Identification of a novel alternative splicing of human FGF receptor 4: soluble-form splice variant expressed in human gastrointestinal epithelial cells

Among four closely related members of the FGF receptor family, FGFR 1, 2, and 3 have alternative splicing forms encoded by different exons for the C-terminal half of the third Ig-like domain, but FGFR 4 has no such alternative exon. Furthermore, FGFR 1, 2, and 3 have another splice variant of nontransmembrane type; however, such a variant has not been reported for FGFR 4. While searching for a novel receptor-type tyrosine kinase by RT-PCR, we identified a non-transmembrane-type receptor of FGFR 4 in human intestinal epithelial cell lines (Intestine 407 and Caco-2). Sequence analysis of this receptor revealed that exon 9 coding the single transmembrane domain was displaced by intron 9. Consequently, this variant form was 120 bp shorter than the normal form and had no transmembrane portion. Moreover, the signal sequence in exon 2 was maintained, suggesting that this splice variant is a soluble receptor. This soluble receptor was detected in human gastrointestinal epithelial cells and pancreas, and also in gastric, colon, and pancreatic cancer cell lines. Single cell RT-PCR showed that this soluble receptor was expressed simultaneously with the transmembrane-type receptor in the same cell. Western blot analysis revealed that this receptor was secreted from the transfected COS7 cells. Thus, a soluble-form splice variant of FGFR 4 was identified in human gastrointestinal epithelial cells and cancer cells. This is the first report of alternative splicing of FGFR 4.     

Structure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant

Several variants of the serotonin 5-HT4 receptor are known to be produced by alternative splicing. To survey the existence and usage of exons in humans, we cloned the human 5-HT4 gene. Based on sequence analysis seven C-terminal variants (a-g) and one internal splice variant (h) were found. We concentrated in this study on the functional characterization of the novel splice variant h, which leads to the insertion of 14 amino acids into the second extracellular loop of the receptor. The h variant was cloned as a splice combination with the C-terminal b variant; therefore, we call this receptor 5-HT4(hb). This novel receptor variant was expressed transiently in COS-7 cells, and its pharmacological profile was compared with those of the previously cloned 5-HT4(a) and 5-HT4(b) isoforms, with the latter being the primary reference for the h variant. In competition binding experiments using reference 5-HT4 ligands, no significant differences were detected. However, the broadly used 5-HT4 antagonist GR113808 discriminated functionally among the receptor variants investigated. As expected, it was an antagonist on the 5-HT4(a) and 5-HT4(b) variant but showed partial agonistic activity on the 5-HT4(hb) variant. These data emphasize the importance of variations introduced by splicing for receptor pharmacology and may help in the understanding of conflicting results seen with 5-HT4 ligands in different model systems.     

Intra-domain Cross-talk Regulates Serine-arginine Protein Kinase 1-dependent Phosphorylation and Splicing Function of Transformer 2β1

Transformer 2β1 (Tra2β1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2β1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2β1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated by a docking groove in the kinase domain. Although SRPK1 readily phosphorylates RS2 in a splice variant lacking the N-terminal RS domain (Tra2β3), RS1 blocks phosphorylation of these serines in the full-length Tra2β1. Thus, RS2 serves two new functions. First, RS2 positively regulates binding of the central RNA recognition motif to an exonic splicing enhancer sequence, a phenomenon reversed by SRPK1 phosphorylation on RS1. Second, RS2 enhances ligand exchange in the SRPK1 active site allowing highly efficient Tra2β1 phosphorylation. These studies demonstrate that SRPK1 is a regulator of Tra2β1 splicing function and that the individual RS domains engage in considerable cross-talk, assuming novel functions with regard to RNA binding, splicing, and SRPK1 catalysis.     

Identification and characterization of a novel human APH-1b splice variant lacking exon 4

APH-1 is one of the four essential components of the presenilin-gamma-secretase complex and has two human homologs, APH-1a, and APH-1b, both of which are seven-pass membrane proteins. Here, we identified a novel splice variant of human APH-1b. This variant lacks exon 4, which encodes the entire fourth transmembrane domain. The mRNA expression of this variant was detected in most tissues at low levels. In transiently transfected cells, protein expression of the APH-1b variant was much lower than that of the wild-type. Furthermore, exogenous expression of the APH-1-interacting protein, nicastrin, significantly increased the variant protein levels. These data suggest that the APH-1b variant protein is destabilized, and implies that the fourth transmembrane domain plays an important role in the protein stability and function of APH-1.     

