HuR binds a cyclic nucleotide-dependent, stabilizing domain in the 3' untranslated region of Na(+)/glucose cotransporter (SGLT1) mRNA.

Differentiation-dependent expression of the Na(+)/glucose cotransporter (SGLT1) is accompanied by a large, cAMP-dependent increase in stability of its mRNA. Stabilization is mediated by protein binding to a critical uridine-rich element (URE) in its 3' untranslated region. In the present study, we demonstrate that HuR, an RNA binding protein of the embryonic lethal abnormal vision family, binds the SGLT1 URE. HuR binding was increased after elevation of intracellular cAMP levels and was dependent on protein phosphorylation. This interaction was prevented by a substitution mutation previously shown to block cAMP-dependent reporter message stabilization. These results implicate HuR as a key mediator of cAMP-dependent SGLT1 mRNA stabilization.

A cis-dominant cyclic nucleotide-dependent regulatory domain in the 3'-untranslated region of Na(+)/glucose cotransporter (SGLT1) mRNA.

A 122 nt uridine-rich sequence (URE) in the Na(+)/glucose cotransporter (SGLT1) mRNA 3'-untranslated region is critical for cAMP-dependent message stabilization. Its function was investigated in LLC-PK(1) cells stably expressing beta-globin reporter transcripts. Insertion of the SGLT1 URE downstream from an unrelated destabilizing sequence, the c-fos ARE, evoked cAMP-dependent message stabilization. Stabilization was blocked by a substitution mutation within the SLGT1 URE. These observations indicate that the SGLT1 URE is sufficient to transmit cAMP-dependent, cis-dominant mRNA stabilization in the presence of appropriate trans-acting factors and appears to function independently of the nature of the destabilizing domain.

Differential regulation of three glucose transporter genes in a renal epithelial cell line.

Three hexose transporter genes, the Na(+)/glucose cotransporters SGLT1 and SGLT3 (formerly SAAT1/pSGLT2) and the facilitative transporter GLUT1, are expressed in a renal epithelial cell line with proximal tubule characteristics. A number of studies have demonstrated that SGLT1 expression is coupled to the cellular differentiation state and is also negatively regulated by its substrate glucose. In the present study, we demonstrate that SGLT3 mRNA expression is relatively unaffected by conditions promoting dedifferentiation (reseeding to a subconfluent density, activation of protein kinase C) or differentiation (confluent cell density, activation of protein kinase A) nor was expression sensitive to hyperglycemic glucose levels in the medium. We further demonstrate that protein kinase A and protein kinase C exert opposing effects on GLUT1 and SGLT1 mRNA levels in polarized cell monolayers, indicating that GLUT1 mRNA is also highly regulated in polarized epithelial cells by agents affecting cell differentiation. The relatively constitutive expression of SGLT3 mRNA suggests a novel role for this low-affinity Na(+)/glucose cotransporter, to provide concentrative glucose uptake under hyperglycemic conditions where expression of high-affinity glucose cotransporter SGLT1 mRNA is significantly downregulated.

Cyclic nucleotide regulation of Na+/glucose cotransporter (SGLT1) mRNA stability. Interaction of a nucleocytoplasmic protein with a regulatory domain in the 3'-untranslated region critical for stabilization.

Expression of the Na(+)-coupled glucose cotransporter SGLT1 is regulated post-transcriptionally at the level of mRNA stability. We have previously demonstrated that cAMP-dependent stabilization of the SGLT1 message was correlated with the protein phosphorylation-dependent binding of cytoplasmic proteins to a uridine-rich sequence (URE) in the 3'-untranslated region (UTR). In the present study, the regulatory role of the URE was demonstrated by inserting it into the 3'-UTR of a beta-globin reporter minigene under the control of the tetracycline-regulated promoter. The resultant chimeric globin/SGLT1 mRNA expressed after transfection into LLC-PK1 cells exhibited a decreased half-life compared with the beta-globin control, indicating that the URE serves a destabilizing function. Activation of protein kinase A stabilized the chimeric message but not the beta-globin control, indicating the presence of a regulatory stabilizing sequence within the URE. A 38-kDa nucleocytoplasmic protein was identified that recognized a 12-nucleotide binding site within the URE. A mutation in this binding site that prevented protein binding assayed in vitro by UV cross-linking also prevented protein kinase A-dependent stabilization of the chimeric message assayed in vivo. These findings identify the interaction between a 38-kDa nucleocytoplasmic protein and a regulatory uridine-rich sequence in the 3'-UTR as critical for cAMP-mediated SGLT1 message stabilization.

