Departments of 1 Internal Medicine and 2 Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0354
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ABSTRACT |
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myo-Inositol is a ubiquitous intracellular organic osmolyte and phosphoinositide precursor maintained at millimolar intracellular concentrations through the action of membrane-associated Na+-myo-inositol cotransporters (SMIT). Functional cloning and expression of a canine SMIT cDNA, which conferred SMIT activity in Xenopus oocytes, predicted a 718-amino acid peptide homologous to the Na+-glucose cotransporter with a potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites. A consistent ~1.0- to 13.5-kb array of transcripts hybridizing with this cDNA are osmotically induced in a variety of mammalian cells and species, yet SMIT activity appears to vary among different tissues and species. An open reading frame on human chromosome 21 (SLC5A3) homologous to that of the canine cDNA (96.5%) is thought to comprise an intronless human SMIT gene. Recently, this laboratory ascribed multiply sized, osmotically induced SMIT transcripts in human retinal pigment epithelial cells to the alternate utilization of several 3'-untranslated SMIT exons. This article describes an alternate splice donor site within the coding region that extends the open reading frame into the otherwise untranslated 3' exons, potentially generating novel SMIT isoforms. In these isoforms, the last putative transmembrane domain is replaced with intracellular carboxy termini containing a novel potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites, and this could explain the heterogeneity in the regulation and structure of the SMIT.
myo-inositol; retinal pigment epithelial cell; Xenopus oocyte expression system; osmoregulated genes; sodium-glucose cotransporter superfamily; SCL5A3 open reading frame
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INTRODUCTION |
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MYO-INOSITOL (MI) is a ubiquitous constituent of living cells and an essential nutrient for most mammalian cells in culture (47, 48). An obligate substrate for phosphoinositide synthesis (11, 32), MI also functions interchangeably with sorbitol, taurine, and betaine as a nonionic intracellular osmolyte in the regulation of cell volume and ionic composition (31, 32). Intracellular concentrations 50- to 1,000-fold greater than that of extracellular fluid (47, 48) are, with a few notable exceptions (31, 42, 60), attributed to membrane-associated Na+-MI cotransporter (SMIT) activity (3, 25). Functional cloning from a Madin-Darby canine kidney (MDCK) cell cDNA library and expression in Xenopus oocytes ascribed this transport activity to a 718-residue polypeptide (25) homologous with Na+-glucose cotransporter 1 (SGLT1) (56) containing potential protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites (16, 25). This cDNA hybridizes to a consistent array of osmotically induced ~1.0- to 13.5-kb transcripts in a variety of mammalian cell types (1, 25, 38, 68). A human genomic library screened with this canine SMIT cDNA revealed a full-length open reading frame (ORF) in the Down's syndrome region of chromosome 21 that potentially encodes human SMIT (SLC5A3) (1). Multiply sized SMIT transcripts were thought to arise either from this intronless sequence through alternate transcript termination and polyadenylation sites or from multiple genes (1).
The various kinetic and biochemical characteristics of SMIT activity among mammalian cell types (2, 4, 5, 8, 10, 35, 38, 42, 46, 61, 63, 65) and tissues (6, 9, 14, 17, 28, 50-53, 59) could imply a more complex gene structure, although small interspecies differences in SGLT1 amino acid sequence are associated with considerable variation in transport kinetics (19). Recently, this laboratory has reexamined the basis for the multiple transcripts ascribed to the human SMIT gene (39, 40) in cultured human retinal pigment epithelial (RPE) cells, an in vitro model for diabetic complications (10). Four overlapping SMIT cDNAs [clones 1-2, 2-1, 3-1, and 4-1 (39, 40)] were cloned from a size-selected, tonicity-induced human RPE cDNA cell library and sequenced. The 5'-untranslated region (UTR) and a 2,154-nt ORF were highly conserved compared with those of the canine sequence. The ORF was virtually identical to that assigned to human chromosome 21 (1). Comparison of the 910-nt 3'-UTR with the canine sequence revealed 100% identity at the stop codon and 15 nt downstream but [except for the poly(A) tails] no significant homology with the remaining 193 nt of the canine 3'-UTR (39, 40). The sequence from the divergent 3'-UTR provided further information with which to isolate several additional human genomic DNA clones [all localized to the same yeast artificial chromosome (YAC 860G11)], which contained the putative SMIT exon 1, exon 2 containing the ORF, and untranslated exons 3-5 (Fig. 1) (39, 40). A probe derived from the exon 1 sequence hybridized to the full ~1.2- to 16-kb array of human SMIT RNA transcripts, and primer extension with several different primers suggested a single transcription initiation site for the SMIT gene, implying a single promoter (39, 40).
