the journal of bioidcical chemistry vol. 268, no. 33, 25 ... · the journal of bioidcical chemistry...

THE JOURNAL OF BIOIDCICAL CHEMISTRY Vol. 268, No. 33, Issue of November 25, pp. 25041-25053, 1993 8 1993 by The American Soeiety for Biochemistry and Molecular Biology, Inc. Printed in V.S.A.

Cloning of a Membrane-associated Protein Which Modifies Activity and Properties of the Na'-D-Glucose Cotransporter*

(Received for publication, March 23, 1993, and in revised form, July 2, 1993)

h i k e Veyhl, Jorg Spangenberg, Bernd Puschel, Robert Poppe, Carmela Dekel, Gunter Fritzsch, Winfried Haase, and Hermann KoepsellS From the Max-Planck-Znstitut fur Biophysik, Frankfurt am Main 60596, Germany

An expression library from porcine kidney cortex was screened with a monoclonal antibody ( W 6 ) which stimulates high-affinity phlorizin binding in kidney and intestine but does not react with the membrane protein (SGLT1) which mediates Na+-coupled transport of D-glU- cose (Hediger, M. A, Coady, M. J., Ikeda, T. S., and Wright, E. M. (1987) Nature 330,379381). A cDNA (RS1) was obtained which codes for a hydrophilic M , 66,832 polypeptide and contains a predicted hydrophobic a-helix at the COOH terminus. After expression in Xenopus oocytes RS1 protein was found associated with the plasma membrane. RS1-homologous mRNAs were detected in renal outer cortex and outer medulla, small intestine, liver, and LLCPKl cells, but not in skeletal muscle, heart muscle, Madin-Darby canine kidney (MDCK) cells, renal inner medulla, and Xenopus oocytes. After nondenaturing gel electrophoresis of renal brush- border membranes comigration of RS1- and SGLT1-homologous proteins as a high molecular weight complex was demonstrated. RS1 altered the expression of Na+- glucose cotransport by SGLTl in Xenopus oocytes. There was no effect on the expression of the nonhomologous transporters for Na+-y-aminobutyric acid cotransport and for Na+-independent glucose transport. However, RS1 also changed the expression of the SGLT1-homologous Na+-myo-inositol cotransporter from MDCK cells. The V,, of methyl-a-D-glucopyranoside (AMG) transport expressed after injection of a small amount of SGLT1-cRNA was increased 40-fold when a stoichiometric amount of RS1-cRNA was coinjected. In addition the voltage and glucose dependence of expressed AMG uptake and the concentration dependence of transport inhibition by phlorizin were changed when stoichiometric amounts of RSl-cRNA were coinjected with SGLT1- cRNA Thus with SGLTl one apparent transport site (&.a about 100 p ~ ) and one apparent phlorizin inhibition site (Ki about 5 p ~ ) was observed whereas with SGLTl plus RS1 two apparent transport sites about 20 +m, about 1 m ~ ) and two apparent phlorizin inhibition sites (Kicl) about 0.3 +m, Kit,) about 30 JIM) were found as has been described in brush-border membrane vesicles of kidney and intestine (see e.g. Koepsell, H., Fritzsch, G., Korn, K., and Madrala, A. (1990) J. Membl: BioL 114,113-132). The data suggest that the Na+-

Grant SFB 169. The costs of publication of this article were defrayed in *This work was supported by Deutsche ForschungsgemeinschaR

part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank*IEMBL Data Bank with accession number(s) X64315 (SSSDCBSR).

tut der Universitat Wiirzburg, Koellikerstr. 6, 97070 Wurzburg, Ger- To whom correspondence should be addressed: Anatomisches Insti-

many. Fax: 0931-572338.

D-glucose cotransporter and possibly also other SGLTl- type Na+-cotransporters contain RSl-type regulatory subunits.

~ ~ ~ ~~~~~~

Last years significant progress has been made to elucidate the sequences of the Na+-symporters. From three genetic fami- lies integral membrane proteins have been cloned (family of the Na+-glucose cotransporter SGLTl (1-41, family of the Na+-y- aminobutyric acid cotransporter GAT1 (5,6), family of the Na+- glutamate cotransporter GLTl (7-9)). The members of the SGLTl family mediate Na+-driven transport of glucose (clone SGLT1, Ref. l), nucleotides (21, myo-inositol (3) and neutral amino acids (41, whereas Na+-dependent transport of neuro- transmitters is mediated by the members of the two other fami- lies. The Na+-D-glucose cotransporter is responsible for glucose absorption in kidney and intestine. This transporter is defec- tive in congenital glucosdgalactose malabsorption (10) and works effectively at glucose concentrations which differ by or- ders of magnitude. Since high and low affinity transport and binding sites of glucose were described in kidney and intestine (11-16), but were not observed after transport expression by SGLTl(l7,18), the existence of SGLTl transporter subtypes or of a regulatory subunit was postulated (16). In our laboratory monoclonal antibodies were raised which alter Na+-D-glucose cotransport andlor Na+-dependent high-affinity phlorizin binding in kidney and intestine, bind to membrane-bound polypeptides with apparent molecular weights around 70,000, and show a similar immunohistochemical distribution as peptide antibodies against SGLTl protein (19-23). The present inves- tigation was initiated when we realized that one of our monoclonal antibodies (R4A6) did not react with expressed SGLTl protein. This antibody was used to screen an expression library of porcine kidney cortex. A cDNA clone (RS1)l was isolated which codes for a protein that alters the kinetics and the voltage dependence of SGLT1-expressed Na+-glucose cotransport in oocytes of Xenopus laevis when the amount of coinjected RS1-cRNAwas stoichiometric to the amount of injected SGLT1- cRNA. Unlike the kinetics obtained after expression of Na+- glucose cotransport by SGLT1-cRNA alone, the kinetics of Na+- glucose cotransport expressed afier injection of SGLT1-cRNA plus RS1-cRNA closely resemble the kinetics of Na+-glucose cotransport measured in brush-border membrane vesicles from

The abbreviations used are: RS1, complete clone encoding the regulatory subunit of the Na+-D-glucose cotransporter (see nucleotides -240-4831 in Fig. 2); RSl', clone with the complete open reading frame (nucleotides 1-2128) containing the additional nucleotides 5'-AG- ATCTCCACC-3' at the 5'-end; RSli, incomplete clone (nucleotides 303- 4831); RSl;, truncated incomplete clone (nucleotides 303-2128); PCR, polymerase chain reaction; DEPC, diethyl pyrocarbonate; AMG, methyl-a-D-glucopyranoside; GABA, y-aminobutyric acid; PBS, phos- phate-buffered saline; MDCK, Madin-Darby canine kidney; mAb, monoclonal antibody.

25041

25042 Protein Which Modifies Na'-D-ghcose Cotransport pig kidney and rabbit or guinea pig intestine (11, 16, 17). Con- trol experiments showed that RS1 does not interact with expressed transporters which have no homology to SGLT1. How- ever, some interaction with Na+-myo-inositol cotransport expressed by the SGLT1-homologous protein SMIT (3) was observed. Since SMIT was cloned from MDCK cells which do not express RS1-homologous proteins and high concentrations of SMIT-type proteins occur in renal inner medulla which does not contain RS1-type proteins, RS1 is not supposed to be associated with SMIT in vivo. Our data suggest that the Na'-D- glucose cotransporter and possibly other SGLT1-type Na+-cotransporters contain regulatory subunits which are identical or homologous to RS1. Part of the results have been reported as an abstract (24).

EXPERIMENTAL PROCEDURES

Methods cDNA Library Construction and Antibody Screening-5-mm sections

from the surfaces of fresh porcine kidneys were frozen in liquid nitrogen, mRNA was isolated, and an oligo(dT)-primed cDNA library in A-Zap phages was prepared by standard procedures (25). For antibody screening Escherichia coli BB4 cells were infected with phages, mixed with 0.6% (w/v) agarose, applied to 1.5% (w/v) agar plates, and incubated 3.5 h at 42 "C as recommended by Stratagene. Then nitrocellulose filters (HAHY 132, Millipore) soaked with 10 mM isopropylthiogalacto- side were applied and the plates were incubated 3.5 h at 37 "C. After cooling to 4 "C the filters were removed, washed twice with PBS containing 0.01% (w/v) Tween 20 (PT buffer), once with PBS, and then dried. To expose more antigenic sites the filters were incubated 15 min (22 "C) with PBS containing 0.4% (w/v) SDS. After three washings with PBS buffer and two with PT buffer, the filters were blocked by incubation (2 h, 22 "C) with PBS containing 0.05% (w/v) Tween 20 and 10% (v/v) newborn calf serum (PTN buffer). Reaction with the monoclonal antibody R4A6 was performed by incubating the filters 14 h (4 "C) with 5 pg/ml of R4A6 dissolved in PBS containing 0.5% (w/v) Tween 20, 5% (v/v) newborn calf serum, 1 M D-glucose and 10% (v/v) glycerol, (PTNG buffer). After washing the filters with PNTG buffer, PT buffer, and PTN buffer, they were incubated (lh, 37 "C) with p-chain-specific alkaline phosphatase-coupled anti-mouse IgM antiserum from goat (diluted 1:lOOO in PTN), washed three times with PT buffer, once with PBS, and once with 100 mM Tris-HC1, pH 9.5, 100 nm NaCl, 5 mM MgC1,. Then alkaline phosphatase activity of colony-related filter spots was measured, and the identified colonies were isolated and added to 500 pl of 50 mM Tris-HC1, pH 7.5, 100 mM NaC1,8.1 mM MgS04, 0.01% (w/v) gelatin (SM buffer) containing 4% (vh) chloroform.

Polymerase Chain Reactions-% investigate the tissue distribution of Rs1 by primer-specific amplification of cDNA, mRNA was prepared from different tissues, oligo(dT)-primed single-stranded cDNA was synthesized (25) and subjected to polymerase chain reactions (PCR) employing the primer pairs Sl', S2-, and S3',S4- (Table I). The PCR reaction was performed in the presence of 67 mM Tris-HC1, pH 8.8, 6.7 mM MgCl,, 16.6 nm (NH4),S04, 0.17 mg/ml bovine serum albumin, 5% (v/v) dimethyl sulfoxide, 0.5 mM each dNTP, and 0.4 p~ of each primer. After initial denaturation and addition of Tag polymerase 35 PCR cycles (1 min, 94 "C; 90 s between 48 and 52 "C; 3 min, 72 "C) were performed. The samples were separated by agarose gel electrophoresis and stained with ethidium bromide or hybridized with the internal primers S5' or S6' (Table I).

