent short-term regulations of hRS1.
Glucose-dependent regulations of SGLT1 have been described in the small intestine ( 8 ). For example, a downregulation of SGLT1 has been described after weaning of sheep that could be reversed within hours after intestinal infusion of monosaccharides ( 43 ). Importantly, in these experiments a different monosaccharide selectivity was observed for the upregulation of SGLT1 in the small intestine compared with the substrate selectivity of SGLT1 ( 7 ). Glucose-dependent regulation also may be important for SGLT1 in other locations, for example, in neurons, where SGLT1 was mainly found intracellularly and may be incorporated into the plasma membrane after ischemia ( 35 ).
ACKNOWLEDGMENTS
This work was supported by the Deutsche Forschungsgemeinschaft Grant SFB 487/C1. The figures were prepared by M. Christof.
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作者单位：Institut fur Anatomie und Zellbiologie, Bayerische Julius-Maximilians-Universität, Würzburg, Germany