Characterization of the human SLC2A11 (GLUT11) gene: alternative promoter usage,function, expression,and subcellular distribution of three isoforms,and lack of mouse orthologue

GLUT11 (SLC2A11) is a class II sugar transport facilitator which exhibits highest similarity with the fructose transporter GLUT5 (about 42%). Here we demonstrate that separate exons 1 (exon 1A, exon 1B, and exon 1C) of the SLC2A11 gene generate mRNAs of three GLUT11 variants (GLUT11-A, GLUT11-B, and GLUT11-C) that differ in the amino acid sequence of their N-termini. All three 5′-flanking regions of exon 1A, exon 1B and exon 1C exhibited promoter activity when expressed as luciferase fusion constructs in COS-7 cells. 5′-RACE-PCR, quantitative real-time PCR, and Northern blot analysis performed with specific probes for exon 1A, 1B and 1C demonstrated that GLUT11-A is expressed in heart, skeletal muscle, and kidney, GLUT11-B in kidney, adipose tissue, and placenta, and GLUT11-C in adipose tissue, heart, skeletal muscle, and pancreas. Surprisingly, mice and rats lack the SLC2A11 gene. When expressed in Xenopus oocytes, all three GLUT11 isoforms transport glucose and fructose but not galactose. There was no apparent difference in the subcellular distribution of the three isoforms expressed in COS-7 cells. Our data indicate that different promoters and splicing of the human SLC2A11 gene generate three GLUT11 isoforms which are expressed in a tissue specific manner but do not appear to differ in their functional characteristics.     

Alternative splicing of delta-like 1 homolog (DLK1) in the pig and human

Delta-like homolog 1 (DLK1), a paternally imprinted gene with several alternative splicing isoforms, is an important regulator of fetal and postnatal development. We report the sequence of porcine DLK1 (pDLK1) and examine the expression and alternative splicing isoforms in the pig (Sus scrofa) and human. DLK1-A was the sole isoform identified in human tissues and has been shown to be present in mouse and cattle. Surprisingly, DLK1-A was undetected in various tissues from fetal and postnatal pigs. Instead, DLK1-C2 was the most abundant isoform while DLK1-B was expressed to a lesser extent. In fractionated adipose tissue, pDLK1 was most highly expressed in the stromal-vascular cell fraction. In addition, total pDLK1 was highly expressed in fetal adipose tissue but dramatically decreased postnatally. Our data suggests that expression of DLK1-B and -C2 isoforms is sufficient for normal pig development. Furthermore, human and pig samples showed no alterations in species-specific splicing, but expression levels decreased with age, suggesting that regulation of expression, not splicing, is the most likely mechanism controlling the biological function of DLK1.     

Alternative splicing produces transcripts encoding four variants of mouse G-protein-coupled receptor kinase 6.

A family of protein kinases, termed G-protein-coupled receptor kinases (GRK1-6), is known to phosphorylate agonist-occupied G-protein-coupled receptors. We have identified mRNAs encoding four distinct mouse GRK6 isoforms (mGRK6), designated mGRK6-A through mGRK6-D. Mouse GRK6-B and mGRK6-C diverge from the known human GRK6 (577 residues) at residue 560 and are 13 residues longer and 16 residues shorter, respectively, than human GRK6, while mGRK6-A very likely represents the mouse equivalent of human GRK6. Mouse GRK6-D is identical to the other mGRK6 variants in the amino-terminal region, but comprises only 59 of the 263 amino acids of the putative catalytical domain. As mGRK6-D retains the region involved in interacting with activated receptors, but most likely lacks catalytic activity, this variant might represent a naturally occurring inhibitor of other GRKs. Analysis of the genomic organization of mGRK6 gene revealed that the four mRNAs are generated by alternative RNA splicing from a single approximately 14. 5-kb gene, made up of at least 17 exons and located on mouse chromosome 13. Similar to human GRK6, mGRK6-A contains three cysteine residues within its carboxyl-terminal region known to serve as substrates for palmitoylation. Mouse GRK6-B lacks these palmitoylation sites, but carries a basic carboxyl-terminus containing consensus sequences for phosphorylation by protein kinases C and cAMP/cGMP-dependent protein kinases. Mouse GRK6-C displays none of these motifs. Thus, mGRK6-A, mGRK6-B, and mGRK6-C are predicted to differ in terms of their regulation by carboxyl-terminal posttranslational modification. Analysis of mRNA expression revealed that the four mGRK6 mRNAs are differentially expressed in mouse tissues, suggesting that the four mGRK6 isoforms are involved in regulating tissue- or cell type-specific functions in vivo.     