The methods of 5'-RACE (26) and inverse PCR (27) were employed to clone the 5'-end of RS1. For 5'-RACE single-stranded cDNA was synthesized after priming with S7- (Table I) and was tailed with poly(dA). The second cDNA strand was synthesized after priming with the oligo(dT)-containing adapter primer S8' (Table I), and cDNA was amplified by 35 PCR cycles employing the RS1-specific primer S9- and the adapter oligonucleotide S10' (Table I). The resulting amplification product was hybridized with oligonucleotide Sl l - (Table I), gel-purified, sequenced, and used for another round of amplification in which S11- and S12' were used as primers. The specificity of the amplification product of this round was verified by hybridization with S13' which was derived from the cDNA of a previous amplification round. For the inverse PCR genomic DNA was isolated from porcine kidney and digested with RsaI. After an intramolecular ligation of the DNAfragments (28). the PCR reaction was performed with the primers S14- and S15'. The amplification product was hybridized with S13', electroeluted, and sequenced.

TABLE I Nucleotide sequences of oligonucleotides which were used

for PCR experiments + and - indicates whether the sequences belong to the plus or minus

DNA strand. Nucleotide sequences derived from Rs1 (see Fig. 2) are printed in italics. Their positions in RSl are indicated in parentheses. A SacI restriction site and a Kozak-type translation initiation sequence in S16' are underlined.

S1': 5"CTTAAT TCA GCA GGC GG-3' (794-810) S2-: 5"GAA GCT GGA TGA CAA GG-3' (1572-1556) S3': GGC TTT ACC TTG CAG GAA G-3' (1768-1786) S4-: 5""TC CAT GGT TAT GTA GG-3' (1880-1864) S5': 5"GT GAT GGC CTG TTA GTG-3' (1367-1383) S6': 5"T CGA GTT GGT GGA AAT GC-3' (1803-1820) S7-: 5"CTT CCT GTC TTG TGT GG-3' (401-385) S8': 5"AAG GAT CCG TCG ACT GCA GAA TTC AAG CTT

S9-: 5"TTT GAG GCC TTG GGT GC-3' (374-359) S10': 5'-AAG GAT CCG TCG ACT GC-3' S11-z 5"CAA TGG CTT CTT CGA GG-3' (325-309) S12': 5'-CGA CTG CAG AAT TCA AGC T-3'

(TIT)-3'

S13': 5'-CAT TAC CAA C'IT CAG ATG-3' (8-25) S14-: 5"CTT GAA AGG GCA GAC TGA G-3' (105-123) S15': 5"CAA TGC TCC AGC AAA CCA G3' (264-282) S16': 5"CGC GAG CTC AGA TCT CCA CCA TGT CAT CAT

TAC CAA CTT C-3' (1-20) - S17-: 5"GAT ATT CGG GAT GCC AAC C-3' (460-478)

Subcloning and DNA Sequencing-Standard genetic procedures were performed as described by Sambrook et al. (25). From single colonies of A-Zap phages identified by mAb R4A6, pBluescript plasmids were excised with helper phage R408, and XL1-Blue cells were trans- fected and selected for ampicillin resistance as recommended by Strata- gene. Liquid cultures from single ampicillin-resistant XL1-Blue cells were used to p u r i ~ the plasmid DNA (RSli, nucleotides 303-4831 in Fig. 2). Rsli plasmid was digested with EcoRI plus HindIII and a EcoRIIHindIII fragment (RSli*, nucleotides 303-2128 in Fig. 2) was purified by electroelution from agarose gels, precipitated and subcloned into pBluescript 11. To prepare a clone which contains the complete open reading frame (Rsl'), the 5'-end of the sequence was synthesized by PCR, using the primers S16' (containing a SacI restriction site and a Kozak-type translation initiation sequence) and S17-. The amplification product was purified by gel elution, digested with SacI plus BglI, and ligated to the BglYHindIII fragment of R s l i (nucleotides 442-2128 in Fig. 2). The resulting construct was subcloned into the restriction sites SacI and HindIII of pBluescript 11.

For subcloning of PCR-amplified cDNAs, the termini of the gel eluted amplification products were filled in with Klenow polymerase. After phosphorylation with T4 polynucleotide kinase the cDNAs fragments were blunt end-subcloned into pBluescriptI1 plasmid.

DNA sequencing of double-stranded DNA in pBluescript11 plasmids was performed by the dideoxynucleotide chain termination method (29) using T7 DNA polymerase and [cx-~~SI~ATP. Both cDNA strands were sequenced using standard sequencing primers and sequence-related oligonucleotides. The oligonucleotides were synthesized in 0.2-pmol scale on a Cyclon Plus DNA synthesizer from MilliGen (Eschborn, Ger- many) using the solid phase phosphoamidite method and purified on Oligo-Pak columns from MilliGen.

Hybridization-For Northern blotting poly(A)-selected RNAs from different tissues were separated by agarose gel electrophoresis in the presence of formaldehyde, transferred to Genescreen" membrane (Du Pont de Nemours), heat-fixed, and hybridized at 42 "C (5 x SSC, 50% (v/v) formamide, 0.5% (w/v) SDS, 10% (w/v) dextran sulfate, 0.5 mg/ml of herring sperm) with Rs1: which was radioactively labeled by nick translation. Washing was performed at 42 "C with 0.2 x SSC containing 0.1% (w/v) of SDS. For Southern blotting, DNAs were separated on agarose gels, transferred to Hybond-NTM membranes (Amersham Buchler) by the alkali method (30) and hybridized with oligonucleotides which were radioactively labeled with T4 polynucleotide kinase. Hy- bridization was performed 7 "C below the melting temperature in the presence of 6 x SSC and 0.5% (w/v) SDS. If required the stringency was increased by washing at higher temperatures.

SGLTl (l), GAT1 (5), GLUT1 (31), SMIT (3), Rsl', RSli, or RSli', In Vitro Synthesis of cRNA-% prepare sense cRNA from the clones

purified plasmids were linearized with Not1 (SGLT1, GAT1, SMIT), XhoI (RSl", RSli, RSl,'), or HindIII (GLUTl), and 5'7meGppp5'G capped cRNA was synthesized by T3 polymerase (SGLT1, Rsl', RSL,

Protein Which Modifies Na+-D-glucose Cotransport 25043

RSl,'), T7 polymerase (GATl, SMIT), or SP6 polymerase (GLUTl) employing the mCAP mRNA Capping Kit of Stratagene (Heidelberg, Ger- many). Antisense cRNAof RSl', RSl,, or RSl,' was synthesized with T7 polymerase after linearization of Bluescript with SacI. The synthesized cRNAs were suspended in double-distilled water which was treated with diethyl pyrocarbonate (DEPC). The concentration of the cRNAs were determined by measuring the optical density a t 260 nm, and the quality of the cRNAs was controlled by evaluating their appearance as single bands and their apparent size aRer denaturating agarose gel electrophoresis. The cRNA samples were stored in liquid nitrogen and thawed directly before use.

In Vitro Danslation-In vitro synthesis of RS1 or RS1,' protein was performed by incubating a mixture of 5 pl containing 0.9 pg of RS1' or RSli cRNA and 45 pl of rabbit reticulocyte lysate with and without [3sSlmethionine (150 pCi) for 1 h at 37 "C. The reaction was stopped by adding 50 pl of 126 mM Tris-HCI, pH 6.8, 4% (w/v) P-mercaptoethanol, 20% (w/v) polyethylene glycol, and 2 0 4 samples were applied to SDS- polyacrylamide slab gels (20). For the detection of methionine-labeled RSli and RS1' polypeptide, the gels were dried and applied to autoradiography. To test for antibody reaction, proteins translated in the absence of [3sSlmethionine were electrotransferred from the polyacrylamide gels to nitrocellulose and then reacted with R4A6 (20).

Expression of Different Z'kansporters in Oocytes ofX. Laeuis-Sections of ovary from anesthetized female X. laeuis clawed toads (kept a t 18 "C) were removed, dissociated, and incubated 6 h (22 "C) in 5 mM Hepes- Tris, pH 7.4, 110 mM NaCI, 3 mM KCI, 2 mM CaCI2, 1 mM MgCI2 (ORi) containing 3 mdml of collagenase, 20 mgll penicillin, and 25 mglliter streptomycin. Full-grown oocytes arrested in the prophase of the first meiotic division (type V or VI aRer Dumont (32)) were selected, incubated 10 min (22 "C) in Ca2+-free ORi to remove follicle cells, and stored overnight at 18 "C in ORi with streptomycin and penicillin. Per oocyte, 50 nl of DEPC-treated water (controls) or 50 nl of DEPC-treated water containing different concentrations of cRNAs were injected. The injec- tions were carried out using either cRNAs of single clones or mixtures of cRNAs (RSl', RSl,, or RSli' with SGLTl, GAT1, GLUTl, or SMIT). For translation the injected oocytes were incubated 48 h at 18 "C in ORi containing 50 mglliter gentamycin which was exchanged after 24 h.

Dansport and Binding Measurements in Oocytes of X. Laeuis-Na+- o-glucose cotransport was measured by incubating the oocytes at 22 "C with ORi containing 14C-labeled o-glucose or 14C-labeled methyl-cr-D- glucopyranoside (AMG) or with 5 mM Hepes-Tris, pH 7.4, 110 mM tetramethylammonium chloride, 3 mM KCI, 2 mM CaCI2, 1 mM MgC12 (HT buffer) plus glucose. Initial o-glucose uptake rates were measured after incubating the oocytes for 1 h. This long incubation time was used since a linear glucose uptake was observed for 90 min when uptake was measured with different AMG concentrations (50 PM and 1 mM) in water-injected oocytes and in oocytes in which glucose transport was partially and maximally expressed by injection of SGLTl-cRNA. To measure phlorizin inhibition of glucose uptake, the phlorizin was added 10 min before the uptake measurement was started by the addition of radioactively labeled glucose. Na+-independent glucose uptake was measured by incubating oocytes (30 min, 22 "C) with HT buffer containing 50 mM mannitol plus 25 PM 2-deoxy-~-[~H]glucose, and nonspecific uptake was subtracted which was measured after replacement of mannitol by 50 mM nonlabeled 2-deoxy-D-glucose. Cotransport of Na+ and y-aminobutyric acid (GABA) was measured by incubating oocytes (15 min, 22 "C) with ORi containing 0.7 p~ 13HlGABA. Nonspecific uptake measured in the presence of 2 mM nonlabeled GABA was subtracted. Na+-dependent myo-inositol uptake was measured by incubating the oocytes 15 min with ORi or HT buffer containing 0.5 PM myo- fHlinosito1. 86Rb uptake mediated by the (Na++K+)-ATPase and ouabain binding to the (Na++K+)-ATPase was measured as described earlier (33). The incubation periods with different substrates were stopped by transferring the oocytes to the respective ice-cold incubation buffers in which the radioactively labeled substrates were omitted and washing them three times at 0 "C. Finally single oocytes were solubilized in 5% (w/v) SDS and analyzed for radioactivity. The data presented in the tables and figures represent means of 8 to 10 determinations in different oocytes f S.E. (S.DJdn). The glucose dependence and phlorizin inhibition of AMG uptake expressed by SGLT1-cRNA or SGLTl- cRNA plus RSli- or RSl'-cRNA (see Figs. 6-9) was described by fitting the data to the Michaelis-Menten equation or to an equation in which two transport sites are assumed (16).