Exon-specific probes ascribed the multiple SMIT transcripts to alternate utilization of the five SMIT exons. The smallest, the 1.2-kb SMIT transcript, was devoid of sequence from exon 2, but contained sequence from exons 1, 4, and 5 (39, 40). The larger transcripts contained sequence from exons 1 and 2 and differing abundances of sequence from downstream exons. Osmotic induction of RPE cells produced an asynchronous time-dependent rise and subsequent fall in SMIT transcripts, with the largest (16 kb) transcript rising and declining in abundance more rapidly than the smaller transcripts (39, 40). Thus the human SMIT gene appeared to be a single transcriptional unit considerably more complex than previously envisioned (1). The low relative abundance of exon 3 sequence in the 3.8-kb transcripts containing exons 1, 2, 4, and 5 suggested possible sequence variability in the region of the exon 2-3 boundary.
This article reports the sequencing of an additional SMIT cDNA clone (Fig. 1; clone 1-3) with an 85-nt deletion beginning within the ORF; this deletion would eliminate the stop codon and the putative COOH-terminal transmembrane domain of the predicted peptide (25, 56). A combination of cloning, RT-PCR, and oligonucleotide hybridization studies ascribes this deletion to an alternate splice donor site within the ORF that gives rise to alternate SMIT isoforms whose COOH termini are encoded by otherwise untranslated downstream exons. In the predicted isoforms, the last transmembrane domain is replaced with large intracellular COOH termini (56) containing novel potential PKA and PKC phosphorylation sites, potentially explaining the variability in SMIT activity and the propensity for tissue MI depletion in diabetes.
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MATERIALS AND METHODS |
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Cell culture. Primary cultures of human RPE cells were established and passaged (10) in isotonic MEM (IMEM) with 20% calf serum (295 mosmol/kgH2O) at 37°C in a humidified 5% CO2 atmosphere. Confluent cells were exposed to MEM with 20% calf serum and made hypertonic by treatment with 300 mM mannitol (595 mosmol/kgH2O) for 24 h, after which RNA was purified (18). SH-SY5Y cloned human neuroblastoma cells and human prostate BPH1 cells were cultured in DMEM with 10% calf serum. The medium for human aortic endothelial cells (HAEC) was endothelial basal medium purchased with 12 µg/ml bovine brain extract, 10 ng/ml human epidermal growth factor, 1 µg/ml hydrocortisone, 2% fetal bovine serum, 0.05 mg/ml gentamicin, and 0.05 µg/ml amphotericin B (Clonetics Corporation, San Diego, CA).
Construction and screening of the cDNA library. An oligo(dT)-primed, size-selected (>1 kb) RPE cDNA library, prepared as previously described (18) with pCDNA1 (Invitrogen, San Diego, CA), was screened at reduced stringency with an EcoR I-BamH I fragment of the canine SMIT cDNA (generously provided by H. M. Kwon, Johns Hopkins Univ.; GenBank accession no. M85068; nt 724-2870) containing most of the ORF and 236 nt of the 3'-UTR (25), labeled as described in Probes. Three unique clones were isolated (39, 40).
Identification of alternate SMIT transcripts by RT-PCR. RT-PCR was performed on total cellular RNA extracted from RPE cell lines 45 and 47, SH-SY5Y cells, HAEC, and BPH1 cells, all of which were grown in IMEM or hypertonic MEM (HMEM) for 12-16 h, and from human kidney cortex and sural nerve tissue. Oligo(dT)-primed cDNA was synthesized from 5 µg of total RNA by using 200 U of Superscript II in 20 µl of the vendor's buffer containing RNase inhibitors (0.5 U/µl) (Life Technologies, Gaithersburg, MD). DNA was amplified with 1 µl of cDNA as the template in 50 µl of 60 mM Tris, pH 10, 15 mM (NH4)2SO4, 2 mM MgCl2, and 0.2 mM deoxynucleoside triphosphates (dNTPs) containing 0.2 µM (each) 5' and 3' primers and 1.25 U of AmpliTaq (Perkin-Elmer, Foster City, CA). After denaturation at 95°C for 3.5 min, the DNA was amplified by 30 cycles of denaturation at 94°C for 1.5 min, annealing at 56°C for 1.5 min, and extension at 72°C for 2 min. A final elongation reaction was conducted at 72°C for 8 min. Five microliters of the PCR products were resolved by electrophoresis on 5% polyacrylamide gels. The primers for RT-PCR were 5'-GTAAGAGCCTCAGCAAGAGG-3', corresponding to sequence 2502-2521, and 5'-CCAAGAAATACCCGCCTCTG-3', complementary to sequence 2904-2923.
DNA sequencing. The nucleotide sequences of both strands of cDNA clone 1-3 were determined by the dideoxy chain termination method (39) using synthetic oligonucleotide primers and Sequenase (US Biochemical, Cleveland, OH) after alkaline denaturation. The nucleotide sequence was analyzed with Genetics Computer Group software.
