Identification of two novel alternatively spliced Neuropilin-1 isoforms

Neuropilin-1 (NRP1) is a coreceptor to a tyrosine kinase receptor for both the vascular endothelial growth factor (VEGF) family and semaphorin (Sema) family members. NRP1 plays versatile roles in angiogenesis, axon guidance, cell survival, migration, and invasion. NRP1 contains three distinct extracellular domains, a1a2, b1b2, and c. We report here the identification of two novel soluble human NRP1 isoforms, which we named sIIINRP1 and sIVNRP1. These soluble NRP1 isoforms were generated by alternative splicing of the NRP1 gene, a common regulatory mechanism occurring in cell surface receptor families. Both sIIINRP1 and sIVNRP1 contain a1a2 and b1b2 domains, but no c domain, and the rest of the NRP1 sequence. Additionally, sIIINRP1 is missing 48 amino acids within the C-terminus of the b2 domain. Both sIIINRP1 and sIVNRP1 are expressed in human cancerous and normal tissues. These molecules are capable of binding to VEGF165 and Sema3A. Furthermore, recombinant sIIINRP1 and sIVNRP1 proteins inhibit NRP1-mediated MDA-MB-231 breast cancer cell migration. These results indicate the multiple levels of regulation in NRP1 function and suggest that these two novel NRP1 isoforms are useful antagonists for NRP1-mediated cellular activities.     

Molecular cloning and functional characterization of two alternatively spliced Ntcp isoforms from mouse liver1

To isolate the murine Na+/taurocholate cotransporting polypeptide (Ntcp), we screened a mouse liver cDNA library and identified Ntcp1, encoding a 362 amino acid protein and Ntcp2, encoding a 317 amino acid protein which had a shorter C-terminal end. Both isoforms mediated saturable Na+-dependent transport of taurocholate when expressed in Xenopus laevis oocytes. Analysis of the gene revealed that Ntcp2 is produced by alternative splicing where the last intron is retained.     

Expression and differential intracellular localization of two major forms of human 8-oxoguanine DNA glycosylase encoded by alternatively spliced OGG1 mRNAs

We identified seven alternatively spliced forms of human 8-oxoguanine DNA glycosylase (OGG1) mRNAs, classified into two types based on their last exons (type 1 with exon 7: 1a and 1b; type 2 with exon 8: 2a to 2e). Types 1a and 2a mRNAs are major in human tissues. Seven mRNAs are expected to encode different polypeptides (OGG1-1a to 2e) that share their N terminus with the common mitochondrial targeting signal, and each possesses a unique C terminus. A 36-kDa polypeptide, corresponding to OGG1-1a recognized only by antibodies against the region containing helix-hairpin-helix-PVD motif, was copurified from the nuclear extract with an activity introducing a nick into DNA containing 8-oxoguanine. A 40-kDa polypeptide corresponding to a processed form of OGG1-2a was detected in their mitochondria using antibodies against its C terminus. Electron microscopic immunocytochemistry and subfractionation of the mitochondria revealed that OGG1-2a locates on the inner membrane of mitochondria. Deletion mutant analyses revealed that the unique C terminus of OGG1-2a and its mitochondrial targeting signal are essential for mitochondrial localization and that nuclear localization of OGG1-1a depends on the NLS at its C terminus.     

Identification and characterization of a spliced C-type lectin-like gene encoded by rat cytomegalovirus

The English isolate of rat cytomegalovirus (RCMV) encodes a 20-kDa protein with a C-type lectin-like domain that is expressed in the delayed-early and late phases of the viral replication cycle. Genomic sequence analysis of the restriction fragment KpnR of RCMV revealed significant homology to several C-type lectin-containing molecules implicated in natural killer (NK) and T-cell interactions, as well as genes from four poxviruses and African swine fever virus. The gene is spliced into five exons and shows a splicing pattern with exon boundaries similar to those observed in the human differentiation antigen CD69. The cap site of the gene was mapped by RNase protection, 5' rapid amplification of cDNA ends, and primer extension experiments. This analysis demonstrated that the core promoter of the RCMV lectin-like gene contains a GATA rather than a TATA box. Splicing patterns were confirmed with isolates from an infected-cell cDNA library. A unique aspect of the protein is that its translation is not initiated by the canonical methionine but rather by alanine. To study its role in virus replication and pathogenesis, a recombinant virus was constructed in which the gene is interrupted. Replication in tissue culture was similar to that of wild-type virus.     

Retroviral expression of alternatively spliced forms of rat fibronectin

We describe the construction in retroviral vectors and the expression of recombinant rat fibronectin (FN) cDNAs corresponding with the various alternatively spliced forms of FN. In NIH 3T3 cells, the exogenous rat FN subunits are efficiently secreted as heterodimers with endogenous mouse subunits. In contrast, in lymphoid WEHI231 cells, there is no endogenous FN synthesis and the recombinant FNs are secreted and can be purified as homogeneous proteins. We show that the purified recombinant FNs are biochemically and biologically functional. In basic assays for adhesion, spreading, cytoskeletal organization, and migration using various established adherent cell lines, different forms of FNs containing the different alternatively spliced segments show no marked differences in activity. We have used these recombinant FNs to investigate three systems in which earlier results had suggested potential differences between different forms of FN. First, all forms tested appear equally active in restoring normal morphology to a transformed cell line. Second, we detect minor differences in their ability to assemble into preexisting extracellular matrices. Finally, we report that only those forms of FN that contain the V segment will promote the spreading of a lymphoid cell line indicating that this segment confers additional biological functions for some cell types, a result that confirms and extends earlier data. These homogeneous, biologically active recombinant FNs will allow further studies of the role of the alternatively spliced segments of FN.     

Identification of a novel alternatively spliced septin.

Septins are a family of cytoskeletal proteins involved in cytokinesis, targeting of proteins to specific sites on the plasma membrane, and cellular morphogenesis. While many aspects of their function in cytokinesis in yeast cells have been investigated, the function of septins in mammalian cells is less well understood. For example, septins are present in post-mitotic neurons, suggesting they have other roles in, for example, establishing cell polarity. The full extent of the septin gene family is not known in mammalian cells. To better understand the septin gene family, we have cloned and characterized a novel mammalian septin.