Electrophysiological Measurements-Measurements of membrane potential and voltage-clamp experiments were performed by conven- tional two-microelectrode techniques, and the Na' concentration in the oocytes was measured by flame photometry. For determination of current-voltage relations, steady-state current was measured during the

last 100 ms of 500-ms rectangular voltage pulses to different potentials. The pulses were applied from the respective resting potential a t a frequency of 0.5 Hz.

Antibody Reactions with Brush-border Membrane Proteins Which Were Fractionated by Nondenaturing Gel Electrophoresis-Brush- border membranes which were purified from porcine renal cortex and resuspended as described earlier (34) were solubilized by 1-h (37 "C) incubation with 2.5% (w/v) Triton X-100, and the particulate material was removed by 1-h centrifugation at 160,000 x g. The supernatant was diluted with sample buffer containing 62.5 mM Tris-HCI, pH 6.8, 10% (w/v) glycerol, and 1% (w/v) Triton X-100, and 40 pg of proteidane was applied to a discontinous (5%/7%) polyacrylamide slab gel which was performed as described by Laemmli (35) with the difference that SDS was replaced by 0.1% (w/v) Triton X-100. After electrophoresis the proteins were stained with silver or electrically transferred to poly(viny1- idene difluoride) membranes after the gel had been incubated with 25% (v/v) isopropanol (2 h ) and 0.2% (w/v) SDS (1 h). The blot was developed with a polyclonal rabbit antiserum against a peptide derived from SGLTl (ASl) or with the monoclonal antibody R4A6 which is directed against RSl. AS1 was raised against residues 565-575 (NS- KEERIDLDA) of SGLTl(1). For immunization the peptide was coupled to keyhole limpet hemocyanin via an NH,-terrninal cystein residue.

ImrnunohistochemistyFor immunofluorescence labeling, oocytes were fixed overnight in ethano1:chloroform:acetic acid (6:3:1, v/v/v). Af- ter further dehydration with ethanol the oocytes were embedded in parafin wax, cut into 5-pm sections, and applied to albumin-coated glass slides. For immunostaining, the sections were dewaxed with xylol, hydrated with PBS, and first blocked (15 min, 22 "C) with PBS containing 1% (w/v) bovine serum albumin, 0.5% (w/v) Tween 20, 0.5% (w/v) Triton X-100, and then transferred to the antibody incubation buffer (PBS containing 0.1% (w/v) bovine serum albumin and 0.05% (w/v) Tween 20). Incubation with the primary antibody (R4A6,50 pglml) was performed for 1 h (22 "C). After washing anti-mouse IgM antibodies from goat coupled to rhodamine (diluted 1:50) were applied (1 h, 22 "C), the sections were washed, enclosed, and inspected by fluorescence microscopy.

For postembedding immunogold staining of ultrathin sections, oocytes were fixed (16 h, 4 "C) in PBS containing 4% (v/v) paraformalde- hyde and 1% (v/v) glutaraldehyde. Then the oocytes were incubated 2 h (22 "C) in PBS containing 2% (w/v) glycine, dehydrated in ethanol, and embedded in LR White resin (19). Ultrathin sectioning, etching, immu-

M r

97 k -

66 k -- - " " *

"

L 5 k -

29 k "

a b c d e f g h RSli - + + - + - + RS1" - +

FIG. 1. In vitro expression of RS1 which reacts specifically with the mAb R4A6. Reticulocyte lysates with (c, d, e) or without ~-[~~Slmethionine (a, 6, f i g, h ) were mixed with water (-1 or with RS1-cRNA (RSli +, RSl' +), incubated a t 37 "C, and the samples were applied to SDS-polyacrylamide gel electrophoresis. The gel was dried and applied to autoradiography (c-e), or the proteins were electrically transferred to nitrocellulose and either stained with Amido Black (a, b) or reacted with R4A6 ( f i g ) or control IgM antibody (h). The positions of the molecular mass marker proteins phosphorylase b (97 m a ) , bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa) are indicated.

25044 Protein Which Modifies Na'-D-glucose Cotransport

-240 l ~ ~ l l l l l l l A l T ~ A l 6 C C T K C l A l l A T l C l Q l G l l A T A T Q l 6 l l ~

- 1 0 l 6 l C 6 A C C ~ 1 6 t i G A I l C 6 l T C l l 6 C A T C ~ l C T K ~ l A T ~ T ~ C ~ T

-120 ll~CT66TTlll6~lCT6lCClGlTl~l~CTlC6TMEC6ClMllMl6~~

-60 T l l ~ K l ~ C C C l A ~ C 6 l l C ~ ~ 6 6 l ~ l ~ ~ ~ ~ ~ ~

1 A T 6 l U l e A l l K W l T ~ l ~ l l ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ M S S L P l S O G F N H Q A H P S 6 Q R 20

61 ~C~~ll~lA6lCCTCCWTCTTGCIUCTlKl~lCl6lCTClKClCffilCl~CCllTC p E 1 6 S P P S L A H S V S A S V C P F 40

121 ~ C c A 6 1 Q c C c A M t A t C A l l ~ C l M A 6 C l 6 T ~ l G T ~ l l l ~

181 K T T U C C T ~ M T ~ C C ~ ~ ~ T ~ T ~ ~ T T C C T C ~ C C A ~ ~ C C ~

K P S O P O S I E P K A V K A V K A L K 60

A S A E F Q I T F E R K E Q L P L Q O P 80

2 4 1 T C T U T T G T K ~ T C T T C A ~ C A ~ M T K T C C T C C A G C U A C U G T C C C ~ ~ T G

u)1 C ~ l l C C C l C 6 U t M K C A l l 6 l l K A U l ~ T C T ~ ~ T C T ~ T ~ ~ K

S O C A S S A D N A P A N Q T P A I P L 100

Q N S L E E A I V A O N L E K S A E G S 120

361 K C C ~ C T C U A T C T C A T C T C C A C A ~ ~ ~ T A G T T T A T C T G T C A C M C ~ T Q 6 L K S H L H T R Q E A S L S V ~ ~ 1 4 0

421 w 1 M t A K C A C A C A G C C T l A T A t C l ~ ~ T T C G C A l C C C G M T M Q E P Q R L I G E K G Y H P E Y Q 160

481 U C C C M 6 T C M G T G A A T C G C C T T C A K A K A C ~ ~ C M ~ T G M C A t C A T U G O P S Q V N G L Q Q H E E P R N E Q H E 180

541 6 T l 6 l ~ K l C C K A l ~ C C A ~ C A l C T G l 6 T M ~ C A ~ C T l ~ V V Q Q M A P H O P E H L C N T G O L E 200

601 C T l C l T 6 6 A G M A 6 6 C M C A t M l C M E ~ G l 6 l l C 6 l T T ~ C T K ~ l G A U L L 6 E R Q Q N Q P K S V 6 L E T A V R 220

6 6 1 CWCACACGCCKAKAGGATGT~CCTTCCTGGTACAWCMTATTCTCCCTTAC G D R P Q Q D V O L P G T E K N l L P Y 240

721 G G A T K T T T C G C T K T C M G T T C A G A " T A C A G T T ~ C A G T C C G C F G C S S S E T F W E l D T V E Q S 260

7 8 1 CTAGTTtCTGTGCTTAATTCAGCACGCtCTCAGMTACCTCTGTCA~CATCAGTtCA L V A V L N S A G G Q N T S V R N I S A 280 *

841 T C T Q l C T C A C C G T A W T M 7 C C C l l M l ~ G T A ~ C A T T ~ T 6 T M T C C T T C C *

S O L T V O N P L M E V E T L K C N P S 300

901 T C l G M T T ~ T M 6 T M l C C C K T T C C A C T e A C M T T T A C A G C T T C T S E F L S N P T S T Q N L Q L P E S S V 320

961 G M A l 6 l C l C W A C M A T M A G M T A l ~ T C A C C C C l C C T C l T T M G T C T C l G T ~ E M S G T N K E Y G N H P S S L S L C G 340

1021 A C T T G T C A K C C T C T G T A T C ~ A C A C t M T C T T K T C A T C T A T A A C ~ A K C T T G T C Q P S V E S A E E S C S S I T A A L 360

1081 M G l i M C T T C A T W K T T T T f f i T C A T T A G T A G l M A C C A t C G T T A ~ T A C A T C T ~ K E L H E L L Y l S S K P A L E $ T S E 380

E V T C R S E l V T E 6 Q T O V K O L S 400 1141 C M C T T A C C T G T C 6 6 T C A G A C A T A G T M C T ~ ~ C A U T G T T M t G A C C l T l C T

1201 6MA6A166ACCEMA6TQ~AlCllK~lKl~lUKAGTGllCACM6TC E R U l Q S E H L l A A Q N E Q C S Q V 4 2 0

1261 T C C T l C T A T C A 6 6 C C A C A l C T G l A l U 6 l 6 M ~ l l M C A U C A C T T C M C ~ S F Y Q A T S V S V K T E E L T D T S T 440

1321 C A T K T C t M C A ~ T G T A ~ T A T T A C C T C C I M L T C C I C T G T T A O A G T E D V E N I T S S 6 P G D 6 L L 460 *