Northern blots.
Denatured total cellular RNA (10 µg) and a 0.24- to 9.5-kb RNA ladder
(Life Technologies) were resolved on 2.2 M formaldehyde-1% agarose
gels; transferred by capillary blotting to ZetaBind filters (Cuno,
Meridian, CT); stained with methylene blue to confirm the integrity,
uniformity of loading, and completeness of transfer; and fixed in a
vacuum at 80°C for 2 h. Filters were prehybridized at 65°C for
4-6 h before an exon 2-specific cDNA probe (2 × 106 dpm/ml) was added (40). After
18 h, the filters were washed at 65°C and exposed to preflashed
film at 70°C. For oligonucleotide hybridization, filters
were prehybridized at 43°C for 4-6 h before a denatured exon
2-specific cDNA probe (2 × 106 dpm/ml) was added. After 18 h,
the filters were washed at 48°C before exposure to preflashed
BioMax film at
70°C. Hybridization was quantitated on a
Molecular Dynamics phosphorimager.
Probes.
A SMIT exon 2 probe consisted of the 1.3-kb
Pst I fragment (nt 1064-2334)
contained within the ORF of the human SMIT cDNA, as previously
described (39, 40). Oligonucleotides (purified by HPLC) complementary
to putative SMIT exon 2 alternate splice junctions or exon-intron
boundaries were end-labeled with
[-32P]ATP (6,000 Ci/mmol; NEN Research Products, Boston, MA), purified from
unincorporated label on NENSORB cartridges (NEN Research Products), and
added at a final amount of 1 × 106 dpm/ml. These "Band-Aid"
oligonucleotide probes spanning a putative alternate splice junction
within the ORF in exon 2 were designed to be complementary both to
~10 nt extending upstream from the putative splice donor site
(2'; position 2590; see below) and to the ~10 nt at the
5' boundaries of either exon 3 (sequence 2A/3) or exon 4 (sequence 2A/4) (Table 1).
Negative controls consisted of probes complementary to only ~10 nt
upstream or downstream from the exon 2 splice donor site (2A/
or
/2B), which would not be expected to hybridize under the
conditions employed. A similar approach was used to investigate
possible alternate splicing in the region of the exon 2 3'
exon-intron boundary. A probe was designed to be complementary to the
~10-nt sequence upstream from the exon 2 3' boundary (position
2675; sequence 2C) and the ~10 nt of putative intronic sequence
downstream from the exon 2 3' boundary (2C/2D; Table 1). The
lengths of the ~20-nt probes were adjusted to give similar
hybridization characteristics. The sequences of the individual Band-Aid
oligonucleotides are given in Table 1.
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Synthetic oligonucleotide synthesis.
All synthetic oligonucleotides were prepared by the DNA Synthesis Core
Facility of the Michigan Diabetes Research and Training Center and the
Univ. of Michigan's Biomedical Research Core Facilities by using
automated DNA synthesizers (Applied Biosystems, Foster City, CA) with
-cyanoethyl phosphoramidite chemistry on a controlled pore glass support.
Functional expression in Xenopus
laevis oocytes.
The transport function of each of the SMIT isoforms was assessed by
[3H]MI uptake in
X.
laevis oocytes microinjected with SMIT
1, 2, or 3 cRNA prepared in vitro. In brief, cDNA expression constructs were prepared by "recombinant" PCR methods (40). The SMIT 1 ORF
was amplified from 100 ng of phage DNA containing exon 2 (1-36) with a "high-fidelity" mixture of
Taq and
Pwo DNA polymerases (Expand High
Fidelity; Boehringer Mannheim, Indianapolis, IN) and 30 pmol of
synthetic oligonucleotide primers that incorporated unique restriction
endonuclease cleavage sites to facilitate subcloning. The 5'
primer contained the translation initiation codon, but the Kozak
sequence (5'-CTTTGGGGATCCTTCCACCATGAGAGCTGTACTGGACACAGC-3'; Fig. 3; bases 501-528) (24) was modified to increase its
efficiency. The 3' primer
(5'-AAGGAATTAAGCGGCCGCAAGTTCATAAGGAGAAATAAACAAAC-3'; Fig. 3; bases 2641-2666) was complementary to the terminal portion of the ORF. The DNA was denatured at 95°C for 3 min before 10 cycles of amplification (95°C for 0.5 min, 60°C for 1.5 min,
and 72°C for 2 min) and a final elongation at 72°C for 7 min.
