Isolation and differential tissue distribution of two human cDNAs encoding PDE1 splice variants.

A cDNA selection technique has been used to isolate full-length human cDNAs of the phosphodiesterase 1 (PDE1) calcium calmodulin (CaM)-regulated phosphodiesterase gene family. We isolated cDNAs representing multiple splice variants of PDE1A, 1B and 1C from a variety of tissues. Included among these were two novel splice variants for PDE1A and 1B. The first, PDE1A5, encodes a 519-residue protein, which is different from PDE1A1 by the insertion of 14 residues, a divergent carboxy terminus and also differs from PDE1A3 through a divergent amino terminus. Our second novel splice variant represents the first occurrence of a splice variant of the PDE1B gene. PDE1B2 encodes a 516-residue protein and diverges from PDE1B1 by the replacement of the first 38 residues by an alternative 18, which is predicted to be functionally significant. Using the splice variant sequence differences to perform comparative Northern analysis, we have demonstrated that each variant has a differential tissue distribution.     

Alternative splicing switches the divalent cation selectivity of TRPM3 channels

TRPM3 is a poorly understood member of the large family of transient receptor potential (TRP) ion channels. Here we describe five novel splice variants of TRPM3, TRPM3alpha1-5. These variants are characterized by a previously unknown amino terminus of 61 residues. The differences between the five variants arise through splice events at three different sites. One of these splice sites might be located in the pore region of the channel as indicated by sequence alignment with other, better-characterized TRP channels. We selected two splice variants, TRPM3alpha1 and TRPM3alpha2, that differ only in this presumed pore region and analyzed their biophysical characteristics after heterologous expression in human embryonic kidney 293 cells. TRPM3alpha1 as well as TRPM3alpha2 induced a novel, outwardly rectifying cationic conductance that was tightly regulated by intracellular Mg(2+). However, these two variants are highly different in their ionic selectivity. Whereas TRPM3alpha1-encoded channels are poorly permeable for divalent cations, TRPM3alpha2-encoded channels are well permeated by Ca(2+) and Mg(2+). Additionally, we found that currents through TRPM3alpha2 are blocked by extracellular monovalent cations, whereas currents through TRPM3alpha1 are not. These differences unambiguously show that TRPM3 proteins constitute a pore-forming channel subunit and localize the position of the ion-conducting pore within the TRPM3 protein. Although the ionic selectivity of ion channels has traditionally been regarded as rather constant for a given channel-encoding gene, our results show that alternative splicing can be a mechanism to produce channels with very different selectivity profiles.     

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.     

Different expression patterns for ubiquitous calpains and Capn3 splice variants in monkey ocular tissues

The purpose of the present investigation was to compare the expression of ubiquitous and tissue-specific calpains in ocular tissues from the Macaca fascicularis monkey. Calpain isoforms in retina and corneal epithelium from adult M. fascicularis monkeys were characterized by RT-PCR, cDNA cloning and sequencing. Calpain isoform activities in ocular tissues were investigated by fractionation on DEAE-HPLC, immunoblotting, and casein zymography. Capn3 splice variants in the ocular tissues from rat, rabbit and monkey were compared after RT-PCR. RT-PCR analysis revealed that numerous splice variants of Capn3 were expressed in the epithelium from monkey cornea. The variants contained deletions or insertions in or around the IS1, IS2, and NS regions. The cDNAs for Capn3 variants were highly conserved, yet the expression patterns of the Capn3 isoforms were widely different among the mammalian species. In contrast, the expression patterns of ubiquitous calpains in ocular tissues were conserved among the mammalian species, and similarities between monkey and human cDNAs for Capn1 (mu-calpain) and Capn2 (m-calpain) were 98 and 99%, respectively. These results suggested that differences in expression patterns of Capn3 variants might be related to the function of each variant in a particular tissue or species.     

Identification of novel splice variants of the human catalytic subunit Cbeta of cAMP-dependent protein kinase.

Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.     

Role of Nova-1 in regulating alpha2N,a novel glycine receptor splice variant,in developing spinal cord neurons

Inhibitory glycine receptor (GlyR) subunits undergo developmental regulation, but the molecular mechanisms of GlyR regulation in developing neurons are little understood. Using RT-PCR, we investigated the regulation of GlyR alpha-subunit splice forms during the development of the spinal cord of the rat. Experiments to compare the amounts of mRNA for two known splice variants of the GlyR alpha2 subunit, alpha2A and alpha2B, in the developing rat spinal cord revealed the presence of an additional, novel variant that lacked any exon 3, herein named "alpha2N." Examination of the RNA from spinal cords of different-aged rats showed a dramatic down-regulation of alpha2N during prenatal development: alpha2N mRNA formed a significant portion of the alpha2 subunit pool at E14, but its relative level was reduced by 85% by birth and was undetectable in adults. Two proteins previously implicated in regulating the splicing of GlyR alpha2 pre-mRNA, the neurooncological ventral antigen-1 (Nova-1) and the brain isoform of the polypyrimidine tract binding protein (brPTB), underwent small changes over the same period that did not correlate directly with the changes in the level of alpha2N, calling into question their involvement in the developmental regulation of alpha2N. However, treatment of spinal cord neurons in culture with antisense oligonucleotides designed selectively to knock down one of three Nova-1 variants significantly altered the relative level of GlyR alpha2N, showing that Nova-1 isoforms can regulate GlyR alpha2 pre-mRNA splicing in developing neurons. These results provide evidence for a novel splice variant of the GlyR alpha2 subunit that undergoes dramatic developmental regulation, reveal the expression profiles of Nova-1 and brPTB in the developing spinal cord, and suggest that Nova-1 plays a role in regulating GlyR alpha2N in developing neurons.     

