Expression of three isoforms of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in human penile cavernosum

Inhibition of cGMP-specific phosphodiesterase type V (PDE5) has been shown to improve penile erection in patients with erectile dysfunction. We report here the cloning of three PDE5 isoforms from human penile tissues. Two of the isoforms were identical to PDE5A1 and PDE5A2, respectively, which had been isolated from nonpenile tissues. The third isoform was novel and hence called PDE5A3. The deduced amino acid sequence of PDE5A3 was the same as the C-terminal 823-residue sequence of PDE5A1 and PDE5A2. While PDE5A1 and A2 isoforms were expressed in all tissues examined, the A3 isoform was confined to tissues with a smooth muscle or cardiac muscle component. When expressed in COS-7 cells, PDE5A1, A2, and A3 isoforms had similar cGMP-catalytic activities with K(m) of 6.2, 5.75, and 6.06 microM, respectively. Their cGMP-catalytic activities were inhibited by zaprinast with IC(50) values of 3.2 microM, 1.3 microM, and 1.6 microM, respectively, and by sildenafil with IC(50) of 28, 14, and 13 nM, respectively.     

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.     

Human Ca2+/calmodulin-dependent phosphodiesterase PDE1A: novel splice variants, their specific expression, genomic organization, and chromosomal localization

We report here the identification of novel human PDE1A splice variants, their tissue distribution patterns, genomic structure, and chromosomal localization of the gene. We identified one N-terminus (N3) and one C-terminus (C3) by cDNA library screening and dbEST database search. These N- and C-termini, including the reported N-termini (N1 and N2) and C-termini (C1 and C2), combined to generate nine different PDE1A cDNAs. N1 and N2 are similar to the 5' ends of the bovine PDE1A proteins of 61 kDa and 59 kDa, respectively, and C1 and C2 are the 3' ends of the reported human PDE1A variants. The results of PCR and Southern blot analysis show that nine PDE1A splice variants exhibit distinctive tissue distribution patterns by the difference of the N-terminus. PDE1As with N2 were widely expressed in various tissues, mainly in the kidney, liver, and pancreas. On the other hand, PDE1As with N1 and N3 were particularly expressed at a high level in the brain and testis, respectively. These findings suggest that the distinct expression patterns among PDE1A variants depend on the several promoters situated upstream of exons encoding 5' ends of the variants. The PDE1A gene spans over 120 kb of genomic DNA, and consists of at least 17 exons and 16 introns. The PDE1A gene was located on human chromosome 2q32 by fluorescent in situ hybridization analysis.     

Identification of rat cyclic nucleotide phosphodiesterase 11A (PDE11A): comparison of rat and human PDE11A splicing variants.

We have isolated and characterized rat cyclic nucleotide phosphodiesterase (PDE)11A, which exhibits properties of a dual-substrate PDE, and its splice variants (RNPDE11A2, RNPDE11A3, and RNPDE11A4). The deduced amino-acid sequence of the longest form of rat PDE11A splice variant, RNPDE11A4, was 94% identical with that of the human variant (HSPDE11A4). Rat PDE11A splice variants were expressed in a tissue-specific manner. RNPDE11A4 showed unique tissue distribution distinct from HSPDE11A4, which is specifically expressed in the prostate. Rat PDE11A splice variants were expressed in COS-7 cells, and their enzymatic characteristics were compared. Although the Km values for cAMP and cGMP were similar for all of them (1.3-1.6 and 2.1-3.9 microM, respectively), the Vmax values differed significantly (RNPDE11A4 > RNPDE11A2 > RNPDE11A3). Human PDE11A variants also displayed very similar Km values and significantly different Vmax values (HSPDE11A4 > HSPDE11A2 > HSPDE11A3 > HSPDE11A1). The Vmax values of HSPDE11A4 for cAMP and cGMP were at least 100 times higher than those of HSPDE11A1. These observations indicate unique characteristics of PDE11A splicing variants.     