1381 G T C G A T A A ~ M T G T C C C C A G C T C T A ~ T C A G l ~ C A ~ K A G l T l A G T C A C T V O K E N V P R S R E S V N E S S L V T 480

1441 CTA6ACTCAKTMMCGTCTMTCMCCACACTKACCTlAf f iTGlA~ lTTCACCC *

L O S A K T S N Q P H C T L G V E I S P 500

1501 ~ l T T T A 6 C ~ T U t G A C G t l G C A t T t A A C l C M C C A ~ C T T C T ~ G C M A C G G A G T C C T T G G L L A G E E G A L N Q T S E O T E S L 520 *

1561 T C A T C C A K T T C A T A C T G C T T A M ~ T T T ~ T C ~ A C A C A C A A T C C A G T C A C A M C S S S F l L V K D L G Q C T Q N P V T N 540

1621 A C G C C T C A G A C C A C A W M A T C T C T G T C C T ~ K T K A ~ T A C G T C A A ~ T T T ~ R P E T R E N V C P E A A G L R O E F E 560

1681 CCACCTACCACECATCCATCATCAAGTCCTTCCTTTCTTGCACCGTTAATTTTTCCTtCT P P T S H P S S S P S F L A P L I F P A 580

1741 tCACACATTCACC~TTCITCGGGCCGCCTTTACCTTGCAGGAAGCTCTTGGGGtTTTG A O I D R I L R A C F T L Q E A C G A L 6 0 0

1801 CATCWGTTCCTCGAAATGCACACCTTGCACTTCTTGTTTTGCTA~AAAGAACATTGTA M R V G C N ~ O L A L L V L L A I N I V 6 2 0

FIG. 2. Nucleotide sequence and predicted amino acid sequence for the longest open reading frame of Rs1. The numbers on the left and on the right refer to the nucleotide and amino acid positions, respectively. The sequence was derived from clone RSli (nucleotides 303-4831), from 5'-RACE experiments (nucleotides -13 to 302), and from inverse PCR experiments (nucleotides -240 to -14). Aputative TATAbox (nucleotides

Protein Which Modifies Na'-D-glucose Cotransport 25045

nostaining (with 1 pg/ml of R4A6, rabbit serum against mouse IgM, and gold-labeled protein A), and electron microscopic inspection were performed as described earlier (19).

Materials

2-Deoxy-~-l-[~H]glucose (640 GBqImmol), D-['~CIAMG (5.7 GBs/ mmol), my0-[2-~H]inositol (677 GBq/mmol), avian myeloblastosis virus reverse transcriptase, and rabbit reticulocyte lysate were obtained from Amersham Buchler (Braunschweig, Germany) and [y-2,3-3Hlaminobu- tyric acid (1.14 GBq/mmol), 86Rb (60 GBqImmol), and ~-[~~Slmethionine (46 TBq/mmol) from Du Pont de Nemours (Dreieich, Germany). A-Zap phages, R408 helper phages, E. coli BB4, T7 RNA polymerase, T3 RNA polymerase, and mRNA capping kit were purchased from Stratagene (Heidelberg, Germany); and DNA polymerase I, proteinase K, and restriction enzymes were from Boehringer (Mannheim, Germany) RNase H, T4 DNA polymerase, T4 DNA ligase, and RNA standards were delivered by GIBCO (Eggenstein, Germany). EcoRI methylase was obtained from New England Biolabs (Schwalbach, Germany), and rhodamine-coupled goat anti-mouse IgM antiserum was from Serva (Heidelberg, Germany). The molecular mass marker urease, collagenase type IAfrom Clostridium histolyticum (1.3 unitdmg), isopropylthio- galactoside, diethyl pyrocarbonate (DEPC), and alkaline phosphatase- coupled anti-mouse IgM antiserum from goat were delivered by Sigma (Miinchen, Germany). All other chemicals were obtained as described earlier (19, 20, 36).

RESULTS

Identification of a cDNA Encoding a n Antigenic Polypeptide of a Monoclonal Antibody against the Na+-D-glucose Cotrans- porter-A &Zap expression library from pig kidney cortex was screened with mAb R4A6 which stimulates Na'-dependent high-affinity phlorizin binding to brush-border membranes from kidney and intestine and binds to a renal and intestinal brush-border membrane polypeptide with an apparent molecular weight of 75,000 (19, 20, 37). Out of 250,000 recombinant phages, four positive clones with inserts between 1.0 and 4.5 kilobase pairs were isolated which showed no hybridization with SGLT1-cDNA (1) and with cDNAs of the Na+-independent glucose transporters GLUT1 (human erythrocyte, Ref. 31) and SNF3 (yeast, Ref. 38). In vitro translation in reticulocyte lysates showed that one cDNA clone (RSli) with an insert of 4.5 kilobase pairs encoded a single polypeptide with an apparent molecular weight of 59,000 which reacted specifically with mAb R4A6 (Fig. 1).

When both DNA strands of the RSli cDNA insert were sequenced a clone with 4529 nucleotides was obtained (RSl;, see nucleotides 3034831 in Fig. 2). RSli contained an open reading frame which starts at the Eco RI cloning site of &Zap and coincides with the open reading frame of p-galactosidase encoded by A-Zap phages (39). Preliminary primer extension experiments showed that the open reading frame of RSli ex- ceeded the 5'-end. However, the first ATG in the sequence of RSli (nucleotide 427 in Fig. 2) may be used as start codon for protein synthesis, since computer analysis (PC-GENE, Genofit) showed that the nucleotides 420-429 may serve as a translation initiation sequence in eukaryotes (40, 41). The 3"noncod- ing region of RSli is 2962 base pairs long. It contains two consensus polyadenylation AATAAA signals (nucleotides 2163- 2168 and 3224-3229). The 5'-nucleotide sequence missing in RSli was cloned by PCR employing the RACE methodology and inverse PCR (see "Experimental Procedures"). Nucleotides -240 to 4831 in Fig. 2 comprise the sequence of the complete clone (RS1). The coding region starts at the first ATG in the open reading frame of RS1. This ATG is not part of a typical eukaryotic translation initiation sequence but is preceded by a

LO a 1

- L O - ( , , , , ) I 100 200 300 LOO 500 600

Residue number b

r I = I I FIG. 3. Hydropathy plot (a) and secondary structure predic-

tion ( b ) of RS1 protein. The hydrophobicity indices were calculated according to Kyk and Doolittle (42) employing a window of 9 amino acids. b shows four a-helices which are predicted according to Gamier et al. (43).

potential TATA box. The predicted protein contains 623 amino acids and has a molecular mass of 66,832 Da. In agreement with the calculated molecular weight, a polypeptide with an apparent molecular weight of 72,000 was obtained after in vitro translation of RS1* in reticulocyte lysates (Fig. 1, lane c) . Com- parison of the DNA sequences of RSli and RS1, and of the amino acid sequence of RS1 with the sequences in GenBank, EMBL, SWISS-Prot, and NBRF-Prot (January 1993) showed no similarities with other sequences. Fig. 3 shows the hydrophobicity plot of the RS1 protein (42). The protein is rather hydrophilic and contains four a-helical regions (amino acids 47-72, 103-122, 348-370, and 607-621) as predicted by the Ganier method (43). The method of Rao and Argos (44, 45) predicted that the carboxyl-terminal a-helix (amino acids 607- 621) may represent a transmembrane domain. The RS1 protein contains about 21.8% of charged amino acids (E, 9.1%; D, 4.3%; R, 2.9%; K, 2.9%; H, 2.6%) which appear to be arranged in clusters. Six N-linked glycosylation sites are predicted (46,47), suggesting that the predominant part of the protein may be exposed on the outside of the brush-border membrane. This interpretation is consistent with the observation that antibody R4A6 binds to the extracellular side of the brush-border membrane (19).

Interaction of RSli with the Endogeneous Na'-D-glucose Co- transport in Oocytes of X. Luevis--To test for an effect of the polypeptide encoded by RSli on Na'-D-glucose cotransport sense cRNAof RSli was synthesized and injected into oocytes of X. laeuis. Consistent with previous observations (48,491 it was observed that oocytes ofX. laevis exhibit Na' gradient-dependent and phlorizin-inhibitable uptake of D-glucose o r AMG and that the glucose uptake varies considerably between different batches of the oocytes which contain SGLT1-homologous mRNA. This was shown by PCR experiments in which specific amplification products were obtained with primer pairs derived from SGLTl (data not shown). In water-injected and noninjected oocytes, Na+ gradient-dependent uptake rates of 50 p~ D-g1UCOSe (AMG) varied between 0.5 2 0.1 (0.4 f 0.05) pmol x oocyte" x h-l and 38 * 4 (1.9 5 0.2) pmol x oocyte" x h-l. Note that in oocytes with high endogeneous uptake, the uptake of 50 1.1~ D-glUCOSe was much more increased than the uptake of 50 p~ AMG. Since we found that the Na' gradient-dependent uptake of 50 p~ glucose expressed after injection of SGLT1-cRNA

-38 to -33), a possible alternate translational start site (nucleotides 420-429) and two consensus polyadenylation signals (nucleotides 2163-2168 and 3224-3229) are un&rlined. At the COOH terminus 15 amino acids, which may form a hydrophobic a-helix, are boxed. Six potential N-glycosylation sites are indicated by asterisk. A stop codon and EcoRI and Hind111 restriction sites are indicated by arrowheads. For cloning the recognition sites for BglI (nucleotides 435445) and for RsaI (nucleotides 544-547) were used.

25046 Protein Which Modifies Na+-D-glucose Cotransport

into the oocytes was the same whether D-glucose or AMG was employed as substrate: the endogeneous Na'-D-glucose cotransporter and the Na'-D-glucose cotransporter expressed by SGLT1-cRNA are functionally different.

After injection of 0.1-0.5 ng of RSli-cRNNoocyte the Na' gradient-dependent uptake of 50 p~ D-glucose and AMG was increased when the endogeneous Na' gradient-dependent D-

glucose (50 p ~ ) uptake of water-injected oocytes was less than 1.5 pmol x oocyte-' x h-', whereas it decreased when the endogeneous uptake was larger than 9 pmol x oocyte-l x h-l (Fig. 4). These effects were also observed when cRNA was injected which was only derived from nucleotides 303-2128 of RS1 (RSli*). No effects on endogeneous Na'-D-glucose cotransport were observed when antisense cRNA of RSli was injected or when the oocytes were only incubated 3 h after the injection of sense RNA of RSli. The data suggest that translation of RSli protein is required for the observed effects on glucose transport.