Fructose transporter in human spermatozoa and small intestine is GLUT5.

We recently reported that the glucose transporter isoform, GLUT5, is expressed on the brush border membrane of human small intestinal enterocytes (Davidson, N. O., Hausman, A. M. L., Ifkovits, C. A., Buse, J. B., Gould, G. W., Burant, C. F., and Bell, G. I. (1992) Am. J. Physiol. 262, C795-C800). To define its role in sugar transport, human GLUT5 was expressed in Xenopus oocytes and its substrate specificity and kinetic properties determined. GLUT5 exhibits selectivity for fructose transport, as determined by inhibition studies, with a Km of 6 mM. In addition, fructose transport by GLUT5 is not inhibited by cytochalasin B, a competitive inhibitor of facilitative glucose transporters. RNA and protein blotting studies showed the presence of high levels of GLUT5 mRNA and protein in human testis and spermatozoa, and immunocytochemical studies localize GLUT5 to the plasma membrane of mature spermatids and spermatozoa. The biochemical properties and tissue distribution of GLUT5 are consistent with a physiological role for this protein as a fructose transporter.     

Bovine NAD+-dependent isocitrate dehydrogenase: alternative splicing and tissue-dependent expression of subunit 1

NAD+-dependent isocitrate dehydrogenase (IDH), a key regulatory enzyme in the Krebs cycle, is a multi-tetrameric enzyme. At least three of the subunits in the core tetramer of mammals are unique gene products. Subunits 1/beta and 2/gamma are considered to be regulatory, while subunits 3,4/alpha, comprising half the tetramer, are catalytic. The full sequence was obtained for the major subunit 1 cDNA in bovine heart, IDH 1-A. A second cDNA, rare in heart, was also identified (IDH 1-B). Differences in the two were confined to the 3'-region, suggesting alternative splicing. Screening of brain, kidney, and liver RNA showed the presence of IDH 1-A and 1-B and a third major species, IDH 1-C. Amplification of bovine genomic DNA by PCR across the regions of difference produced a single product. Comparison of the genomic and mRNA sequences showed that IDH 1-A resulted from splicing of exon W to exon Y, eliminating intron w, exon X, and intron x. IDH 1-B was formed by splice junctions between exon W, exon X, and exon Y. IDH 1-C resulted from splicing of exon W to exon X and subsequent retention of intron x. The 2 proteins predicted from these 3 mRNAs are identical over their first 357 residues. Protein IDH 1-A, resulting from a termination codon within exon Y, contains an additional 26 residues. Proteins IDH 1-B and 1-C derive from a common termination codon within exon X and contain an additional 28 residues. The two C-terminal regions differ notably in the number and nature of charged residues, resulting in proteins with a charge difference of 3.2 at pH 7.0. Subunit 1 sequences previously reported from other species grouped with one or the other of the bovine proteins. No evidence was found for alternative splicing in subunit 3,4/alpha. The results of the present study, together with recent work on the 2/gamma subunit [Brenner,V., Nyakatura, G., Rosenthal, A., and Platzer, M. (1998) Genomics 44, 8], indicate that the regulatory subunits of the enzyme, but not the catalytic, possess alternatively spliced forms varying in C-terminal properties with tissue-specific expression. The finding is suggestive of a mechanism for modulation of allosteric regulation tailored to the needs of different tissues.     