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.     

Human PDE4A8, a novel brain-expressed PDE4 cAMP-specific phosphodiesterase that has undergone rapid evolutionary change

We have isolated cDNAs encoding PDE4A8 (phosphodiesterase 4 isoform A8), a new human cAMP-specific PDE4 isoform encoded by the PDE4A gene. PDE4A8 has a novel N-terminal region of 85 amino acids that differs from those of the related 'long' PDE4A4, PDE4A10 and PDE4A11 isoforms. The human PDE4A8 N-terminal region has diverged substantially from the corresponding isoforms in the rat and other mammals, consistent with rapid evolutionary change in this region of the protein. When expressed in COS-7 cells, PDE4A8 localized predominantly in the cytosol, but approx. 20% of the enzyme was associated with membrane fractions. Cytosolic PDE4A8 was exquisitely sensitive to inhibition by the prototypical PDE4 inhibitor rolipram (IC(50) of 11+/-1 nM compared with 1600 nM for PDE4A4), but was less sensitive to inhibition by cilomilast (IC(50) of 101+/-7 nM compared with 61 nM for PDE4A4). PDE4A8 mRNA was found to be expressed predominantly in skeletal muscle and brain, a pattern that differs from the tissue expression of other human PDE4 isoforms and also from that of rat PDE4A8. Immunohistochemical analysis showed that PDE4A8 could be detected in discrete regions of human brain, including the cerebellum, spinal cord and cerebral cortex. The unique tissue distribution of PDE4A8, combined with the evolutionary divergence of its N-terminus, suggest that this isoform may have a specific function in regulating cAMP levels in human skeletal muscle and brain.     

Molecular cloning and characterization of CHM1L, a novel membrane molecule similar to chondromodulin-I

Chondromodulin-I (ChM-I) is a cartilage-specific glycoprotein that stimulates the growth of chondrocytes and inhibits the tube formation of endothelial cells. In the present study, we identified a novel ChM-I like molecule, designated ChM1L. Cloning of full length cDNAs of human, mouse, and rat ChM1L revealed that ChM1L encodes 317 amino acids novel type II transmembrane protein. ChM1L protein was expressed on the cell surface as N-glycosylated and non-N-glycosylated protein with molecular mass of 45 and 40 kDa, respectively. In adult mouse tissues, ChM1L mRNA was highly expressed in eye, skeletal muscle, and whole rib. The temporal pattern of ChM1L mRNA was examined using whole embryo at day 10 to 19 of gestation. After day 11, ChM1L mRNA was detected and its level was progressively elevated in association with development of mouse embryo. These data suggest that ChM1L is a novel membrane molecule which is similar to ChM-I that plays a regulatory role in eye, skeletal muscle, and development of embryo.     

Isolation, expression and analysis of splice variants of a human Ca2+/calmodulin-stimulated phosphodiesterase (PDE1A).

The PDE1A gene encodes a Ca2+/calmodulin-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE). We have performed 5' and 3' RACE and identified two additional 5'-splice variants and one additional 3'-splice variant of the human PDE1A gene. The three known 5'-splice variants and the two known 3'-splice variants combine to generate six different PDE1A mRNAs. However, one of the 5'-splice variants exhibits alternate splicing in the 5' untranslated region. Thus the six mRNAs encode four different PDE1A proteins. Recombinant forms of the different human PDE1A isoforms were expressed in Sf9 cells. The kinetic properties and inhibitor sensitivities of the four PDE1A isoforms are very similar to one another.     

Differential expression of human nicotinic acetylcholine receptor alpha subunit variants in muscle and non-muscle tissues.