The GIT family of ADP-ribosylation factor GTPase-activating proteins. Functional diversity of GIT2 through alternative splicing

We recently characterized a novel protein, GIT1, that interacts with G protein-coupled receptor kinases and possesses ADP-ribosylation factor (ARF) GTPase-activating protein activity. A second ubiquitously expressed member of the GIT protein family, GIT2, has been identified in data base searches. GIT2 undergoes extensive alternative splicing and exists in at least 10 and potentially as many as 33 distinct forms. The longest form of GIT2 is colinear with GIT1 and shares the same domain structure, whereas one major splice variant prominent in immune tissues completely lacks the carboxyl-terminal domain. The other 32 potential variants arise from the independent alternative splicing of five internal regions in the center of the molecule but share both the amino-terminal ARF GTPase-activating protein domain and carboxyl-terminal domain. Both the long and short carboxyl-terminal variants of GIT2 are active as GTPase-activating proteins for ARF1, and both also interact with G protein-coupled receptor kinase 2 and with p21-activated kinase-interacting exchange factors complexed with p21-activated kinase but not with paxillin. Cellular overexpression of the longest variant of GIT2 leads to inhibition of beta(2)-adrenergic receptor sequestration, whereas the shortest splice variant appears inactive. Although GIT2 shares many properties with GIT1, it also exhibits both structural and functional diversity due to tissue-specific alternative splicing.     

Afadin: A Novel Actin Filament–binding Protein with One PDZ Domain Localized at Cadherin-based Cell-to-Cell Adherens Junction

A novel actin filament (F-actin)–binding protein with a molecular mass of ~205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin–binding domain at 1,631–1,829 aa residues and one PDZ domain at 1,016– 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009–1,093 aa residues but lacking the F-actin–binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin–cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.     

Dual subcellular localization in the endoplasmic reticulum and peroxisomes and a vital role in protecting against oxidative stress of fatty aldehyde dehydrogenase are achieved by alternative splicing

Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought to be involved in the degradation of phytanic acid, a saturated branched chain fatty acid derived from chlorophyll. However, the identity, subcellular distribution, and physiological roles of FALDH are unclear because several variants produced by alternative splicing are present in varying amounts at different subcellular locations. Subcellular fractionation experiments do not provide a clear-cut conclusion because of the incomplete separation of organelles. We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody. One variant, FALDH-V, was exclusively detected in peroxisomal membranes. Human FALDH-V with an amino-terminal Myc sequence also localized to peroxisomes. The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum. A gas chromatography-mass spectrometry-based analysis of metabolites in FALDH-expressing cells incubated with phytol or phytanic acid showed that FALDH-V, not FALDH-N, is the key aldehyde dehydrogenase in the degradation pathway and that it protects peroxisomes from oxidative stress. In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal. These results suggest that FALDH variants are produced by alternative splicing and share an important role in protecting against oxidative stress in an organelle-specific manner.     

A novel murine PDGF-D splicing variant results in significant differences in peptide expression and function

Platelet-derived growth factor (PDGF) is a potent mesenchymal cell mitogen and chemoattractant involved in the pathogenesis of fibroproliferative diseases. There are four known PDGF ligand isoforms designated A-D, two of which, C and D, were only recently discovered. We have identified a splicing variant in the PDGF-D isoform that occurs in mice, but not in humans. The presence of the splicing variant in murine PDGF-D appears to be due to an aberration in the splicing site at the junction of exons 5 and 6. The splicing variant results in a deletion predicted to have significant effects on peptide activity since it results in the deletion of bases within the cysteine knot domain that are important for peptide dimerization and receptor binding. It is important to appreciate differences between murine and human PDGF gene expression because PDGF is a key mitogen in the pathogenesis of fibrosis and mice are commonly employed as models for human disease.     

Genomic organization and identification of a novel alternative splicing variant of mouse mitochondrial thioredoxin reductase (TrxR2) gene

Eukaryotic mitochondria are equipped with a complete thioredoxin system, composed of thioredoxin and thioredoxin reductase, which has been implicated in the protection against the reactive oxygen intermdiates generated during the respiratory process in this organelle. Like its cytosolic counterpart, mammalian mitochondrial thioredoxin reductase is a homodimeric selenoprotein. We report here the genomic organization of the mouse mitochondrial thioredoxin gene (TrxR2) that spans 53 kb and consists of 18 exons ranging from 20 to 210 bp. All splicing sites conformed to the GT/AG rule with the exon-intron boundaries located exactly at the same position as the human TrxR2 gene, the only mammalian mitochondrial thioredoxin reductase gene whose genomic structure has been elucidated to date. In addition, we have identified a novel mRNA splicing variant lacking intron 14 resulting in a protein subunit with a shorter interface domain. This new splicing variant provides a framework for further analysis of this important enzyme as its predicted homodimeric conformation can now be expanded to a putative heterodimeric structure as well as a small subunit homodimer with the obvious implications at the regulatory level.     