Interaction of RSl with Na'-o-glucose Cotransport Which Can Be Expressed by SGLTl-Next we investigated whether the expression of Na+-D-ghcose cotransport by injection of SGLT1-cRNAintoXenopus oocytes was altered by coinjection of RSli-cRNA or RSl*-cRNA. The injection of only SGLT1-cRNA into oocytes led to a drastic stimulation of AMG and D-glucose uptake in the presence of 110 m~ Na' (see also Ref. l), and the expressed uptake rates were reduced by more than 95 and 98%, respectively, when Na' was replaced by tetramethylammonium or when 100 VM phlorizin was added. Since the expressed rates of Na' gradient-dependent D-glUCOSe or AMG transport (measured in the presence of 110 m~ Na' and 50 p~ of D-glucose or AMG) were not significantly different (data not shown), the transport expressed by SGLTl is supposed to have a similar

for D-glucose and AMG. Measuring the dependence of expressed AMG transport on the amount of injected SGLT1-cRNA a sigmoidal type of correlation was observed (Fig. 5u). Maximal expression of Na' gradient-dependent AMG transport was observed after injection of 2-4 ng of SGLT1-cRNNoocyte (three different cRNA preparation, five different batches of oocytes). The maximal Na' gradient-dependent uptake rates of 50 VM AMG varied between 142 * 13 and 206 * 23 pmol x oocyte-l x h-'.

The effect of coinjection of RSli-cRNAwith SGLT1-cRNAwas investigated with many different batches of oocytes over a pe- riod of 2 years, and it was observed consistently that the expression of AMG transport by SGLTl was significantly altered by RSli. It turned out that the effects of RSli were dependent on the total amount of injected SGLT1-cRNA and on the ratio between the amounts of injected SGLT1-cRNA and RSli-cRNA. Thus RSli-cRNA stimulated the expressed transport of 50 p~ AMG between 2- and 25-fold when the amount of injected SGLT1-cRNA per oocyte was 0.2 ng or less (10 experiments, molar RSli-cRNNSGLT1-cRNA ratios 1.8 to 4.5). These low amounts of injected SGLT1-cRNA lead to an expression of less than 7% of AMG transport which could be maximally expressed. When the amount of injected SGLT1-cRNA per oocyte ranged between 0.7 and 1.5 ng, which lead to an expression of 3541% of maximal expressable AMG transport, the coinjection of RSli-cRNA(15 experiments, molar RSli-cRNNSGLT1-cRNA ratios 1.6 to 3.1) inhibited the expressed transport of 50 p~ AMG between 39 and 87% (S.E. c 7%) by RSli (see e.g. Fig. 5c).

It was tested whether the stimulation and inhibition of AMG uptake by RSli observed after injection of small and large amounts of SGLT1-cRNA was dependent on the molar ratio of the injected RSli-cRNA and SGLT1-cRNA. Fig. 5 shows that maximal stimulation and inhibition ofAMG uptake (measured at 50 p~ AMG) was observed at a stoichiometry of about 2

M. Veyhl and H. Koepsell, unpublished data.

D-glucose

a 10

C RSl,

6 1 *IAG

FIG. 4. Different effects of RSli on Na+-glucose cotransport in Xenopus oocytes with low and high endogeneous transport activity. Oocytes of X. Zaevis with low activity of endogeneous Na+ gradient-dependent o-glucose or AMG transport (a, b ) and those with high activity of endogeneous transport (c, d ) were injected with 50 nl of water ( C ) or with 50 nl of water containing 0.3 ng of RSl,-cRNA (RS1,). After 48-h (18 "C) incubation, uptake of 50 J.IM D-glucose and of 50 PM AMG was measured in the presence and absence of Na'. A representative experiment is shown.

I

a

b

b u- I a 0 2 L 6 15 F molor RSli-cRNA /SGLTl-cRNA rotlo 3

C

0' I 1

molor RSl , -CRNA/UiLTl-CRNA ratio 0 2 L 6 B

oocytes on the amounts of injected SGLTl-cRNA(a) and of R S l i - FIG. 5. Dependence of AMG transport expressed in Xenopus

cRNA which was coiqjected with SGLT1-cRNA (21, c). In a different amounts of SGLT1-cRNAwere injected, in b 0.15 ng of SGLT1-cRNA were injected per oocyte together with different amounts of RSli-cRNA,

RSli-cRNA. Transport of 50 PM AMG into oocytes ofx. Zaevis was meas- and in c 1.2 ng of SGLT1-cRNA were injected plus different amounts of

ured in the presence of 110 m~ Na' and corrected for AMG uptake measured in water-injected oocytes.


between the injected cRNAs and that no alteration of AMG uptake was observed if the molar ratios of RSl,-cRNA and SGLT1-cRNA were larger than 7. The data demonstrate a stoichiometric interaction between both clones which is assumed to occur at the level of translated proteins (see below). Since the injected cRNAs are probably translated and degraded differentially the observed stoichiometry need not to be identical to the stoichiometry of the interacting polypeptides. To elucidate whether the interaction between SGLTl and

RSli occurs in the cytoplasm andor in the plasma membrane we compared functional properties of Na+-glucose cotransport expressed by SGLTl and by SGLTl plus RSli. Fig. 6 shows the

I 0

6.0 I b

4.0

c 2.0

7 I c

'I [bmol . Oocyt." .h"]

FIG. 6. Comparison of glucose dependence of Na+-glucose cotransport expressed by SGLTl-cRNA or by stoichiometric amounts of SGLTl-cRNA and R!311-cRNA. Different amounts of SGLT1-cRNA per oocyte (a , 0.145 ng; b, 0.5 ng; c, 1.2 ng) were injected alone or together with a stoichiometric amount of RSli-cRNA (a, 0.51 ng; b, 1.75 ng; c, 4.2 ng). In the presence of Na+ and different AMG concentrations, AMG uptake was measured in cRNA-injected and water-injected oocytes, and the expressed uptake rates were calculated. A representative of three experimental series is shown. The straight lines were fitted by assuming one transport site, whereas the curves were calculated by assuming the existence of two glucose transport sites (16). Open symbols represent data obtained after injection of SGLT1-cRNA

(b ) , 570.2 f 10.5 (e); in pa: 117 f 6 (a inset), 122 f 3 (b ) , 126 f 5 (e ) ) . alone. V,, in pmol x oocyte-' x h-l: 15.5 f 0.35 (a, inset), 176.9 f 2.2

Closed symbols represent data obtained after coinjection of SGLTl- cRNAand RS1,-cRNA. V,,,=(high as) in pmol x oocyte" x h-l: 116.0 f 2.5 (a) , 56.5 f 1.0 (b ) , 66.5 f 3.5 (c); V,,,, in pmol x oocyte-' x h-l: 454.0 f 1.0 (a), 513.5 * 1.5 ( b ) , 502.0 * 44.0 (c); &s(high am in PM: 28.5 f 0.6 (a ) , 16.7 f 1.1 ( b ) , 21.4 f 3.0 (c); Ko.6(low am in mM: 1.88 f 0.08(a), 0.91 f 0.03 ( b ) , 1.37 f 0.14 (e ) .

glucose-dependence of expressed AMG uptake obtained after injection of three different amounts of SGLT1-cRNA alone and after coinjection of SGLT1-cRNA with a stoichiometric amount of RSl,-cRNA. After injection of various amounts of SGLT1- cRNA Michaelis-Menten kinetics with apparent K,,, values between 96 * 5 and 125 * 5 p were obtained. With increasing amounts of injected SGLT1-cRNA the V,, of transport increased, whereas the apparent K , values remained constant, suggesting that the properties of the SGLT1-expressed transporter are independent from the transporter density in the membrane. Coinjection of stoichiometric amounts of RSl i -

cRNA and SGLT1-cRNA has two effects: 1) an increase of V,, suggesting an increase of the number of functional transporters in the membrane when small amounts of SGLT1-cRNA were injected (Fig. 6, a and b); 2) a functional alteration of the transporter in the membrane indicated by the generation of high and low affinity transport sites. Thus with different amounts of a stoichiometric mixture of SGLT1-cRNA and RSli-cRNA high- and low-affinity transport sites with apparent values of 17-29 p and 0.9-1.9 m were obtained (Figs. 6, a-c). Theo- retically, the kinetics obtained after coinjection of stoichiometric amounts of SGLT1-cRNA and RSli-cRNA should contain a third component representing a fraction of SGLTl polypeptides which are not associated with RS1,. Since this component has an intermediate value of about 100 p, it cannot be re- solved.

To determine whether the low- and high-affinity transport sites expressed by coinjection of SGLTl plus R S l i represent high- and low-affinity glucose binding sites, the interaction of the competitive inhibitor phlorizin with glucose binding sites on the extracellular side of the transporter (16) was investigated. This was performed by measuring phlorizin inhibition of Na'-D-ghcose cotransport rather than phlorizin binding, since binding measurements showed in agreement to theoretical con- siderations that the number of phlorizin binding sites per oocyte which could be expressed by SGLT1-cRNA was not high enough to allow accurate binding measurements. M e r injection of SGLT1-cRNA, one site for inhibition of AMG transport by phlorizin was observed (Ki = 5.0+0.1 p, see also Ref. 171, whereas high- and low-affinity inhibition sites (Kj(high am = 0.27 * 0.01 p, Kiclow am = 29 * 6 PM) were distinguished after injection of a stoichiometric amount of SGLT1-cRNA and RS1,- cRNA (Fig. 7). The affinities of the phlorizin inhibition sites expressed by SGLTl plus RSli are about the same as the affinities of low- and high-affinity phlorizin binding sites measured in brush-border membrane vesicles from pig kidney outer cortex and outer medulla (16). The data show that expressed SGLTl protein and RSli protein interact in the oocyte plasma membrane. The theoretical possibility that RSli alters the function of SGLTl indirectly via the Na' concentration in the oocytes or via the membrane potential (16) was excluded, since intracellular Na' (data not shown) and the membrane potential measured in the absence of glucose were not changed by RSli- cRNA. The membrane potential was measured in oocytes (ORi buffer on the outside, absence of glucose) which were (a) water- injected, ( b ) injected with 1.2 ng of SGLT1-cRNA, or (c) with 1.2 ng of SGLT1-cRNA plus 4.2 ng of RSli-cRNA and incubated for 48 h. The oocytes showed similar effects on transport as demonstrated in Fig. 6c. The membrane potentials in the three groups of oocytes were -43.8 * 1.8 mV (a), -44.5 * 1.3 mV (b ) , and -44.7 * 1.0 mV (c).