The X.
laevis
-globin gene
5'-UTR was amplified from pSP64T (generous gift from L. Isom, Ann
Arbor, MI) with a 5' primer that incorporated the T7 RNA
polymerase promoter
(5'-TTTAAGCTTAATACGACTCACTATAGCACTTGTTCTTTTTGCAGAAGC-3') and a 3' primer complementary to both the 3' end of the
-globin 5'-UTR and the 5' portion of the 5' SMIT 1 primer
(5'-CATGGTGGAAGGATCCCCAAAGTTGAGCGTTTATTCTGAG-3'). After
purification from the primers, the amplification products were combined
in equimolar amounts and amplified with the 5'
-globin primer
and 3' SMIT 1 primer as described above. The novel ORFs contained
in the SMIT 2 and 3 isoforms were amplified from clone 1-3, and
canine SMIT was amplified from the cDNA kindly provided by Dr. H. M. Kwon. Full-length expression constructs were prepared by recombinant
PCR as described above. Capped RNA was prepared by using T7 RNA
polymerase according to the vendor's protocols (Ambion, Austin, TX).
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RESULTS |
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Evidence for alternate splicing within the SMIT ORF.
Of the various cDNA clones derived from a human RPE cell
library (39, 40) containing sequence for a SMIT ORF (14, 37), clone
1-3 differed from the other clones by a reduction in the size of a
Pst I fragment (Fig.
1). Sequencing clone 1-3 revealed the
deletion of an 85-nt segment (from nt 2590 to 2675) corresponding to a
putative COOH-terminal transmembrane domain (see below) (39, 40, 56).
The resulting transcript was predicted to encode a potentially novel
SMIT polypeptide sequence with a distinct COOH-terminal structure (25,
56). The presence of these distinct transcripts in RPE RNA was
established by RT-PCR with oligonucleotide primers flanking the
deletion. Two bands migrating with apparent sizes of 240 and 337 nt
were amplified from cDNA prepared from RPE 47 cells in IMEM or HMEM
(Fig. 2,
lanes
3 and
4). The larger product comigrated
with the single band obtained from clone 1-3 (Fig. 2,
lane
7), but no product corresponding to
the size obtained with clone 1-2 was evident by ethidium bromide
staining (Fig. 2, lane
6). When the 337-nt RT-PCR product
was subcloned and the resulting subclones were individually sequenced,
all of the 337-nt products were found to be identical in sequence to
that derived from clone 1-3. The sequence of the more abundant
240-nt band corresponded to a deletion of the 182-nt sequence between
positions 2590 and 2772; this led to the prediction of yet a third
COOH-terminal polypeptide sequence (25, 56). The sequences for the 240- and 337-nt RT-PCR products are compared with those of the human SMIT
cDNA in Fig. 3. A larger
RT-PCR product >1,353 nt in apparent size was also evident in
hypertonically stressed RPE 47 cell RNA (Fig. 2,
lane
4), potentially corresponding to
additional alternate splicing in the distal portion of exon 2 (see
below).
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Inferred splice donor and acceptor sites for alternate SMIT isoforms. The three potential isoforms SMIT 1, SMIT 2, and SMIT 3 most likely represent alternate splice products because both of the internal deletions begin at position 2590 (Fig. 3; 2'), a putative alternate 3' exon boundary within the ORF, as depicted in Fig. 4A. The SMIT 1 isoform corresponding to clone 1-2 arises from a splice donor site at the 3' boundary of exon 2 (position 2675) downstream from the stop codon (position 2661) and a splice acceptor site at the 5' boundary of exon 3 (39, 40). The SMIT 2 isoform corresponding to clone 1-3 utilizes an alternate splice donor site (Fig. 3; 2') upstream from the exon 2 stop codon and the same splice acceptor site on the 5' boundary of exon 3, with a new stop codon (position 2856) in exon 4. The SMIT 3 isoform utilizes the same 2' alternate splice donor site, with a splice acceptor site at the 5' boundary of exon 4 and a new stop codon (position 3103) in exon 5.
Confirmation of splice donor and acceptor sites and relative
quantitation of alternate SMIT isoforms by Band-Aid oligonucleotide
hybridization.
Transcripts for putative alternately spliced SMIT 2 and SMIT 3 isoforms
were localized to variously sized RPE
poly(A)+ RNA with end-labeled
synthetic oligonucleotides complementary to the ~10 nt immediately
upstream, and the ~10 nt immediately downstream, from the putative
alternate splice sites as described in MATERIALS AND
METHODS (Fig. 4).
Hybridization conditions were empirically determined to permit the
annealing of ~20-nt probes (20-mers) but not ~10-nt
probes (10-mers). These 20-mer Band-Aid oligonucleotides were
hybridized with poly(A)+ RNA
isolated from RPE cells cultured under hypertonic conditions and used
to assign the alternate spliced isoforms to individual SMIT transcripts
and to determine their relative abundances (Fig. 4). A positive control
consisted of the SMIT exon 2 probe (Fig. 4C,
lane
1) (39, 40). Negative controls
comprised oligonucleotides complementary to the individual ~10-nt
sequences immediately upstream or immediately downstream from the
putative alternate splice sites. The two separate 10-mers complementary
to the sequence immediately upstream (2A/) or downstream
(
/2B) from the putative alternate splice site in exon 2 exhibited no hybridization under these conditions, as expected (Fig.