Sugar binding to Na+/glucose cotransporters is determined by the carboxyl-terminal half of the protein.

d-Glucose is absorbed across the proximal tubule of the kidney by two Na+/glucose cotransporters (SGLT1 and SGLT2). The low affinity SGLT2 is expressed in the S1 and S2 segments, has a Na+:glucose coupling ratio of 1, a K0.5 for sugar of approximately 2 mM, and a K0.5 for Na+ of approximately 1 mM. The high affinity SGLT1, found in the S3 segment, has a coupling ratio of 2, and K0.5 for sugar and Na+ of approximately 0.2 and 5 mM, respectively. We have constructed a chimeric protein consisting of amino acids 1-380 of porcine SGLT2 and amino acids 381-662 of porcine SGLT1. The chimera was expressed in Xenopus oocytes, and steady-state kinetics were characterized by a two-electrode voltage-clamp. The K0.5 for alpha-methyl-d-glucopyranoside (0.2 mM) was similar to that for SGLT1, and like SGLT1 the chimera transported D-galactose and 3-O-methylglucose. In contrast, SGLT2 transports poorly D-galactose and excludes 3-O-methylglucose. The apparent K0.5Na was 3.5 mM (at -150 mV), and the Hill coefficient ranged between 0.8 and 1.5. We conclude that recognition/transport of organic substrate is mediated by interactions distal to amino acid 380, while cation binding is determined by interactions arising from the amino- and carboxyl-terminal halves of the transporters. Surprisingly, the chimera transported alpha-phenyl derivatives of D-glucose as well as the inhibitors of sugar transport: phlorizin, deoxyphlorizin, and beta-D-glucopyranosylphenyl isothiocyanate are transported with high affinity (K0.5 for phlorizin was 5 microM). Thus, the pocket for organic substrate binding is increased from 10 x 5 x 5 (A) for SGLT1 to 11 x 18 x 5 (A) for the chimera.

Regulation of Na(+)-coupled glucose transport in LLC-PK1 cells. Message stabilization induced by cyclic AMP elevation is accompanied by binding of a M(r) = 48,000 protein to a uridine-rich domain in the 3'-untranslated region.

In an exploration of the molecular basis of cyclic AMP-induced stabilization of Na+/glucose cotransporter mRNA (SGLT1 isoform) accompanying cell differentiation in the pig kidney cell line LLC-PK1, we have identified a 48-kDa cytoplasmic protein factor, designated SG-URBP, which specifically binds a 120-nucleotide sequence within the 3'-untranslated region of the SGLT1 message. A 46-nucleotide uridine-rich element within this region appears necessary for specific binding, and the presence of the 3'-untranslated region is necessary for message stabilization by cyclic AMP. The binding activity of SG-URBP is up-regulated after cyclic AMP elevation and protein kinase A activation, whereas protein dephosphorylation either in vivo or in vitro is associated with loss of binding activity. The increase in SG-URBP binding activity correlates with an increase in the half-life of the SGLT1 message, suggesting a cause and effect relationship.

Post-transcriptional regulation of Na+/glucose cotransporter (SGTL1) gene expression in LLC-PK1 cells. Increased message stability after cyclic AMP elevation or differentiation inducer treatment.