ERRβ splice variants differentially regulate cell cycle progression

Orphan receptors comprise nearly half of all members of the nuclear receptor superfamily. Despite having broad structural similarities to the classical estrogen receptors, estrogen-related receptors (ERRs) have their own unique DNA response elements and functions. In this study, we focus on 2 ERRβ splice variants, short form ERRβ (ERRβsf) and ERRβ2, and identify their differing roles in cell cycle regulation. Using DY131 (a synthetic agonist of ERRβ), splice-variant selective shRNA, and exogenous ERRβsf and ERRβ2 cDNAs, we demonstrate the role of ERRβsf in mediating the G1 checkpoint through p21. We also show ERRβsf is required for DY131-induced cellular senescence. A key novel finding of this study is that ERRβ2 can mediate a G2/M arrest in response to DY131. In the absence of ERRβ2, the DY131-induced G2/M arrest is reversed, and this is accompanied by p21 induction and a G1 arrest. This study illustrates novel functions for ERRβ splice variants and provides evidence for splice variant interaction.     

Genomic organization of mouse orexin receptors: characterization of two novel tissue-specific splice variants

In humans and rat, orexins orchestrate divergent actions through their G protein-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R). Orexins also play an important physiological role in mouse, but the receptors through which they function are not characterized. To characterize the physiological role(s) of orexins in the mouse, we cloned and characterized the mouse orexin receptor(s), mOX1R and mOX2R, using rapid amplification of cDNA (mouse brain) ends, RT-PCR, and gene structure analysis. The mOX1R cDNA encodes a 416-amino acid (aa) receptor. We have identified two alternative C terminus splice variants of the mOX2R; mOX2 alpha R (443 aa) and mOX2 beta R (460 aa). Binding studies in human embryonic kidney 293 cells transfected with mOX1R, mOX2 alpha R, and the mOX2 beta R revealed specific, saturable sites for both orexin-A and -B. Activation of these receptors by orexins induced inositol triphosphate (IP(3)) turnover. However, human embryonic kidney 293 cells transfected with mOXRs demonstrated no cAMP response to either orexin-A or orexin-B challenge, although forskolin and GTP gamma S revealed a dose-dependent increase in cAMP. Although, orexin-A and -B showed no difference in binding characteristics between the splice variants; interestingly, orexin-B led to an increase in IP(3) production at all concentrations in the mOX2 beta R variant. Orexin-A, however, showed no difference in IP(3) production between the two variants. Additionally, in the mouse, we demonstrate that these splice variants are distributed in a tissue-specific manner, where OX2 alpha R mRNA was undetectable in skeletal muscle and kidney. Moreover, food deprivation led to a greater increase in hypothalamic mOX2 beta R gene expression, compared with both mOX1R and mOX2 alpha R. This potentially implicates a fundamental physiological role for these splice variants.     

At least three alternatively spliced mRNAs encoding two alpha subunits of the Go GTP-binding protein can be expressed in a single tissue

Hybridization blot (Northern) analysis of mRNA coding for alpha subunits of the Go signal-transducing protein detects three bands at 5.7, 4.2, and 3.2 kilobases (kb). We showed previously that the largest is a splice variant coding for the type 2 form of the polypeptide (alpha o2) and the two smaller RNAs react with a probe specific for the seventh of the eight exons that code for the type 1 form (alpha o1). In the present work we demonstrate that the 3.2- and 4.2-kb mRNAs also result from alternative splicing, the splice site being located 31 nucleotides downstream from the termination codon of the open reading frame, and that therefore the alpha o mRNA is made up of at least nine exons. All three alpha o mRNAs are expressed in both heart and brain, more in the latter than the former, as well as in the hamster insulin-secreting tumor (HIT) cell from which the cDNAs encoding the splice variants had been cloned. In contrast, in lung and testis we found only the 5.7-kb alpha o2 mRNA. The same analysis was unable to detect alpha o-specific sequences in either kidney, pancreas (whole), spleen, or liver, while at the same time detecting strong bands for alpha s mRNA. A comparison of the nucleotide sequences of the 5'- and 3'-untranslated regions of the hamster cDNAs cloned here indicated that previously cloned alpha o cDNAs all belong to the same alpha o1A slice subclass derived from 3.2-kb mRNA. The comparison also revealed that the sequences of the untranslated regions are highly conserved among three species (rat, hamster, and brain). Their 3' tails are 99.1% (HIT versus bovine, 200 known bases) and 99.7% (HIT versus rat, 229 bases) identical, and their 5' leader sequences are 92.7% (HIT versus bovine, 165 known bases) and 90.7% (HIT versus rat, 670 bases) identical. This indicates that untranslated regions of mRNAs need not exhibit high degrees of species variation.     