Novel variant of p230 trans-Golgi network protein identified by serum from Sjögren's syndrome patient

Trans-Golgi network (TGN) protein p230 is a peripheral membrane protein associated with the cytoplasmic face of the TGN. TGNp230 is an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, associates with non-clathrin-coated vesicles arising from the TGN, and is implicated in vesicle biogenesis. Here we used an autoimmune serum from a patient with S ogren's syndrome to clone partial cDNAs from a human hepatoma HepG2 expression library. The partial cDNAs encoded a novel amino-terminal splice variant of TGNp230. Specific reactivity of the autoimmune serum for p230 is supported by immunofluorescene staining of the Golgi apparatus, immunoblotting of a > 200-kDa HeLa cell protein, and reactivity with a bacterially expressed GST-p230 fusion protein. The alternative splicing occurs within the first proline-rich domain of p230. It comprises a deletion of 30 bp followed immediately by an additional 66 bp absent in the published sequence. RT-PCR analysis indicated that the splicing occurs independently of previously reported carboxyl-terminal splicing, and that this novel splice variant is more frequent than the previously reported p230. The novel splice variant of p230 is also located at the TGN. We propose that p230 splice variants may be implicated in selection of cargo molecules for vesicles arising from the TGN.     

Alpha1 soluble guanylyl cyclase (sGC) splice forms as potential regulators of human sGC activity

Soluble guanylyl cyclase (sGC), a key protein in the NO/cGMP signaling pathway, is an obligatory heterodimeric protein composed of one alpha- and one beta-subunit. The alpha(1)/beta(1) sGC heterodimer is the predominant form expressed in various tissues and is regarded as the major isoform mediating NO-dependent effects such as vasodilation. We have identified three new alpha(1) sGC protein variants generated by alternative splicing. The 363 residue N1-alpha(1) sGC splice variant contains the regulatory domain, but lacks the catalytic domain. The shorter N2-alpha(1) sGC maintains 126 N-terminal residues and gains an additional 17 unique residues. The C-alpha(1) sGC variant lacks 240 N-terminal amino acids, but maintains a part of the regulatory domain and the entire catalytic domain. Q-PCR of N1-alpha(1), N2-alpha(1) sGC mRNA levels together with RT-PCR analysis for C-alpha(1) sGC demonstrated that the expression of the alpha(1) sGC splice forms vary in different human tissues indicative of tissue-specific regulation. Functional analysis of the N1-alpha(1) sGC demonstrated that this protein has a dominant-negative effect on the activity of sGC when coexpressed with the alpha(1)/beta(1) heterodimer. The C-alpha(1) sGC variant heterodimerizes with the beta(1) subunit and produces a fully functional NO- and BAY41-2272-sensitive enzyme. We also found that despite identical susceptibility to inhibition by ODQ, intracellular levels of the 54-kDa C-alpha(1) band did not change in response to ODQ treatments, while the level of 83 kDa alpha(1) band was significantly affected by ODQ. These studies suggest that modulation of the level and diversity of splice forms may represent novel mechanisms modulating the function of sGC in different human tissues.     

Identification of a variant form of PZR lacking immunoreceptor tyrosine-based inhibitory motifs

PZR is an immunoglobulin superfamily protein that specifically binds tyrosine phosphatase SHP-2 through its intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Here we report a novel isoform of the protein designated PZR1b. PZR1b shares the same extracellular region with PZR, but it lacks intracellular ITIMs and thus the ability to recruit SHP-2. Genomic sequence analysis revealed that PZR1b is resulted from alternative gene splicing of the PZR gene localized at chromosome 1q24. Like PZR, PZR1b is widely expressed. However, the relative ratio of two forms varies in different human tissues and cells. More importantly, overexpression of PZR1b in human HT-1080 cells had a dominant negative effect by blocking concanavalin A-induced tyrosine phosphorylation of full-length PZR and recruitment of tyrosine phosphatase SHP-2. Therefore, PZR1b may have an important role in cell signaling by counteracting with PZR.