4C,
lanes
2 and
5). In contrast, the 20-mer spanning
both the same alternate splice donor site in exon 2 and the splice
acceptor site in exon 4 (2A/4) exhibited strong preferential hybridization to the 3.8-kb transcript (Fig.
4C,
lane
4), suggesting that the population
of 3.8-kb SMIT transcripts is enriched in SMIT 3 transcripts.
Hybridization with the 20-mer complementary to the sequence spanning
the alternate splice donor site and the splice acceptor site at the
5' boundary of exon 3 (2A/3) was undetectable (Fig.
4C,
lane
3), despite the fact that this
transcript was identified by RT-PCR amplification of RPE RNA and gave
rise to clone 1-3 (Fig. 1). This suggests that
transcripts encoding the SMIT 2 variant are present but at relatively
low abundances in hypertonically stressed RPE 47 cells, as suggested in
Fig. 2, and would explain the reported poor hybridization of an exon 3 probe with the 3.8-kb transcript (39, 40).
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Identification of alternate splicing at the distal boundary of exon 2 by Band-Aid oligonucleotide hybridization. The recently reported human SMIT exon structure (39, 40) does not readily explain the larger-molecular-weight SMIT transcripts, which have been attributed to alternate chain termination and/or polyadenylation sites (1). The >1,353-nt RT-PCR product in Fig. 2 suggested the presence of additional sequence between the primers in distal exon 2 and distal exon 4 (Fig. 3). The inclusion of sequence distal to the putative 3' boundary of exon 2 (39, 40) was explored with a 20-mer Band-Aid oligonucleotide spanning the ~10-nt sequence upstream from the putative exon 2 3' boundary and the adjacent ~10-nt "intronic" sequence immediately downstream (2C/2D) (Fig. 4B). This oligonucleotide hybridized with RPE poly(A)+ RNA only weakly to the 3.8-kb band but much more strongly to the higher-molecular-weight SMIT bands (Fig. 4C, lane 6), suggesting that a "distal exon 2" splice donor site [i.e., distal to the previously reported 3' boundary of exon 2 (39, 40)] may be selectively enriched in the larger transcripts. The downstream splice acceptor site(s) remains unidentified. The size of the >1,353-nt RT-PCR product (Fig. 2, lane 4) could account for transcripts slightly larger than 3.8 kb (Fig. 4C, lane 1) but not for the much larger transcripts, because the presence of additional sequence would likely diminish PCR efficiency. Because the alternate splice donor site at the exon 2 3' boundary is distal to the exon 2 stop codon, the resulting family of transcripts would encode the SMIT 1 isoform but would differ in 3'-UTR (Fig. 4B).
Identification of transcripts for alternate SMIT isoforms in a
variety of cultured human cell lines and tissues by RT-PCR.
To determine whether the alternately spliced SMIT isoforms were unique
to RPE 47 cells, total RNA from a variety of other cultured
human cell lines was reverse transcribed and amplified with the same
primers illustrated in Fig. 3. As shown in Fig. 5A, RT-PCR
products virtually identical in apparent size to those induced in RPE
47 cells by HMEM were observed in a second human RPE cell line (RPE
45), in a cloned human neuroblastoma cell line (SH-SY5Y), in a human
prostate cell line (BPH1), and in HAEC. As in RPE 47 cells, the
abundance of these alternately spliced SMIT transcripts was
increased by exposure to hypertonic conditions in RPE 45 cells, SH-SY5Y neuroblastoma cells, HAEC, and BPH1 cells. An
amplification product corresponding in size to the SMIT 1 isoform was
clearly evident only in hypertonically stressed cells. Thus osmotically
induced transcripts for alternately spliced SMIT isoforms are widely
distributed among human cell lines. Moreover, hypertonic stress was
associated with a tendency for greater relative induction of SMIT 2 than of SMIT 3 isoforms, except in SH-SY5Y neuroblastoma cells. As in
Fig. 2, large RT-PCR products were osmotically induced in multiple cell
types, although the sizes and relative abundances of these products
appeared to differ from cell type to cell type. Figure
5B illustrates similar RT-PCR products
in total RNA isolated from a human sural nerve specimen, which
expresses primarily SMIT 2, and from a human renal cortex, which
expresses primarily SMIT 3.
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Functional expression of SMIT isoforms in
X. laevis
oocytes and regulation by PKA.
