Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms

The existence of mouse H3-receptor isoforms was investigated by PCR analysis and cDNA cloning. Splicing mechanisms previously reported in various species are conserved in the mouse. The retention/deletion of a fragment in the third intracellular loop of the mouse receptor leads to the existence of three isoforms designated mH(3(445)), mH(3(413)) and mH(3(397)) according to the length of their deduced amino acid sequence. PCR analysis showed that mouse H3-receptor isoforms display different expression patterns in the brain. Following expression in Cos-1 cells, [125I]iodoproxyfan binding indicated similar pharmacological profiles of the mH(3(445)), mH(3(413)) and mH(3(397)) isoforms. The pharmacological profile of the mouse H3 receptor is more similar to the rat receptor than to the human receptor, although some differences were also observed between the mouse and rat receptors. For example, the potency of thioperamide and ciproxifan is slightly higher at the mouse receptor than at the rat receptor but 40-100-fold higher than at the human receptor. In situ hybridization histochemistry showed that the distribution of H3-receptor mRNAs in the mouse brain is rather similar to that previously reported in the rat brain. However, the autoradiographic and cellular expression patterns observed in several brain areas such as the thalamus or hippocampus reveal important differences between the two species.     

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor

The H(4)R (histamine H(4) receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H(4)R is primarily expressed in eosinophils and mast cells and has the highest homology with the H(3)R. The occurrence of at least twenty different hH(3)R (human H(3)R) isoforms led us to investigate the possible existence of H(4)R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H(4)R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H(4)R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H(4)R-ligand induced signalling or constitutive activity for these H(4)R splice variants. However, when co-expressed with full-length H(4)R [H(4)R((390)) (H(4)R isoform of 390 amino acids)], the H(4)R splice variants have a dominant negative effect on the surface expression of H(4)R((390)). We detected H(4)R((390))-H(4)R splice variant hetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H(4)R splice variants were detected in various cell types and expressed at similar levels to the full-length H(4)R((390)) mRNA in, for example, pre-monocytes. We conclude that the H(4)R splice variants described here have a dominant negative effect on H(4)R((390)) functionality, as they are able to retain H(4)R((390)) intracellularly and inactivate a population of H(4)R((390)), presumably via hetero-oligomerization.     

Structural and functional properties of two human FXYD3 (Mat-8) isoforms

Six of 7 FXYD proteins have been shown to be tissue-specific modulators of Na,K-ATPase. In this study, we have identified two splice variants of human FXYD3, or Mat-8, in CaCo-2 cells. Short human FXYD3 has 72% sequence identity with mouse FXYD3, whereas long human FXYD3 is identical to short human FXYD3 but has a 26-amino acid insertion after the transmembrane domain. Short and long human FXYD3 RNAs and proteins are differentially expressed during differentiation of CaCo-2 cells. Long human FXYD3 is mainly expressed in nondifferentiated cells and short human FXYD3 in differentiated cells and both FXYD3 variants can be co-immunoprecipitated with a Na,K-ATPase antibody. In contrast to mouse FXYD3, which has two transmembrane domains for lack of cleavage of the signal peptide, human FXYD3 has a cleavable signal peptide and adopts a type I topology. After co-expression in Xenopus oocytes, both human FXYD3 variants associate stably only with Na,K-ATPase isozymes but not with H,K-ATPase or Ca-ATPase. Similar to mouse FXYD3, short human FXYD3 decreases the apparent K(+) and Na(+) affinity of Na,K-ATPase over a large range of membrane potentials. On the other hand, long human FXYD3 decreases the apparent K(+) affinity only at slightly negative and positive membrane potentials and increases the apparent Na(+) affinity of Na,K-ATPase. Finally, both short and long human FXYD3 induce a hyperpolarization activated current, similar to that induced by mouse FXYD3. Thus, we have characterized two human FXYD3 isoforms that are differentially expressed in differentiated and non-differentiated cells and show different functional properties.     

Multiple forms of mouse vascular endothelial growth factor-D are generated by RNA splicing and proteolysis

The secreted glycoprotein vascular endothelial growth factor-D (VEGF-D) is angiogenic, lymphangiogenic, and promotes metastatic spread of tumor cells via lymphatic vessels. VEGF-D consists of a receptor-binding domain (VEGF homology domain) and N- and C-terminal propeptides. Proteolytic processing produces numerous forms of human VEGF-D, including fully processed derivatives (containing only the VEGF homology domain), partially processed, and unprocessed derivatives. Proteolysis is essential to generate human VEGF-D that binds the angiogenic receptor VEGF receptor-2 (VEGFR-2) and the lymphangiogenic receptor VEGFR-3 with high affinity. Here, we report that alternative use of an RNA splice donor site in exon 6 of the mouse VEGF-D gene produces two different protein isoforms, VEGF-D(358) and VEGF-D(326), with distinct C termini. The two isoforms were both expressed in all adult mouse tissues and embryonic stages of development analyzed. Both isoforms are proteolytically processed in a similar fashion to human VEGF-D to generate a range of secreted derivatives and bind and cross-link VEGFR-3 with similar potency. The isoforms are differently glycosylated when expressed in vitro. This study demonstrates that RNA splicing, protein glycosylation, and proteolysis are mechanisms for generating structural diversity of mouse VEGF-D.     