The observation that RSli-cRNA did not alter SGLT1-expressed AMG uptake when an excess of RSli-cRNA was coinjected (see Fig.5, b and c ) suggests that in this case more than one RSli polypeptide interact with SGLTl protein which may be present as dimer or tetramer (16,501. The experiment in Fig.

25048 Protein Which Modifies Na+-D-glucose Cotransport

i . L.0 c

fractional inhibition

F~G. 7. Comparison of phlorizin inhibition of Na+-D-ghcose cotransport expressed by SGLT1-cRNA (0) or by stoichiometric

with water, with 0.5 ng of SGLT1-cRNA/oocyte or with 0.5 ng of SGLT1- amounts of SGLT1-cRNAand RSlpcRNA (0). Oocytes were injected

cRNA plus 1.75 ng of RSli-cRNA/oocyte. After 48-h incubation, uptake of 50 ~ M A M G was measured in the presence of 110 m~ Na+, and varying

lated. In the absence of phlorizin the uptake rates of 50 VM AMG ex- concentrations of phlorizin and the expressed uptake rates were calcu-

pressed by SGLT1-cRNAor by SGLT1-cRNAplus RSli-cRNAwere 4.7 f 0.3 and 26.3 * 1.1 pmol x oocyte" x h-l, respectively. In the graph the fractional phlorizin inhibition of AMG uptake expressed by SGLTl (0) and by SGLTl plus RSli (0) is plotted against the ratio between fractional inhibition and phlorizin concentration. The straight line was fitted by assuming one phlorizin inhibition site and the curve by assuming two inhibition sites (16).

\ \ 1

0 I

50 lm v[pmol x O O C ~ ~ O " xh"]

FIG. 8. Comparison of glucose dependence of Na*-glucose cotransport expressed by SGLT1-cRNA or by SGLT1-cRNA plus excess of RSli-cRNA 0.3 ng of SGLT1-cRNA (0) or 0.3 ng of SGLT1- cRNA plus 8.5 ng of RSl,-cRNA (0) were injected per oocyte. The expressed uptake rates were measured and presented as described in the legend to Fig. 6. Injection of SGLT1: V,, = 110 f 6 pmol x oocyte-' x h-l, K,,, = 96 f 5 VM; injection of SGLTl plus RSli: V,, = 124 f 7 pmol x oocyte" x h-l, K,,, = 92 f 5 PM.

8 is consistent with this hypothesis; after coinjection of SGLT1- cRNA and an excess of RSli-cRNA no significant stimulation of V,, was observed and the glucose dependence of AMG transport was about the same as after injection of SGLT1-cRNA alone.

After the missed 5'-end of the RSli-cDNA was identified, a clone with the complete open reading frame and a Kozak-type translation initiation sequence, termed RSl", was synthesized (see "Experimental Procedures"), and it was tested to determine whether the derived cRNA showed the same effects as the incomplete clone RSli. RSl*-cRNA stimulated and inhibited endogeneous AMG uptake in oocytes with low and high endogeneous Na+-D-glucose cotransport, respectively. Also, AMG transport expressed by injection of small amounts of SGLT1-

>

0 , 0 260 &bo

v [pmol I oocyte" I h"]

FIG. 9. Comparison of glucose dependence of Na+-glucose cotransport expressed by SGLTl-cRNA (0) or by SGLT1-cRNAplus a stoichiometric amount of RSl'-cRNA (0). 0.5 ng of SGLT1-cRNN oocyte was injected alone or together with 1 ng of RS1'-cRNA. The measurements were performed and the data calculated as described in the legend to Fig. 6. M e r injection of SGLTl alone: V,, = 115.5 * 1.8 pmol x oocyte-1 x h-l, = 127.5 3.9 PM; aRer injection of SGLTl PlUS B1': Vm,(high am = 83.6 f 1.6 pm01 X Oocyte-' X h-', Vmar(law = 316.4 f 2.3 PmOl X O O C ~ " X h-', KO.E(high = 28.0 f 1.7 +lM, K0.5(lOw

= 947.6 f 68.8 PM.

( m V ) -150 -100 -50 0 (mV) -150 -100 -50 0

A

L-BO(nA)

FIG. 10. Voltage dependence of current which is mediated by Na*-n-glucose cotransport expressed by SGLTl-cRNA (A) or by stoichiometric amounts of SGLT1-cRNA and RSl'-cRNA (B) . 00- cytes were injected with 0.5 ng of SGLT1-cRNAloocytes (A) or with 0.5 ng of SGLT1-cRNA plus 1 ng of RSl'-cRNA ( B ) . After 48-h incubation the voltage dependence of membrane current was measured in the absence and presence of different AMG concentrations (0.05 m ~ , (W, 0.1 m M (a), 0.2 m M (01, 1 mM (O)), and the AMGinduced membrane current was calculated. Mean values of three independent measurements are shown.

cRNA (0.5 ndoocyte or less) was stimulated if appropriate amounts of RS1'-cRNA were coinjected (molar ratios of W1*- cRNNSGLT1-cRNA between 1 and 4). Fig. 9 shows that also the substrate dependence of AMG-transport expressed by SGLTl was altered by RSl*. The data suggest that the incomplete protein translated by RSli and the complete protein translated by RSI' interact with SGLTl protein in the same way and that the first 101 amino acids of RS1 protein are not involved in the interaction with SGLT1.

Next the voltage dependence of sodium-glucose cotransport was measured in the presence of different glucose concentrations when transport was expressed by SGLT1-cRNA or by a stoichiometric amount of SGLT1-cRNAand RSl'-cRNA. Fig. 10 shows the currents at various clamped membrane potentials which were induced by different concentrations of AMG. In water-injected control oocytes, clamped to -150 mV, inward currents of less than 1.5 nA were induced by 1 m~ AMG. Coin- jection of RS1*-cRNA does not only increase the glucose-induced currents, but alters the shapes of the glucose-induced current voltage curves compared with SGLT1-injected oocytes (Fig. 10). After coinjection with RSl', steeper glucose-dependent current-voltage curves are obtained at negative membrane potentials indicating a more pronounced effect of the mem-


brane potential on the cotransport of Na+ and glucose. Immunohistochemical Demonstration That Expressed RSli

Protein Is Incorporated into the Plasma Membrane-To confirm by morphological methods that RSli protein is incorporated into the plasma membrane, oocytes were injected with water, 1.2 ng of SGLT1-cRNA, or 1.2 ng of SGLT1-cRNA plus 4.2 ng of RSli-cRNA. After 48-h incubation AMG uptake was measured in each group and some oocytes were fixed, sectioned, and immunostained with R4A6. Uptake of 50 PM AMG was increased by SGLT1-cRNA and inhibited after coinjection of RSli-cRNA as described in the legend to Fig. 6c. No significant immunostaining was observed in water-injected oocytes (Fig. 11, a and c) and in oocytes injected with SGLT1-cRNA (data not shown). After injection of SGLTl-cRNA plus RSli-cRNA, diffuse staining below the plasma membrane and intense staining of the plasma membrane were observed (Fig. 11, b and d). The data show that the protein translated by RSli-cRNA is inserted into the plasma membrane in the presence of SGLT1.

Attempts to Define the Specificity of RSl-To test the specificity of RS1 for SGLTl we measured the effect of RSli-cRNAon other transporters. Studying the effect of RSli-cRNA on the endogeneous Na+-L-glutamate cotransport (51) and the endo-

K

.: 7

:-

- A

. # I

'*.,

FIG. 11. Demonstration that expressed RS1, protein is incorporated into the Xenopus oocyte plasma membrane. Oocytes were injected with water (a, c ) or with 1.2 ng of SGLT1-cRNA plus 4.2 ng of RSli-cRNA/oocyte (b , d) . After 48-h incubation the oocytes were embedded, sectioned, immunostained with the monoclonal antibody R4A6, and inspected by light microscopy (a, b ) and electron microscopy (c , d ) as described under "Experimental Procedures." The arrows point to gold particles which indicate the reaction of R4A6 at the plasma membrane. Bars: 50 pm ( a ) and 0.5 pm (c) .

geneous (Na++K+)-pump (33) in oocytes of X. laeuis, it was found that RSli-cRNA concentrations, which changed the endogeneous Na+-D-glucose cotransport activity, did influence neither Na' gradient-dependent uptake of L-glutamate nor ouabain binding to the (Na'+K+)-ATPase and ouabain-inhibitable Rb+ flux through the (Na++K+)-ATPase (Table 11). Table I11 demonstrates that the coinjection of RSli-cRNA with cRNAs encoding the Na'-y-aminobutyric acid cotransporter (5) or a Na+-independent glucose transporter (31) showed no effects on the expression of the transport activity. To detect RSli effects on the insertion of expressed transporters into the oocyte plasma membrane, the amounts of injected transporter cRNAs (16.5 or 33 ngloocyte of GLUT1 (31) and 25 ngloocyte of GAT1 (5)) were selected to be in a range where the expression of transport was correlated to the amount of injected cRNA. The amounts of coinjected RSli-cRNA tested covered (i) the range in which RSli effects on SGLT1-expressed Na+-D-glucose cotransport were observed (0.5-4.3 ng of RSli-cRNA) and (ii) the same molar 2:l ratio of injected cRNAs which was required to ob- serve effects of RSli-cRNA on SGLT1-expressed glucose transport (that is 66 ng of RSli-cRNA plus 16.5 ng of GLUT1-cRNA, 100 ng of RSli-cRNA plus 25 ng of GAT1-cRNA). For the respective transport measurements, substrate concentrations far below the K, values of transport were employed in order to detect RSli effects on the K, values. Next we tested the effect of RS1 on the expression of Na+-myo-inositol cotransport by SMIT-cRNA (3). Fig. 12 shows that the expression of Na+-myo- inositol cotransport was signifcantly stimulated if an about 2-fold molar excess of RS1-cRNA was coinjected with SMIT- cRNA. The data suggest that RS1 interacts with transporters (transporter subunits) which are homologous to SGLT1.