Initial studies established that
Na+-dependent uptake of 10 µM
2-[3H]MI by oocytes
injected with a construct containing T7 and the 5'-UTR of the
-globin gene was indistinguishable from that in uninjected Xenopus oocytes.
Preliminary experiments explored the effect of the cRNA injection dose
(10-60 ng), incubation time (2-6 days), and SMIT isoform on
Na+-MI cotransport (Fig.
6).
Na+-MI cotransport activity after
a 3-day incubation increased with the aliquot of injected SMIT 1 cRNA
(Fig. 6A) and with the duration of
incubation after a 20-ng SMIT 1 cRNA injection (Fig.
6B). A comparison of
Na+-MI cotransport activity in
oocytes 3 days after 10- to 20-ng injections of cRNA containing
sequence for SMIT 1, SMIT 2, or SMIT 3 with that in oocytes injected
with a construct containing T7 and the
-globin gene 5'-UTR
devoid of SMIT sequence consistently revealed greater activity in the
former oocytes, with SMIT 1 > SMIT 3 > SMIT 2 (for each,
P < 0.01 vs. T7-
-globin gene
5'-UTR injection; Fig. 6C, with
results replicated in 2 additional experiments). The
basis for the reported divergent effects of PKA activation on
Na+-MI cotransport in canine MDCK
cells (41) and human RPE cells (21) was explored with
Xenopus oocytes selected for a high
level of SMIT functional expression. Figure
7 illustrates a representative experiment in which Na+-MI
cotransport activity was measured in SMIT 1-, SMIT 2-, and canine
SMIT-expressing oocytes in the presence and absence of IBMX-forskolin.
Transport activity was significantly increased by IMBX-forskolin in
SMIT 1-expressing oocytes but decreased significantly in SMIT
2-expressing and canine SMIT-expressing oocytes. This result was
confirmed in two additional experiments. The pooled data from all three
experiments, expressed as a percentage of the relevant control value
for each experiment, demonstrated that IBMX-forskolin increased SMIT 1 Na+-MI cotransport activity to 160 ± 9% of control (n = 18) but
decreased SMIT 2 and canine SMIT transport activities to 48 ± 7%
(n = 16) and 58 ± 5%
(n = 16) of those for their respective
controls; in all cases, P was <0.01.
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DISCUSSION |
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Despite the clear physiological (3, 26, 62, 64) and long-postulated pathophysiological (3, 49, 55) significance of Na+-MI cotransport, its molecular basis and regulation are only superficially understood. The functional cloning of a canine SMIT cDNA in Xenopus oocytes (25) led to the prediction of a 718-amino acid SMIT polypeptide with potential PKA and PKC phosphorylation sites and an array of putative hydrophobic "membrane-spanning" domains, resembling those of the SGLT family (56). When expressed in Xenopus oocytes, the canine SMIT cDNA endowed Na+-MI cotransport activity with substrate specificity and kinetic characteristics similar to those in MDCK cells (16). The full-length cDNA hybridizes with a consistent array of hypertonically induced ~1.0- to >13-kb transcripts in kidney (25), lens (68), and neural cells (38) from a variety of mammalian species. The origin of these multiple transcripts is just now being clarified. A single, full-length ORF (SLC5A3) 97% homologous with the canine sequence at the amino acid level has been cloned from a human genomic library by using the canine cDNA as a probe and is thought to encode a human SMIT (1). It was originally speculated that this intronless sequence gave rise to multiple SMIT transcripts through alternate transcript termination and polyadenylation sites (1). To some extent, the tissue-, cell-, and organ-specific variability of MI metabolism and transport contrasts with a widely conserved pattern of SMIT expression and nucleotide sequence and its reportedly uncomplicated intronless gene structure (1). For example, the kidney synthesizes, concentrates, excretes, and reabsorbs MI and uniquely expresses the specific mammalian cytoplasmic oxygenase for MI degradation (20) as well as SMIT. Brisk Na+-MI cotransport activity in the small intestine (6), choroid plexus (53), ocular ciliary body (45), and RPE (10) accounts for rapid intestinal absorption and the higher MI levels in cerebrospinal fluid (53) and vitreous humor (45). Human RPE cells (10) and bovine lens epithelial cells (5) exhibit more than one kinetic class of Na+-MI cotransport activity, presumably reflecting either translationally distinct SMITs or posttranslational modification. Moreover, mammalian Na+-MI cotransport activity exhibits somewhat different apparent affinities for MI (6, 14, 17, 65), differential sensitivity to sulfhydryl agents (14, 51), different kinetic interactions with glucose (4-6, 10, 12, 14, 17, 22, 23, 61, 66, 67) and other carbohydrates (66, 67), and differential acute and chronic responses to protein kinase activation and Ca2+ (13, 15, 21, 22, 41, 43, 44) in a tissue- and/or species-specific pattern. For example, PKA activators have been reported to acutely decrease Na+-MI cotransport activity in MDCK cells (41) and to acutely increase Na+-MI cotransport activity in human RPE cells (21) despite the conservation of the putative PKA phosphorylation site across species (1, 25). Various iterations of the diabetic state appear to exaggerate the variability in the regulation of Na+-MI cotransport activity (29, 35, 54), increasing transport activity in freshly isolated renal glomerular cells (61), cultured mesangial cells (7), and cultured porcine aortic endothelial cells but decreasing transport activity in incubated endoneurial preparations from streptozotocin-diabetic rats (12), cultured mouse neuroblastoma cells (66), incubated rat lens (30), cultured bovine lens epithelial cells (4, 5), cultured bovine retinal capillary pericytes (27), cultured human skin fibroblasts (34), and cultured aortic smooth muscle cells (33). The relationship between glucose and Na+-MI cotransport may be complex and multifactorial even within a single cell type: in cultured bovine lens epithelial cells, glucose appears to simultaneously decrease high-affinity Na+-MI cotransport directly and reduce low-affinity Na+-MI cotransport indirectly via the aldose reductase pathway (5).