We have further investigated the molecular basis of increased differentiation-regulated expression of SGTL1, a Na+/glucose cotransporter, in the renal epithelial cell line LLC-PK1. Treatment of confluent monolayers either with the differentiation inducer hexamethylene bisacetamide (HMBA) or with cyclic AMP-elevating agents promoted increased levels of the SGLT1 mRNA, the immunodetectable 75-kDa cotransporter subunit, and the transport activity. Two molecular species of SGLT1 mRNA (2.2 and 3.9 kilobases (kb)) are transcribed from the same gene in LLC-PK1 cells and differ only in the length of the 3'-untranslated region. The larger transcript is less stable (t1/2 = 2 h) than the smaller one (t1/2 = 10 h) in control, confluent monolayers. The 3.9-kb species was stabilized from degradation after either cyclic AMP elevation (t1/2 = 14 h) or HMBA addition (t1/2 = 8 h), with negligible effects on the stability of the 2.2-kb species (t1/2 = 11 h). Inhibition of translation by cycloheximide resulted in a 10-fold increase in the t1/2 of the 3.9-kb transcript and a 2-fold increase in that of the 2.2-kb species in control monolayers. Our results demonstrate that post-transcriptional regulation of message stability plays a major role in differentiation-dependent SGTL1 expression promoted by either HMBA or cyclic AMP.

SAAT1 is a low affinity Na+/glucose cotransporter and not an amino acid transporter. A reinterpretation.

Recently a member of the Na+/glucose (SGLT1) gene family of cotransporters was isolated from a pig renal cell line and was thought to be the neutral amino acid transporter System A. This cDNA (Kong, C. T., Yet, S. F., and Lever, J. E. (1993) J. Biol. Chem. 268, 1509-1512) encodes a 660-amino acid protein with 76% identity to SGLT1. To confirm and extend the kinetic characterization of SAAT1, we have expressed this clone in Xenopus oocytes and measured transport using both radiotracer and electrophysiological techniques. SAAT1 did not stimulate either 50 microM 2-(methylamino)isobutyrate uptake or 2-(methylamino)isobutyrate-evoked inward Na+ currents, but instead stimulated 50 microM alpha MG (alpha-methyl-D-glucopyranoside) uptake 27-fold from 2 +/- 1 pmol.h-1/oocyte (n = 9) to 55 +/- 6 pmol.h-1/oocyte (n = 9) and alpha MG-evoked inward Na+ currents (I) by up to 1000 nA/oocyte. The apparent affinity constant for alpha MG (K alpha MG 0.5) was approximately 2 mM and was independent of membrane potential from -30 to -150 mV but was voltage-sensitive between -30 and +30 mV. The relative sugar specificity for the transporter was alpha MG > or = D-glucose >> D-galactose >>> 3-O-methyl-D-glucopyranose, L-glucose. The sugar-evoked currents were Na(+)-dependent (KNa 0.5 approximately 10 mM at -50 mV) and the Hill coefficient was 1. KNa 0.5 decreased with hyperpolarization of the membrane from -50 to -150 mV. Phlorizin inhibited the alpha MG-evoked current with apparent Ki of 18 microM at -50 mV. We conclude that the SAAT1 cDNA encodes a renal low affinity Na+(1)/glucose(1) cotransporter and propose that pig SAAT1 be renamed pSGLT2.

N-linked glycosylation is not required for Na+/glucose symport activity in LLC-PK1 cells.

The role of N-linked glycosylation in Na+/glucose symporter function was investigated using LLC-PK1 cell cultures. Tunicamycin treatment did not inhibit Na(+)-dependent glucose transport or phlorizin binding activity assayed in intact LLC-PK1 cells. However apical membrane vesicles derived from tunicamycin-treated cells had no detectable Na(+)-dependent glucose transport activity but retained unchanged phlorizin binding to the symporter. These observations suggest that N-linked glycosylation is not required for transport function or insertion in the membrane in intact cells but may play a role in maintaining symporter transport activity in isolated membranes.

Regulation of Na+/glucose cotransporter (SGLT1) mRNA in LLC-PK1 cells.