Tandem promoters and developmentally regulated 5'- and 3'-mRNA untranslated regions of the mouse Scn5a cardiac sodium channel

The SCN5A gene encodes a voltage-sensitive sodium channel expressed in cardiac and skeletal muscle. Coding region mutations cause cardiac sudden death syndromes and conduction system failure. Polymorphisms in the 5'-sequence adjacent to the SCN5A gene have been linked to cardiac arrhythmias. We identified three alternative 5'-splice variants (1A, 1B, and 1C) of the untranslated exon 1 and two 3'-variants in the murine Scn5a mRNA. Two of the exon 1 isoforms (1B and 1C) were novel when compared with the published human and rat SCN5A sequences. Quantitative real time PCR results showed that the abundance of the isoforms varied during cardiac development. The 1A, 1B, and 1C mRNA splice variants increased 7.8 +/- 1.7-fold (E1A), 6.0 +/- 1.0-fold (E1B), and 20.6 +/- 3.7-fold (E1C) from fetal to adult heart, respectively. Promoter deletion and luciferase reporter gene analysis using cardiac and skeletal muscle cell lines demonstrated a pattern of distinct cardiac-specific enhancer elements associated with exons 1A and 1C. In the case of exon 1C, the enhancer element appeared to be within the exon. A 5'-repressor preceded each cardiac enhancer element. We concluded that the murine Na(+) channel has both 5'- and 3'-untranslated region mRNA variants that are developmentally regulated and that the promoter region contains two distinct cardiac-specific enhancer regions. The presence of homologous human splicing suggests that that these regions may be fruitful new areas of study in understanding cardiac sodium channel regulation and the genetic susceptibility to sudden death.     

Phylogenesis and Biological Characterization of a New Glucose Transporter in the Chicken (Gallus gallus), GLUT12

In mammals, insulin-sensitive GLUTs, including GLUT4, are recruited to the plasma membrane of adipose and muscle tissues in response to insulin. The GLUT4 gene is absent from the chicken genome, and no functional insulin-sensitive GLUTs have been characterized in chicken tissues to date. A nucleotide sequence is predicted to encode a chicken GLUT12 ortholog and, interestingly, GLUT12 has been described to act as an insulin-sensitive GLUT in mammals. It encodes a 596 amino acid protein exhibiting 71% identity with human GLUT12. First, we present the results of a phylogenetic study showing the stability of this gene during evolution of vertebrates. Second, tissue distribution of chicken SLC2A12 mRNA was characterized by RT-PCR. It was predominantly expressed in skeletal muscle and heart. Protein distribution was analysed by Western blotting using an anti-human GLUT12 antibody directed against a highly conserved region (87% of identity). An immuno-reactive band of the expected size (75kDa) was detected in the same tissues. Third a physiological characterization was performed: SLC2A12 mRNA levels were significantly lowered in fed chickens subjected to insulin immuno-neutralization. Finally, recruitment of immuno-reactive GLUT12 to the muscle plasma membrane was increased following 1h of intraperitoneal insulin administration (compared to a control fasted state). Thus insulin administration elicited membrane GLUT12 recruitment. In conclusion, these results suggest that the facilitative glucose transporter protein GLUT12 could act in chicken muscle as an insulin-sensitive transporter that is qualitatively similar to GLUT4 in mammals.     

The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members (review).

During the last 2 years, several novel genes that encode glucose transporter-like proteins have been identified and characterized. Because of their sequence similarity with GLUT1, these genes appear to belong to the family of solute carriers 2A (SLC2A, protein symbol GLUT). Sequence comparisons of all 13 family members allow the definition of characteristic sugar/polyol transporter signatures: (1) the presence of 12 membrane-spanning helices, (2) seven conserved glycine residues in the helices, (3) several basic and acidic residues at the intracellular surface of the proteins, (4) two conserved tryptophan residues, and (5) two conserved tyrosine residues. On the basis of sequence similarities and characteristic elements, the extended GLUT family can be divided into three subfamilies, namely class I (the previously known glucose transporters GLUT1-4), class II (the previously known fructose transporter GLUT5, the GLUT7, GLUT9 and GLUT11), and class III (GLUT6, 8, 10, 12, and the myo-inositol transporter HMIT1). Functional characteristics have been reported for some of the novel GLUTs. Like GLUT1-4, they exhibit a tissue/cell-specific expression (GLUT6, leukocytes, brain; GLUT8, testis, blastocysts, brain, muscle, adipocytes; GLUT9, liver, kidney; GLUT10, liver, pancreas; GLUT11, heart, skeletal muscle). GLUT6 and GLUT8 appear to be regulated by sub-cellular redistribution, because they are targeted to intra-cellular compartments by dileucine motifs in a dynamin dependent manner. Sugar transport has been reported for GLUT6, 8, and 11; HMIT1 has been shown to be a H+/myo-inositol co-transporter. Thus, the members of the extended GLUT family exhibit a surprisingly diverse substrate specificity, and the definition of sequence elements determining this substrate specificity will require a full functional characterization of all members.     