Apparent Association of Proteins with Homology to SGLTl and to RSI in the Renal Brush-border Membrane-Trying to investigate the association of SGLT1-type protein and RS1- type protein in vivo, purified porcine renal brush-border membranes were solubilized with 2.5% (w/v) Triton X-100, the proteins were separated by native polyacrylamide gel electrophoresis, transferred to poly(viny1idene difluoride) membranes, and probed with antibodies which are directed against RS1 protein or SGLTl protein. The antibody against RS1 was our monoclonal antibody R4A6 which also binds to the brush-border membrane in the S1, S2, and S3 segments of the renal proximal tubule (37), but does not react with SGLTl protein. The antibody against SGLTl (AS1) was a polyclonal peptide antibody raised against the amino acids NS- KEERIDLDA (amino acid 565-575 in Ref. 1). As concluded from the primary structure, AS1 may cross-react with the SGLT1-homologous Na+-cotransport proteins for nucleosides and amino acids (2, 4) but not with the Na+-myo-inositol co-

TARLE I1 Demonstration that the endogeneous activities of the Na*-L-gZutamate cotransporter and the (Nu' + K+)-ATPase in Xenopus oocytes are not

altered by RSl ,

50 nl of water containing 0.3 ng of RSli-cRNA were incubated 48 h at Noninjected ooyctes ofX. laeuis and oocytes which were injected with

18" C. In 10 oocytes of each group ( i ) Na' gradient-dependent uptake of 0.1 m M L-[:'H]glutamate, ( i i ) Na' gradient-dependent uptake of 1 mM ~-[~H]glutamate, ( i i i ) ouabain-inhibitable uptake of 1 mM '"Rb', and (iv)

"Experimental Procedures." The transport and binding data of one ex- specific binding of 1.2 p~ ["]ouabain was measured as described under

perimental series (mean values * S.E.) are presented. With two other batches of oocytes the same results were obtained.

Uptake Injection Binding,

0.1 mH ~.-Clu 1 m M L-GIu 1 my Rb' pH Ouabain

pmof x mqte- ' x min" fmol x oocyfe" None 0 .9+0 .1 3 .6*0 .6 3 .8k0.3 21.4k0.5 RS1,-cRNA 1.0*0.2 3 . 4 e 0 . 6 4.1 i 0 . 3 22.0*0.5

25050 Protein Which Modifies Na'-D-glucose Cotransport TABU 111

Demonstration that the expression of a Na*-in&pendent mglucose transporter and of the Nu*-GABA-cotransporter is not altered by RSl,

Oocytes were injected with water (control) and with the indicated amounts of GLUT1-cRNA, GAT1-cRNA and RSl,-cRNA. After 48-h incubation the expressed uptake rates were determined. In GLUTl- cRNA-injected oocytes glucose-inhibitable uptake of 25 PM 2-deoxy-D- glucose uptake was measured in the absence of Na' and corrected for the uptake in water-injected oocytes. In GAT1-cRNA-injected oocytes GAFJA-inhibitable uptake of 0.7 PM GABA was measured in the presence of Na' and corrected for the uptake in water-injected oocytes. Mean values of 6 1 0 oocytes f S.E. are presented.

Iqiected cRNA Expressed uptake

2-Deoxv-~~lumse GABA

33 ng of GLUT1 33 ng of GLUTl plus 0.5 ng of RSli 33 ng of GLUTl plus 4.3 ng of RSli 33 ng of GLUTl plus 8.5 ng of RSli 33 ng of GLUTl plus 66 ng of RSli 16.5 ng of GLUTl 16.5 ng of GLUTl plus 66 ng of RSli 25 ng of GATl 25 ng of GATl plus 0.5 ng of RSli 25 ng of GATl plus 4.3 ng of RSli 25 ng of GATl plus 8.5 ng of RSli 25 ng of GATl plus 50 ng of RSl, 25 ng of GATl plus 100 ng of RSll

T

50.3 f 4.3 51.5 f 5.4 51.7 f 6.5 50.3 f 8.0 50.9 f 9.6 29.9 f 5.4 33.5 f 6.0

fmol x oocyte" x min"

10.0 f 1.2 9.9 f 1.6

10.3 f 1.5 10.1 f 1.3 10.0 f 1.2 11.0 f 1.6

= o 1 I 1 I I I 1 I - O 1 2 3 L 5 6

mdar RSI'-cRNA/SMIT-cRNA ratio F I G . 12. Dependence of Na*-dependent myo-inositol transport

expressed by SMIT-cRNA on the amount of coiqjected RS1'- cRNA. 5 ng of SMIT-cRNA were injected per oocyte together with different amounts of RS1'-cRNA. After 72 h (18 "C) incubation, uptake of 0.5 PM myo-inositol was measured in the absence and presence of Na+ and the Na+-dependent uptake calculated. In water-injected control oocytes, Na+-dependent uptake of 0.5 PM myo-inositol was smaller than 10 fmol x oocyte" x h-'. The uptake of 0.5 PM myo-inositol measured in the absence of Na' in injected oocytes was smaller than 3 fmol x oocyte" x h".

transporter (3). AS1 was used in this experiment, since it reacted strongly, and also with peptide antibodies with an apparent selectivity for SGLT1, the performed experiment can only demonstrate an apparent association of SGLT1- and Rs1-type proteins rather than the association of the specific proteins SGLTl and RS1, since it is probable that brush-border membranes contain additional nonidentified proteins with homology to SGLTl and Rsl. Light microscopic immunohistochemistry in rat kidney showed that AS1 reacts with the brush- border membrane in the three segments of the proximal tubule where Na+-D-glucose cotransport has been demonstrated (data not shown, see also Ref. 21). Na+-Dglucose cotransport in the S3 segment is probably mainly mediated by SGLT1, whereas a SGLT1-homologous protein is supposed to be the main Na+-D- glucose transporting protein in the S1 and the S2 segment (52).

M r

116k - 9 7 k - Y

r

L5k -a

29k -

A

a b c d e

B

Mr 5 L 5 k - m 272k -

116k -

66k -

a b c d e FIG. 13. Immune reactions of antibadicm .grlad SGLTl and

RS1 with brush-border membrane proteins which were *pa- rated by denaturing (A) and nondenaturing polyacrylamide gel electrophoresis ( E ) . Brush-border membranes of porcine proximal tubules were solubilized with 2% (w/v) SDS ( A ) or with 2.5% (w/v) Triton X-100 ( E ) and applied to polyacrylamide gel electrophoresis which was performed in the presence of 0.1% (w/v) SDS or 0.1% (w/v) Triton X-100, respectively. f i r electrophoresis the proteins were stained with silver (lane a ) or blotted and probed either with peptide antiserum AS1 directed against SGLTl (lane 6 ) or with the monoclonal IgM antibody R4A6 directed against RS1 (lane d). Controls were performed with preimmune serum (lane c) or mouse myeloma IgM (lane e). The lines indicate the positions of the molecular mam marker proteins a t 545 kDa (urease, hexamer). 272 kDa (urease, trimer), 116 kDa (& galactosidase), 97 kDa (phosphorylase b) . 66 kDa (bovine serum albumin), 45 kDa (ovalbumin), and 29 kDa (carbonic anhydrase).

A localization in the brush-border membrane of the S1, S2, and S3 segment was obtained for the antigeds) of mAb T4B2 which inhibits Na+-Dglucose cotransport (371, binds to SGLT1." and probably cross-reacts with the SGLT1-homologous Na+-D-glucose cotransporting protein in the S1 and S2 segment.

After separation of porcine brush-border membrane proteins by polyacrylamide gel electrophoresis performed in the presence of SDS, antibody R4A6 and the peptide antibody AS1 reacted with polypeptide monomers with apparent molecular weights of about 70,000 (see lanes b and d in Fig. 13A). How- ever, when Triton-solubilized brush-border membranes were separated by nondenaturing gel electrophoresis, both antibodies bound to a protein with an apparent molecular weight of about 300,000 (see lanes b and d in Fig. 13B). AS1 showed an additional reaction with a polypetide which migrated in the gel as was expected for the SGLTl monomer. The data suggest that SGLT1- and RS1-type proteins are associated in the brush- border membrane where they may form heterotetramers.

Distribution RSl-related mRIVAa-The distribution of Rsl- related RNAs was investigated by Northern blots and by amplification of Rsl-specific polynucleotide fragments. When mRNA from different porcine tissues or from Xenopus oocytes were probed with R s l i ' , hybridization was observed with small intestine, renal outer cortex, renal outer medulla, liver, and spleen, but not with skeletal muscle, heart muscle, brain, and Xenopus oocytes (Fig. 14). The hybridization occurred mainly with a 3.6-kilobase mRNA band, but some hybridization was also observed with a 7.3-kilobase band. This suggests that most mRNAs use the polyadenylation signal at nucleotides 3224-

Western blob of SGLTl- or RS1-injected Xenopus oocytes which were probed with mAb T4B2 showed that this antibody reacts with SGLTl (M. Veyhl and H. Koepsell. unpublished data).

Protein Which Modifies Na+-D-ghcose Cotransport 2505 1

7.3 k b -

x c al

1.76 - 1.03 - 0.6 5- 0.39 -

A

3.6kb -

0.27,

0.12 - 0.1 8 - a-

IJ

FIG. 14. Didbution of -1-related mFtNAm analyzed by Northern blot. Samples (10 pg) of poly(A*) RNA from porcine tissues and Xenopus oocytes were separated by agarose gel electrophoresis, blotted, and hybridized with RSli'. The mRNA was extracted from oocytes, whole organs (muscle, liver, spleen), cerebral cortex (brain), jejunal mucosal scrapings (small intestine), the outer 3 mm of renal cortex (outer renal cortex), or the outer stripe of the outer medulla (16) (outer renal medulla). Size standards are indicated.

3229. For further experiments on the distribution of RS1-homologous mRNAs, the PCR methodology was used. From different mRNAs cDNAs were synthesized and cDNA fragments from two regions of RS1 (nucleotides 794-1556 and 1768-1864) were amplified by employing RS1-specific primers. Specific amplification products were identified by hybridization with respective RS1-specific internal primers. Fig. 15 shows that specific cDNAs were amplified in renal cortex of pig and man, in small intestine of pig and man, in pig liver, LLCPKl cells, but not in pig renal inner medulla, pig skeletal muscle, pig heart muscle, MDCK cells, and oocytes ofX. laevis. Unlike the North- e m analysis, no amplification produds were observed in porcine spleen.