The porcine kidney epithelial cell line LLC-PK1 expresses a sodium-coupled glucose cotransporter (SGLT1) together with other differentiation markers of renal proximal tubule such as trehalase and gamma-glutamyltranspeptidase. Expression is regulated by cell density and exogenous differentiation inducers such as hexamethylene bisacetamide (HMBA). Northern blot and PCR analysis of clonal cell populations indicated SGLT1 mRNA was not detectable in subconfluent cultures, but 2.2 and 3.9 kb SGLT1 mRNA species appeared after cell confluence, accompanying expression of the transport activity. SGLT1 mRNA levels were significantly increased after treatment of confluent cultures with HMBA, paralleling increases in the transport activity and immunodetectable 75 kD cotransporter subunit. SGLT1 mRNA was also increased after treatment of cultures with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), an inducer of Na+/glucose cotransport activity. The 3.9 kb SGLT1 transcript showed the largest increase after either HMBA or IBMX treatment. HMBA treatment also resulted in increased mRNA levels of two other differentiation markers--trehalase and gamma-glutamyltranspeptidase. By contrast, trehalase and gamma-glutamyltranspeptidase mRNA levels were not increased by IBMX. Regulation of Na+/glucose symporter expression by either cell density, cyclic AMP elevation, or differentiation inducer treatment occurs, at least in part, at the level of SGLT1 mRNA and can be dissociated from regulation of other differentiation markers.

Targeting of recombinant Na+/glucose cotransporter (SGLT1) to the apical membrane.

A full-length Na+/glucose cotransporter cDNA (SGLT1) from rabbit intestine was subcloned into the pMAMneo mammalian expression vector and transfected by Ca2+ precipitation into Madin-Darby canine kidney (MDCK) cells. Stable MDCK transfectants isolated after clonal isolation and selection in G418 exhibited dexamethasone-inducible Na+/glucose cotransport activity under regulation of the MMTV promoter of the vector. Transfectants expressed the recombinant 75 kDa Na+/glucose cotransporter subunit as shown by Western blot, and SGLT1 mRNA as shown by Northern blot, but these were undetectable in untransfected MDCK cells. Over 93% of total recombinant transport activity was targeted to the apical membrane. This indicates that the primary amino acid sequence of SGLT1 contains the information necessary to target this transporter to the apical membrane.

Polyamine regulation of Na+/glucose symporter expression in LLC-PK1 cells.

Addition of polyamines or their analogs to newly confluent LLC-PK1 cells resulted in down-regulation of Na(+)-dependent glucose transport (symport) activity. Polyamines prevented the induction of this symporter by the differentiation inducer hexamethylene bisacetamide (HMBA) but did not influence induction by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Partial depletion of endogenous polyamines after addition of alpha-difluoromethylornithine (DFMO) resulted in a 4 to 5-fold increase in symporter expression. Symporter induction by either HMBA or DFMO was inhibited by the protein kinase inhibitor H-7 but H-7 did not affect symporter induction by IBMX. Changes in symporter activity were accompanied by changes in levels of the 75 kD symporter subunit detected by Western blot. Cultures exposed to HMBA exhibited reduced levels of ornithine decarboxylase activity. Our results suggest that induction of symporter expression by HMBA may be mediated in part by its effects on polyamine metabolism, and point to parallel roles of polyamines and cyclic AMP in regulating the expression of this physiologically important renal transport system.

Cloning and expression of a mammalian Na+/amino acid cotransporter with sequence similarity to Na+/glucose cotransporters.

We describe the full-length sequence and functional expression of a cDNA cloned from LLC-PK1 cells, which appears to encode a mammalian Na(+)-dependent neutral amino acid transporter with properties characteristic of system A. This sequence, designated SAAT1, is 76% identical and 89% similar in amino acid sequence to the Na(+)-dependent glucose transporter SGLT1 of the same species. A leucine zipper region was detected in both SAAT1 and SGLT1. The message for SAAT1 was a single 2.4-kilobase species in kidney, but mRNA species of 2.4 and 3.7 kilobases were observed in LLC-PK1 cells as well as in intestine. Transcripts were also found in spleen, liver, and muscle. Expression of SAAT1 in COS-7 cells resulted in increased levels of Na(+)-dependent uptake of 2-(methylamino)isobutyric acid, a specific substrate for the system A amino acid transporter. Uptake due to cDNA expression was inhibited by a range of amino acids that are transported by system A and exhibited a km of 0.8 +/- 0.2 mM. These results suggest that the system A amino acid transporter is closely related to the Na+/glucose transporter SGLT1.