Expression of functionally different dectin-1 isoforms by murine macrophages

Dectin-1 is a specific receptor for beta-glucans and a major receptor for fungal particles on macrophages (Mphi). It is a type II membrane receptor that has a C-terminal, NK-like, C-type lectin-like domain separated from the cell membrane by a short stalk region and a cytoplasmic immunoreceptor tyrosine-based activation-like motif. We observed functional differences in dectin-1-dependent recognition of fungal particles by Mphi from different mouse strains. RT-PCR analysis revealed that mice have at least two splice forms of dectin-1, generated by differential usage of exon 3, encoding the full-length dectin-1A and a stalkless Mphi dectin-1B. Mphi from BALB/c mice and genetically related mice expressed both isoforms in similar amounts, whereas Mphi from C57BL/6 and related mice mainly expressed the smaller isoform. NIH-3T3 fibroblast and RAW264.7 macrophage cell lines stably expressing either isoform were able to bind and phagocytose zymosan at 37 degrees C. However, binding by the smaller dectin-1B isoform was significantly affected at lower temperatures. These properties were shared by the equivalent human isoforms. The relative ability of each of the isoforms to induce TNF-alpha production in RAW264.7 Mphi was also found to be different. These results are the first evidence that dectin-1 isoforms are functionally distinct and indicate that differential isoform usage may represent a mechanism of regulating cellular responses to beta-glucans.     

Characterization and in vivo functional analysis of splice variants of cypher

Previously, we reported two splice variants of Cypher, a striated muscle-specific PDZLIM domain protein, Cypher1 and Cypher2. We have now characterized four additional splice isoforms, two of which are novel. The six isoforms can be divided into skeletal or cardiac specific classes, based on the inclusion of skeletal or cardiac specific domains. Short and long isoforms share an N-terminal PDZ domain, but the three C-terminal LIM domains are unique to long isoforms. By RNA and protein analysis, we have demonstrated that Cypher isoforms are developmentally regulated in both skeletal and cardiac muscle. We have previously shown that knockout of Cypher is neonatal lethal. To investigate the function of splice variants in vivo, we have performed a rescue experiment of the Cypher null mutant by replacing the endogenous Cypher gene with cDNAs encoding either a short or long skeletal muscle isoform. In contrast to Cypher null mice, a percentage of mice that express only a short or a long skeletal muscle-specific isoform can survive to at least 1 year of age. Although surviving mice exhibit muscle pathology, these results suggest that either isoform is sufficient to rescue the lethality associated with the absence of Cypher.     

Genomic organization and tissue-specific expression of splice variants of mouse organic anion transporting polypeptide 2

cDNAs that code for mouse organic anion transporting polypeptide 2 (oatp2) have been cloned. At least three forms of mouse oatp2 cDNAs containing the same coding sequence were isolated. The common coding sequence is for a protein of 670 amino acids with 12 putative transmembrane domains. The deduced amino acid sequence of the mouse oatp2 shares 89% identity with the reported rat oatp2. Cloning and analysis of mouse oatp2 gene indicates that these isoforms are alternatively spliced products from the same gene. Heterogeneity was observed in the 5'-untranslated region of the cDNAs. Two of the three isoforms lacked the noncoding exon 3 sequence. Northern-blot hybridization analysis using the exon 3-specific probes demonstrated that mouse oatp2 mRNA containing exon 3 sequence is expressed in heart and lung, whereas exon 1-, 2-, and 17-specific probes detected mRNA only in brain and liver. The mouse oatp2 gene consists of 17 exons, including three noncoding exons, and 16 introns. All of the introns are flanked by GT-AG splice sequences except for intron 10 that is flanked by GC-AG splice sequence.     

OPA1 processing controls mitochondrial fusion and is regulated by mRNA splicing, membrane potential, and Yme1L

OPA1, a dynamin-related guanosine triphosphatase mutated in dominant optic atrophy, is required for the fusion of mitochondria. Proteolytic cleavage by the mitochondrial processing peptidase generates long isoforms from eight messenger RNA (mRNA) splice forms, whereas further cleavages at protease sites S1 and S2 generate short forms. Using OPA1-null cells, we developed a cellular system to study how individual OPA1 splice forms function in mitochondrial fusion. Only mRNA splice forms that generate a long isoform in addition to one or more short isoforms support substantial mitochondrial fusion activity. On their own, long and short OPA1 isoforms have little activity, but, when coexpressed, they functionally complement each other. Loss of mitochondrial membrane potential destabilizes the long isoforms and enhances the cleavage of OPA1 at S1 but not S2. Cleavage at S2 is regulated by the i-AAA protease Yme1L. Our results suggest that mammalian cells have multiple pathways to control mitochondrial fusion through regulation of the spectrum of OPA1 isoforms.     

Dynamic expression of alternate splice forms of D-cbl during embryogenesis

The Cbl family of proteins act as E3 ubiquitin-protein ligases and have been associated with the down regulation of a variety of receptor tyrosine kinases. Cbl proteins associate with many different cell signalling molecules suggesting that they may have functions outside of the RING finger-mediated ubiquitin ligase activity. The Drosophila melanogaster cbl gene (D-cbl) encodes two splice forms (Oncogene 19 (2000) 3299). Here we report on the differential expression of these isoforms during Drosophila embryogenesis. Both isoforms are maternally expressed but the long isoform of D-cbl is also transiently expressed in the invaginating mesoderm and later is specifically expressed in neurons of the central nervous system (CNS). Cbl protein is shown to be localised to axons of the longitudinal connectives and commissures in the central nervous system.