DISCUSSION

The results suggest that the Na+-D-glucose cotransporters in kidney and intestine contain catalytic and regulatory subunits. The catalytic subunit of the Na+-D-ghcose cotransporter in intestine and renal outer medulla is probably the previously cloned SGLTl protein which also occurs in LLCPKl cells (1, 52-55). The catalytic subunit in outer renal cortex is supposed to be a SGLT1-homologous protein which could be identical to the recently cloned protein Hu14 (52, 56). The regulatory subunit is a protein which is identical or homologous to RS1. RS1 has been cloned from renal cortex, but homologous mRNAs have been identified in renal outer medulla, small intestine, and LLCPKl cells. Furthermore we have cloned a 1500-base pairs-long cDNA fragment from rabbit intestine (RS2) which

FIG. 15. Distribution of -1-related mFtNh analyzed by PCR. RNA was isolated from total organs, different kidney regions, intestinal mucosal scrapings, tissue culture cells, or Xenopus oocytes and single- stranded cDNA was synthesized after oligddT) priming. Employing two primer pairs derived from RS1 (A, Sl*-S2-; B . S3'-S4-). RS1-related cDNAs were amplified. The amplification products were separated by agarose gel electrophoresis and analyzed by staining with ethidium bromide (pictures with dark background) and by hybridization with the radioactively labeled internal primers S5' ( A ) and S6' ( B ). In the control the reaction was performed without addition of RNA. Size standards are indicated.

has a high homology to RSl." Immunohistochemistry with a monoclonal antibody directed against RS1 (R4A6) showed that RS1 or homologous proteins are associated with the brush- border membrane in the S1, S2, and S3 segments of the renal proximal tubule and in small intestine (19,37). Preliminary in situ hybridization experiments revealed the same localization of RS1-homologous mRNAs in kidney and intestine as obtained by immunohistochemistry (data not shown).

SGLTl is a membrane polypeptide with 11 putative membrane-spanning a-helices and contains binding sites for glucose and Na+. Expression experiments in different systems (1, 18, 57,581 suggest that SGLTl is sufficient to form a cotransporter for Na' and D-glucose. However, although cotransport expressed by SGLTl always exhibits a Michaelis-Menten type glucose dependence (17, 18). a more complex glucose dependence of Na+ gradientdependent D-glucose transport with two apparent D-glucose transport sites has been often observed in kidney and intestine (11, 16, 17). Thus. low- and high-affinity transport was demonstrated in guinea pig small intestine, which was influenced differently by diet or temperature (11, 59). Similarily in outer cortex and outer medulla of porcine kidney apparent high- and low-affinity transport was measured, which was differentially influenced by temperature and membrane potential (16). Phlorizin binding measurements suggested that low- and high-affinity transport in pig kidney

unpublished data. ' J. Reinhardt, S. Gambaryan, M. Veyhl. C. Dekel. and H. Kwpsell,

25052 Protein Which Modifies Na'-D-glucose Cotransport occurred at high- and low-affhity glucose binding sites on the extracellular side of the Na+-D-glucose cotransporter (16).

The following arguments suggest that the Na+-D-glucose cotransporter contains a regulatory subunit which is identical or homologous to RS1: 1) Na+-D-glucose cotransport exhibiting apparent low- and high-affinity glucose transport sites and low- and high-affinity phlorizin inhibition sites of glucose transport could be expressed by stoichiometric amounts of SGLT1-cRNA and RS1-cRNA, but not by SGLTl alone. Furthermore, a different voltage dependence of Na+-D-glucose cotransport was observed when the transport was expressed by SGLTl plus RS1 and by SGLTl alone. 2) RS1 may increase the membrane insertion of SGLTl protein. This is suggested by the finding that the V,, of Na+-glucose cotransport activity in Xenopus oocytes, which was expressed by injection of small amounts of SGLT1-cRNA, was increased when stoichiometric amounts of RS1-cRNA were coinjected. 3) The interaction of RS1 with SGLTl appears to be specific for SGLT1-homologous proteins. This is suggested by the finding that RS1 did not alter the expression of the Na+-independent glucose transporter (GLUT11 nor the expression of the Na+-GABA cotransporter (GATl), but altered the expression of Na+-D-glucose cotransport and Na+-myo-inositol cotransport by SGLTl (1) and SMIT (31, respectively. The missing interaction of RS1 with transporters without homology to SGLTl is also supported by the observations that injection of RS1-cRNA altered the endogeneously expressed Na+-dependent glucose uptake in the Xenopus 00-

cytes, but does not alter the endogeneously expressed Na+- dependent transport of L-glutamate or the endogeneously expressed (Na++K+)-pump activity. 4) Since the effects of RS1 on the expression and kinetics of Na+-D-glucose cotransport expressed by SGLTl (and on expression of Na+-myo-inositol cotransport by SMIT) were only observed with a fixed molar ratio of injected SGLT1-cRNA (SMIT-cRNA) and RS1-cRNA, it has been demonstrated that stoichiometric amounts of SGLTl (SMIT) and RS1 polypeptides interact. The numbers of interacting polypeptides cannot be determined from these experiments, since the efficiency of translation and the stability of the cRNAs may be different. 5) The cDNA encoding for RS1 has been identified by the monoclonal antibody R4A6 which stimulates Na+-dependent high-affinity phlorizin binding to the Na+- D-glucose cotransporter in kidney and intestine (19, 201, and it was verified that R4A6 binds specifically to expressed RSl protein. 6) The existence of high molecular weight complexes of SGLT1- and RS1-homologous proteins in porcine renal brush- border membranes was demonstrated in nondenaturing polyacrylamide gels employing antibodies directed against SGLTl or RS1, and a functional molecular weight around 300,000 was determined for Na+-D-glucose cotransport in kidney and intestine (60-62). 7) RS1-homologous mRNAs were detected in renal outer cortex, renal outer medulla, small intestine, and LLCPKl cells, where Na+-D-glucose cotransport and SGLT1-homologous mRNAs have been demonstrated, but were not found in skeletal muscle and heart muscle where neither Na+-D-glucose cotransport activities nor SGLT1-homologous mRNA have been detected. The existence of a RS1-related mRNA in liver and spleen where Na+-D-glucose cotransport has not been described but SGLT1-homologous mRNAs found (2,4) suggests that RS1- homologous proteins interact also with other SGLT1-type Na+- cotransporters. The coexpression experiments of RS1 with the Na+-myo-inositol show that such an interaction is possible. However, since MDCK cells and the inner renal medulla contain SMIT-type Na+-myo-inositol cotransporters but no RS1- homologous proteins, the Na+-myo-inositol cotransporter may not contain an SGLT1-type regulatory subunit. The observed interaction of RS1 with SMIT may be explained by the structure of SMIT. For the same reason the P-subunit of the

(H++K+)-ATPase is supposed to interact with the a-subunit of the (Na++K+)-ATPase which is homologous to the a-subunit of the (H++K+)-ATPase (63). 8) Recently experiments were started in cooperation with Dr. Shirazi-Beechey where RSl-homologous mRNA was analyzed in small intestine of sheep which exhibit drastic down-regulation of Na+-D-glucose cotransport and SGLTl protein after weaning and drastic up-regulation of transport and SGLTl protein by intestinal infusion of adult sheep with D-glucose, but only small differences in SGLT1- homologous mRNA (64, 65). Preliminary experiments support the regulatory role of an m1-type protein in Na+-D-glucose cotransport, since in newborn sheep and in D-glucose-infused adult much more RS1-homologous mRNA was found than in noninfused adult sheep.

Radiation inactivation experiments suggest that the Na+-D- glucose cotransporter is an oligomeric protein with an apparent molecular weight betweeen 288,000 and 345,000 (50, 60-62); however, the subunit composition of transporter has not been established. Our present results and recent phlorizin binding measurements (16) suggest that the functional Na+-D-glucose cotransporter contains two to four SGLT1-type catalytic subunits and one or two regulatory RS1-type subunits. Thus at least two catalytic subunits have to be assumed, since two coexisting glucose binding sites with a stoichiometry of one were detected by phlorizin binding measurements (161, and since more than one glucose binding site was required to ex- plain phlorizin-inhibitable labeling of the Na+-D-glucose cotransporter by a covalently binding cyclosporin analog (34). In addition the sigmoidal dependence between the expressed AMG transport and the amount of injected SGLT1-cRNA (Fig. 5a) suggests that more than one catalytic subunit is required to express Na+-D-glucose cotransport. The above described finding that injection of stoichiometric amounts of RS1-cRNA and SGLT1-cRNA lead to effects on expressed Na+-D-glucose cotransport, whereas no effects were observed when excess of RS1-cRNA was coinjected, suggests that two RS1-type regulatory subunits may be associated with SGLT1-type catalytic subunits of the transporter. Apparently a significant fraction of the transporter from pig kidney outer cortex and from rat intestine contains two regulatory subunits, since the monoclonal antibody against RS1 (R4A6) appears to have two epitopes on the Na+-D-glucose cotransport system. This is suggested by the observation that high-affinity phlorizin binding to brush-border membranes was only increased by a distinct concentration of R4A6, whereas no increase of phlorizin binding was observed when the concentrations of R4A6 were higher (19, 20).

The physiological function of the proposed RS1-type regulatory subunit and its mechanistic interaction with SGLT1-type catalytic subunit of the Na+-D-glucose cotransporter deserves future investigations, since this may help us to understand function and regulation of Na+-D-glucose cotransport. The proposed regulatory subunit may play a role in the short term regulation of Na+-D-glucose cotransport which may be mediated by D-glucose, unidentified compounds, or by the membrane potential (16, 66-68). Since antibody R4A6, which binds to RS1 protein, stimulates phlorizin binding, it may be speculated that RS1 is an inhibitory modulator which may be inactivated by R4A6. The proposed RS1-homologous regulatory subunit may also mediate long tern effects of diet or hormones on Na+-D- glucose cotransport activity and/or on the concentration of SGLT1-type protein in the membrane (64,69-73). Finally various transporter properties in different tissues or tissue regions (12,16,74,75) may be due to different combinations of SGLT1- and RS1-type proteins.

for expert technical assistance. The supply of the cDNA clones SGLT1, Acknowledgments-We thank Prof. W. Schwarz for help and D. Ollig

Protein Which Modifies N GAT1, GLUTI, SMIT, and SNF3 by Prof. E. M. Wright, Prof. B. Kanner, Prof. M. Mueckler, Prof. J. S. Handler, and Prof. M. Carlson is gratefully acknowledged.

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