Antibodies to a renal Na+/glucose cotransport system localize to the apical plasma membrane domain of polar mouse embryo blastomeres.

Mouse preimplantation embryos were examined for the cell surface expression of epitopes that cross-react with antibodies to a 75-kDa subunit of a purified porcine renal brush border Na+/glucose cotransport system. A Na+ cotransport system is hypothesized to reside in the apical plasma membrane domain of mouse polar blastomeres and to be associated with the induction of their apical-basal polarity. Western blot analysis showed that unfertilized oocytes as well as preimplantation embryos contain a cross-reacting antigen with an apparent molecular weight of about 75,000. Embryos and their isolated blastomeres were double-labeled and assayed by indirect immunofluorescence (IIF) for the expression of epitopes (visualized by labeling with rabbit antiserum or mouse monoclonal IgG to cotransporter followed by the appropriate rhodamine-conjugated second antibodies) and for the development of cell surface polarity (visualized by the apical restriction of fluoresceinated succinylated concanavalin A binding; FS Con A). IIF did not detect these epitopes until after the second cleavage when 4-cell embryos expressed low-to-moderate levels. Although epitopes were expressed on all surfaces of 4-cell blastomeres, some blastomeres expressed more epitopes on their apical surfaces than on their basolateral ones. All precompaction 8-cell embryos expressed epitopes, with expression being greater apically on some blastomeres. The level of expression appeared to reach a maximum on morulae and to decline on cavitating embryos. Assays performed on isolated blastomeres from postcompaction embryos showed that by the 16-cell stage epitope expression appeared to become restricted to FS Con A-labeled apical plasma membrane domains and was no longer evident on basolateral domains. This apparent apical restriction of epitope expression was confirmed by electron microscopic examination of immunogold-labeled isolated polar 16-cell blastomeres. These results demonstrate that preimplantation mouse embryos contain an antigen(s) that is immunologically and structurally similar to a 75-kDa renal Na+/glucose cotransporter. The onset of cell surface expression of this antigen precedes development of the stable polar phenotype.

Developmentally regulated 75-kilodalton protein expressed in LLC-PK1 cultures is a component of the renal Na+/glucose cotransport system.

Na+/D-glucose symport is a secondary active glucose transport mechanism expressed only in kidney proximal tubule and in small intestine. A monoclonal antibody that recognized the Na+/glucose symporter of pig renal brush border membranes also recognized a 75-kD protein in apical membranes isolated from highly differentiated LLC-PK1 cultures, an epithelial cell line of pig renal proximal tubule origin. The 75-kD antigen was enriched from solubilized LLC-PK1 apical membranes by means of high-pressure liquid chromatography. The symporter antigen became apparent on the apical membrane surface after the development of a confluent monolayer in correlation with the expression of transport activity. Long-term treatment of cultures with the differentiation inducer hexamethylene bisacetamide was accompanied by a dramatically increased expression of the symporter antigen as detected quantitatively by Western blot analysis and qualitatively by immunofluorescence staining. The number of symporter-positive cells was dramatically increased after inducer treatment as predicted for differentiation-regulated expression. These results identify a 75-kD protein as a component of a developmentally regulated renal Na+/glucose symporter expressed in cell culture.

Photoaffinity labeling by [3H]-N5-methyl-N5-isobutylamiloride of proteins which cofractionate with Na+/H+ antiport activity.