Recent studies from this laboratory suggest that the human SMIT gene is
considerably more complex than previously reported (1). It appears to
comprise an intricate multiexon transcriptional unit giving rise to
multiple SMIT transcripts, primarily through a complex process of
alternate utilization of a coding exon and several noncoding exons (39,
40) rather than through alternate transcript termination and
polyadenylation sites (1). The studies reported in this article propose
an even higher order of complexity, consisting of an alternate splice
donor site within the ORF upstream from the most COOH-terminal
membrane-spanning domain (56). Interactions with splice acceptor sites
at the 5' boundaries of exons 3 and 4 extend the ORF downstream
to novel stop codons within previously untranslated exons 4 and 5, respectively. These alternately spliced transcripts predict novel
peptides, SMIT 2 and SMIT 3, in which the most distal membrane-spanning
domain is replaced with unique cytoplasmic (56) COOH-terminal domains
containing a novel potential PKA phosphorylation site and several novel
potential PKC phosphorylation sites (Fig.
8). Another alternate splice donor site
distal to the stop codon in the exon 2 ORF (position 2660 in Fig. 3)
may result in an extended 3'-UTR, contributing to a family of
larger-molecular-weight transcripts presumably encoding SMIT 1.
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Alternately spliced transcripts predicting novel SMIT 2 and SMIT 3 isoforms are identifiable in human RPE RNA through a combination of cloning, RT-PCR, and filter hybridization. The SMIT 3 transcript appears to be selectively enriched and appears to be the most abundant form in the 3.8-kb band. SMIT 2, the initial alternately spliced transcript originally identified through cloning, appears to be present in much lower abundance. These alternately spliced transcripts cannot be dismissed as cloning artifacts or the effects of mispriming or the amplification of vanishingly rare transcripts. SMIT 2, originally identified by cloning, was present in repeated isolates, and its presence was subsequently confirmed by the consistent appearance of an RT-PCR product of the expected size from total RNA from multiple human cell types and tissues. SMIT 3, originally identified through RT-PCR amplification from RPE RNA, was amplified from RNA from multiple human cells and tissues. The presence of alternately spliced SMIT transcripts in RPE RNA was also inferred from the relative paucity of exon 3 sequence vs. that of exons 1, 2, 4, and 5 in the 3.8-kb band (40). Band-Aid oligonucleotide hybridization studies confirmed SMIT 3 as the most abundant transcript in the 3.8-kb band in RNA from hypertonically stressed RPE cells. The relative abundance of SMIT 2 and SMIT 3 RT-PCR products was shifted in a reproducible cell-specific pattern by exposure to hypertonic stress. These products are not derived from processed pseudogenes, because RT-PCR of genomic human DNA yielded no similarly sized products (not shown). Thus the presence of alternate SMIT isoforms, originally detected by cloning, has been confirmed by a combination of RT-PCR and hybridization with multiple cDNA and oligonucleotide probes in a variety of human cells and tissues, and these isoforms have been shown to respond appropriately to the physiological stress of hypertonicity in multiple cell types.