GLUT14, a duplicon of GLUT3, is specifically expressed in testis as alternative splice forms

We have identified and cloned GLUT14, a novel member of the glucose transporter family. GLUT14 (SLC2A14) maps to chromosome 12p13.3 (17.1M), about 10 Mb upstream of GLUT3, with which it shares remarkable identity. Until now GLUT14 was thought to be a pseudogene. It consists of 11 exons with a genomic organization similar to that of GLUT3 and likely resulted from a duplication of GLUT3. GLUT14 has two alternatively spliced forms; the shorter form of GLUT14 (GLUT14-S) consists of 10 exons and produces a 497-amino-acid protein that is 94.5% identical to GLUT3. The long form (GLUT14-L) has an additional exon and codes for a protein with 520 amino acids that differs from GLUT14-S only at the N-terminus. GLUT14-S/L contain 12 putative membrane-spanning helices along with sugar-transporter signature motifs that have previously been shown to be essential for sugar transport activity. The putative glycosylation sites of GLUT14-S/L are present in loop 1. In contrast to the expression of GLUT3 in many tissues, both isoforms of GLUT14 are specifically expressed in testis. The mRNA level of GLUT14 in testis is about four times higher than that of GLUT3. Interestingly, the ortholog of GLUT14 is not found in mice. The multiple duplications of GLUT genes suggest that the GLUT family probably emerged by gene duplications and mutations during evolution in different lineages.     

Glucose transporters: structure, function, and regulation

Glucose is transported into the cell by facilitated diffusion via a family of structurally related proteins, whose expression is tissue-specific. One of these transporters, GLUT4, is expressed specifically in insulin-sensitive tissues. A possible change in the synthesis and/or in the amount of GLUT4 has therefore been studied in situations associated with an increase or a decrease in the effect of insulin on glucose transport. Chronic hyperinsulinemia in rats produces a hyper-response of white adipose tissue to insulin and resistance in skeletal muscle. The hyper-response of white adipose tissue is associated with an increase in GLUT4 mRNA and protein. In contrast, in skeletal muscle, a decrease in GLUT4 mRNA and a decrease (tibialis) or no change (diaphragm) in GLUT4 protein are measured, suggesting a divergent regulation by insulin of glucose transport and transporters in the 2 tissues. In rodents, brown adipose tissue is very sensitive to insulin. The response of this tissue to insulin is decreased in obese insulin-resistant fa/fa rats. Treatment with a beta-adrenergic agonist increases insulin-stimulated glucose transport, GLUT4 protein and mRNA. The data suggest that transporter synthesis can be modulated in vivo by insulin (muscle, white adipose tissue) or by catecholamines (brown adipose tissue).     

Structure of the gene for human coagulation factor V.

Activated factor V (Va) serves as an essential protein cofactor for the conversion of prothrombin to thrombin by factor Xa. Analysis of the factor V cDNA indicates that the protein contains several types of internal repeats with the following domain structure: A1-A2-B-A3-C1-C2. In this report we describe the isolation and characterization of genomic DNA coding for human factor V. The factor V gene contains 25 exons which range in size from 72 to 2820 bp. The structure of the gene for factor V is similar to the previously characterized gene for factor VIII. Based on the aligned amino acid sequences of the two proteins, 21 of the 24 intron-exon boundaries in the factor V gene occur at the same location as in the factor VIII gene. In both genes, the junctions of the A1-A2 and A2-A3 domains are each encoded by a single exon. In contrast, the boundaries between domains A3-C1 and C1-C2 occur at intron-exon boundaries, which is consistent with evolution through domain duplication and exon shuffling. The connecting region or B domain of factor V is encoded by a single large exon of 2820 bp. The corresponding exon of the factor VIII gene contains 3106 bp. The 5' and 3' ends of both of these exons encode sequences homologous to the carboxyl-terminal end of domain A2 and the amino-terminal end of domain A3 in ceruloplasmin. There is otherwise no homology between the B domain exons.(ABSTRACT TRUNCATED AT 250 WORDS)