N5-Methyl-N5-isobutylamiloride (MIA) is one of a series of 5-N-substituted amiloride analogues which exhibit high affinity and specificity for inhibition of Na+/H+ antiport. Amiloride-sensitive [3H]MIA binding to renal brush border membranes exhibited a Kd of 250 nM and a Bmax of 8.6 pmol/mg of protein. Specific binding was optimal at pH 7.5 and inhibited in the presence of Na+ and Li+. Inhibition by amiloride exhibited biphasic kinetics. After resolution of solubilized membranes by high-pressure liquid chromatography, MIA binding activity cofractionated together with Na+/H+ antiport activity, measured after reconstitution in asolectin vesicles, into a major and a minor peak. When fractions containing the major peak of Na+/H+ antiport activity were incubated with [3H]MIA and then photolyzed with a mercury arc lamp, covalent incorporation of label into polypeptides of apparent molecular mass 81 and 107 kDa was observed. These photolabeled bands were also observed in intact brush border membranes in addition to labeled polypeptides of apparent molecular mass 60 and 46 kDa, respectively. Labeling was inhibited by amiloride, reduced in the presence of Na+, and not observed in the absence of photolysis. These data point to the 81- and 107-kDa polypeptides as candidates for identification as components of a Na+/H+ antiport system in renal brush border membranes.

Chemical inducers of differentiation in a long-term renal cell line.

The long-term renal epithelial cell line LLC-PK1 expresses at confluence several differentiated characteristics of renal proximal tubule including Na/glucose cotransport and several brush border membrane hydrolases. The differentiation-inducing chemical hexamethylene bisacetamide (HMBA) triggers a dramatic induction of Na+/glucose symport, trehalase and maltase, expressed as an increase in the number of cells in the culture that express the differentiated phenotype. Characteristics of the induction response are reviewed in terms of proposed mechanisms of inducer action. New evidence suggests that in addition to elevation of intracellular Na levels mediated by partial inhibition of the sodium pump, HMBA treatment also alters polyamine levels via effects on ornithine decarboxylase. These responses may be mediated by HMBA effects on protein kinase C activity. The possible role of polyamine fluctuations and DNA demethylation in mediating HMBA effects on differentiated gene expression is currently being investigated.

Increased trehalase expression after glucose limitation of LLC-PK1 clones.

Expression of the apical membrane disaccharidase trehalase correlates with the development of a proximal tubule-specific differentiated phenotype in the LLC-PK1 cell line derived from renal epithelia. Substitution of galactose for glucose in the culture medium of confluent cultures resulted in a significant trehalase induction; a smaller induction was observed after replacement by fructose, mannose, or the nonmetabolizable sugar alpha-methyl-D-glucose. Induction was specific for trehalase among other microvillar hydrolases, reversible after replacement of glucose, and was increased as a function of decreasing glucose concentration. Stable clonal cell lines that were inducible by glucose limitation and noninducible clones were isolated nonselectively. Both types exhibited trehalase induction after exposure to the differentiation inducer N,N'-hexamethylene-bis-acetamide (HMBA). These results suggest that trehalase induction after glucose deprivation occurs by a different mechanism than that mediated by HMBA.

Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification.

Phlorizin is a specific, high-affinity ligand that binds the active site of the Na+/glucose symporter by a Na+-dependent mechanism but is not itself transported across the membrane. We have isolated a panel of monoclonal antibodies that influence high-affinity, Na+-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK1, a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG2b, reproducibly stimulated Na+-dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na+-dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, taken together with results in the following paper [Wu, J.-S.R., & Lever, J.E. (1987) Biochemistry (following paper in this issue)], provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na+/glucose symporter.

Purification and reconstitution of a 75-kilodalton protein identified as a component of the renal Na+/glucose symporter.

A 75-kilodalton (kDa) protein was purified from solubilized renal brush border membranes by using high-pressure liquid chromatography (HPLC) and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Functional and immunological properties identified the 75-kDa protein as a component of the Na+/glucose symport system. The purified protein was specifically recognized by a monoclonal antibody that functionally interacts with the Na+/glucose symporter. Na+-dependent phlorizin binding activity was associated with fractions containing the 75-kDa protein during HPLC fractionation on the anion exchanger Mono-Q and was greatly increased after reconstitution into egg yolk phosphatidylcholine vesicles. The final purified preparation contained glucosamine and a blocked N-terminus.