Recent elegant structure-function studies of the homologous SGLT1 in the Xenopus oocyte expression system (19, 36, 56, 58) provide new insight into the potential functional significance of these newly predicted SMIT isoforms. On the basis of the analogy with the then-current SGLT1 model (57), canine SMIT was originally described as having an intracytoplasmic NH2 terminus, 12 membrane-spanning domains, the last 2 of which embrace a long extracytoplasmic loop, and a short intracytoplasmic COOH terminus (25). This model would predict that substitution of the last membrane-spanning domain in human SMIT 2 and SMIT 3 isoforms with sequence derived from exons 3, 4, and 5 would orient these novel COOH-terminal domains extracellularly, replacing the large extracellular loop (25). This orientation would sequester the resulting novel potential PKA and PKC phosphorylation sites away from intracellular kinases, rendering these sites functionally irrelevant from the standpoint of regulatory phosphorylation. Recent refinement of the topological model for SGLT1 by meticulous N-glycosylation mutagenesis studies and extensive computer modeling (56) reorients the NH2 terminus extracellularly and reclassifies two relatively hydrophobic Kyte-Doolittle domains distal to the original third and ninth transmembrane domains as membrane-spanning domains, raising the number of transmembrane domains from 12 to 14 (56). Extrapolation of this new SGLT1 model to SMIT, supported by computer modeling (10), would reorient the COOH-terminal domains of the predicted SMIT 2 and SMIT 3 isoforms to the cytoplasmic compartment (Fig. 8), rendering their putative novel PKA and PKC phosphorylation sites accessible to intracellular kinases.
Deletion of the putative 14th membrane-spanning domain in SMIT 2 and SMIT 3 is entirely consistent with the retention of Na+-MI cotransport because bacterial homologs of SGLT1 retain Na+-glucose cotransport activity but lack this transmembrane domain (56). Indeed, recent studies with epitope-tagged (58) and chimeric SGLTs (37) suggest that the COOH terminus, including the intracytoplasmic loop between the 13th and 14th membrane-spanning domains, may influence the kinetic characteristics of sugar transport and the intracellular trafficking of transporter molecules. Xenopus expression studies of recombinant SGLT1 constructs localize the sugar permeation pathway of human SGLT1 to the 10th-13th membrane-spanning domains (36). Thus it is reasonable to speculate that the predicted alternate SMIT isoforms devoid of the 14th membrane-spanning domain would nevertheless retain Na+-MI cotransport activity but with potentially different kinetic and regulatory properties and/or with different subcellular localization or intracellular trafficking patterns. Expression studies of Xenopus oocytes with SMIT isoforms suggest that they retain the ability to accumulate 2-[3H]MI in a Na+-dependent fashion but that they respond differently to posttranslational regulation by PKA: SMIT 1 exhibits an increase in apparent transport activity mirroring the response in intact human RPE cells (21), whereas SMIT 2 exhibits a decrease in transport activity, similar to that observed with canine SMIT in intact MDCK cells (41) or in the Xenopus expression system (Fig. 7). Finally, a striking homology between the intracytoplasmic tail of SMIT 3 and the predicted 14-kDa peptide encoded by the smallest 1.2-kb SMIT transcript (40) deserves comment. This recently described 1.2-kb SMIT transcript (actually ~0.95 kb when cloned and sequenced) (40) lacks exon 2 sequence comprising the SMIT ORF but instead contains a novel ORF with a transcription initiation site in exon 1, 44 nt upstream from the 5' boundary of exon 4. Because the remainder of this 14-kDa 125-amino acid peptide (40) is encoded by exon 4 and exon 5 sequences in-frame with respect to SMIT 3, it would share primary structure with the SMIT 3 cytoplasmic COOH-terminal domain for most of its length (Fig. 8). The functional implications of this predicted structural homology remain to be explored.
In summary, these studies have identified a family of predicted human SMIT isoforms arising from an alternate splice donor site within the ORF whose members differ in their COOH-terminal sequences, potential PKA and PKC phosphorylation sites, and numbers of predicted transmembrane domains. They are expressed in a number of human cells and tissues and are induced by hypertonic stress. Extrapolation of the topological model and structure-function studies of SGLT1 predicted that these isoforms would retain Na+-MI cotransport activity but could differ in transport kinetics, posttranslational regulation, and perhaps intracellular trafficking. Initial functional expression studies are consistent with the hypothesis that the predicted isoforms could explain some of the heterogeneity in the regulation of Na+-MI cotransport activity observed in a variety of cell types under physiological and pathophysiological conditions. Further characterization of predicted SMIT isoforms at the peptide level and in expression systems such as Xenopus oocytes, and at the tissue level will be required before their physiological or pathophysiological implications can be fully appreciated.
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ACKNOWLEDGEMENTS |
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F. Porcellati and Y. Hosaka contributed equally to this work.
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FOOTNOTES |
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This study was supported by the Michigan Diabetes Research and Training Center Grants P60-DK20572, RO1-DK38304 (to D. A. Greene), and RO1-DK44848 and P50-DK39255 (to P. D. Killen).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. A. Greene, Univ. of Michigan, 3920 Taubman Center, Box 0354, Ann Arbor, MI 48109-0354 (E-mail: dgreene{at}medmail.med.umich.edu).
Received 23 September 1998; accepted in final form 18 February 1999.
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