Existence of multiple novel Gs alpha splice variants in acute leukemia patients

The alpha subunit of the stimulatory G protein, Gs alpha, is involved in stimulation of the adenylate cyclase pathway of signal transduction. In this study, we investigated the status of the Gs alpha gene in 29 acute leukemia patients and identified three novel splice variants (designated Gs alpha L-1, Gs alpha L-2, and Gs alpha L-3), possibly derived from aberrant splicing. All of the splice variants have in-frame deletions, removing the functional domain responsible for GTPase activity of Gs alpha, and would encode truncated proteins of 160(Gs alpha L-1), 90(Gs alpha L-2) and 70(Gs alpha L-3) amino acids, respectively. The data suggest that these novel products may be implicated in an as-yet-unidentified signal transduction pathway in hematopoietic cells.     

Changes in adhesive and migratory characteristics of hepatocellular carcinoma (HCC) cells induced by expression of alpha3beta1 integrin

The invasive and metastatic potentials of hepatocellular carcinoma are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms including laminin-5. In this study, we investigated changes in the adhesive and invasive behaviors of human HCC HepG2 cells after transfection with cDNA for alpha3 integrin in order to elucidate the direct involvement of this integrin in these cellular processes. We introduced cDNA for splice variants of alpha3 integrin (alpha3A and alpha3B) into the cells, and selected two transfectant clones (HepG2-3A and HepG2-3B), which express the alpha3A and alpha3B integrins, respectively. Both transfectant cells adhered almost equally to laminin-5-coated plates in an alpha3 integrin-dependent manner, indicating that transfected alpha3Abeta1 and alpha3Bbeta1 integrins were functionally active in these cells. The migratory and invasive potentials of the transfectant cells were assessed by scratch wound assay and in vitro chemoinvasion assay. The results demonstrated that the migration of HepG2-3A and HepG2-3B cells but not of mock transfectant (HepG2-M) cells was stimulated on the plates coated with laminin-5. Furthermore, HepG2-3A and HepG2-3B cells were found to be more invasive into laminin-5-containing matrices than were HepG2-M cells. These results strongly suggest that enhanced expression of alpha3beta1 integrin on HCC cells is directly involved in their malignant phenotypes such as invasion and metastasis.     

Cloning, characterization and expression of CDK5RAP1_v3 and CDK5RAP1_v4, two novel splice variants of human CDK5RAP1

Two novel splice variants of CDK5RAP1, named CDK5RAP1_v3 and CDK5RAP1_v4, were isolated through the large-scale sequencing analysis of a human fetal brain cDNA library. The CDK5RAP1_v3 and CDK5RAP1_v4 cDNAs are 1923bp and 1792bp in length, respectively. Sequence analysis revealed that CDK5RAP1_v4 lacked 1 exon, which was present in CDK5RAP1_v3, with the result that these cDNAs encoded different putative proteins. The deduced proteins were 574 amino acids (designated as CDK5RAP1_v3) and 426 amino acids (CDK5RAP1_v4) in length, and shared the 420 N-terminal amino acids. RT-PCR analysis showed that human CDK5RAP1_v3 was widely expressed in human tissues. The expression level of CDK5RAP1_v3 was relatively high in placenta and lung, whereas low levels of expression were detected in heart, brain, liver, skeletal muscle, pancreas, spleen, thymus, small intestine and peripheral blood leukocytes. In contrast, human CDK5RAP1_v4 was mainly expressed in brain, placenta and testis.     

The opioid ligand binding of human mu-opioid receptor is modulated by novel splice variants of the receptor

The pharmacological actions of morphine and morphine-like drugs, such as heroin, mediate primarily through the mu-opioid receptor (MOR). It has been proposed that the functional diversity of MOR may be related to alternative splicing of the MOR gene. Although a number of MOR mRNA splice variants have been reported, their biological function has been controversial. In this study, two novel splice variants of the human MOR gene were discovered. Splice variants 1 and 2 (here called the SV1 and SV2) retain different portions of intron I. In vitro translation of SV1 and SV2 produced proteins with the predicted molecular weights. The splice variant proteins were identical to the wild-type MOR-1 up to the first transmembrane domains, but were different after the first intracellular loop domains. SV1 and SV2 of hMOR were present in human neuroblastoma NMB cells and human whole brain confirmed by RT-PCR. In a receptor binding assay, cells expressing the SV1 and SV2 do not exhibit binding to [(3)H]diprenorphine. The formations of MOR.SV1 and MOR.SV2 heterodimers were demonstrated by co-immunoprecipitation and bioluminescence resonance energy transfer between MOR and splice variants. Co-transfection of MOR-GFP and SV-DsRed gene showed that MOR and SV protein co-localized at the cytoplasmic membrane. In NMB cells expressing human MOR gene, transfection of SV1 or SV2 reduced binding activity of the endogenous MOR. These data support a potential role of SV1 and SV2 proteins as possible biological modulator of human mu-opioid receptor.