The nicotinic acetylcholine receptor (nAChR) is an oligomeric transmembrane glycoprotein consisting of four homologous subunits in stoichiometry of alpha 2, beta (gamma or epsilon). Recently the presence of a novel exon (P3A) in human alpha AChR gene has been reported. Two variants of the human alpha subunit arise from alternate RNA splicing, one with and one without the P3A exon. However, the evolutionary origin of the P3A exon and the regulation of the expression of the two variants in human muscle and non-human tissues is currently unknown. Examination of genomic DNA from various species shows that the P3A exon sequence is present only in hominoids, old world and new world primates species and is absent in the muscle cDNA or genomic DNA from rat, mouse or dog, indicating that P3A exon is evolutionary conserved for at least 50 million years. The P3A+ variant of alpha subunit was found to be constitutively expressed in skeletal muscle, brain, heart, kidney, liver, lung and thymus, while P3A-variant was differentially expressed only in skeletal muscle. Thus it appears that the P3A+ variant is generated by 'default' selection by the splicing machinery, while expression of the P3A- variant is regulated by tissue-specific factors in the skeletal muscle. Mechanisms regulating differential expression of the alpha subunit variants may be pertinent to the pathophysiology of myasthenia gravis.     

Pyridoxamine (pyridoxine) phosphate oxidase activity in mammalian tissues

1. Vitamin B6-sufficient rats had moderate pyridoxamine-P oxidase specific activities in heart, brain, kidney and liver, but no detectable activity in skeletal muscle. Vitamin B6-deficiency in rats resulted in a decreased oxidase activity in liver but no change in the activities in other tissues. 2. The pyridoxamine-P oxidase activity in vitamin B6-sufficient mice was high in liver, moderate in brain and kidney, and not measurable in skeletal muscle and heart. Vitamin B6-deficient, compared with control mice, had decreased oxidase activities in brain, kidney and liver. 3. Mouse erythrocytes took up pyridoxine more rapidly than did rat and human erythrocytes. 4. Mouse and human erythrocytes rapidly converted pyridoxine to pyridoxal-P. Rat, hamster and rabbit erythrocytes had appreciably lower pyridoxamine-P oxidase activity than did mouse and human erythrocytes.     

Specificity of glucose 1,6-bisphosphate synthesis in rabbit skeletal muscle.

1. To compare glucose 1,6-bisphosphate synthesis in different types of cells, we partially purified (2000-fold) a glycerate 1,3 P2-dependent glucose 1,6-bisphosphate synthase from rabbit skeletal muscle. 2. In agreement with the results reported by others for mouse brain and pig skeletal muscle, the enzyme can be separated from bulk phosphoglucomutase (PGM) activity by DEAE-cellulose chromatography of crude cellular extract. This cannot be achieved on human hemolysates where glycerate 1,3-P2-dependent glucose 1,2-bisphosphate synthesis is displayed only by multifunctional PGM2 isoenzymes. 3. The Km values for glycerate 1,3-P2 (0.50 microM), glucose 1-phosphate (90 microM), Mg2+ (0.22 mM), and also pH optimum (7.8) and mol. wt (70,000) of the rabbit skeletal muscle enzyme are similar to those of the enzymes from mouse brain and human red blood cells, but they differ from those reported for the pig skeletal muscle enzyme.     

Isolation, characterization, and chromosome mapping of a human A-C1 Ha-Ras suppressor gene (HRASLS)

Recently, we cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines, embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. Mouse A-C1 has homology with a ras-responsive gene, rat Ha-rev107 (Hrasls), and modulates a Ha-ras-mediated signaling pathway. Here, we report a cDNA encoding a human homolog of mouse A-C1. The deduced amino acid sequence of human A-C1 consists of 168 amino acids, and shows 83% identity with that of mouse A-C1. Human A-C1 mRNA was expressed in skeletal muscle, testis, heart, brain, and thyroid in vivo. Moreover, expression of human A-C1 mRNA was detected at a high level in human osteosarcoma-derived U2OS cells in vitro. By FISH analysis the human A-C1 gene (HRASLS) was mapped to human chromosome 3q28--> q29.