title: Structural and functional characterization of two alternative splicing variants of mouse Endothelial Cell-Specific Chemotaxis Regulator (ECSCR).
abstract: Endothelial cells (ECs) that line the lumen of blood vessels are important players in blood vessel formation, and EC migration is a key component of the angiogenic process. Thus, identification of genes that are specifically or preferentially expressed in vascular ECs and in-depth understanding of their biological functions may lead to discovery of new therapeutic targets. We have previously reported molecular characterization of human endothelial cell-specific molecule 2 (ECSM2)/endothelial cell-specific chemotaxis regulator (ECSCR). In the present study, we cloned two mouse full-length cDNAs by RT-PCR, which encode two putative ECSCR isoform precursors with considerable homology to the human ECSCR. Nucleotide sequence and exon-intron junction analyses suggested that they are alternative splicing variants (ECSCR isoform-1 and -2), differing from each other in the first and second exons. Quantitative RT-PCR results revealed that isoform-2 is the predominant form, which was most abundant in heart, lung, and muscles, and moderately abundant in uterus and testis. In contrast, the expression of isoform-1 seemed to be more enriched in testis. To further explore their potential cellular functions, we expressed GFP- and FLAG-tagged ECSCR isoforms, respectively, in an ECSCR deficient cell line (HEK293). Interestingly, the actual sizes of either ECSCR-GFP or -FLAG fusion proteins detected by immunoblotting are much larger than their predicted sizes, suggesting that both isoforms are glycoproteins. Fluorescence microscopy revealed that both ECSCR isoforms are localized at the cell surface, which is consistent with the structural prediction. Finally, we performed cell migration assays using mouse endothelial MS1 cells overexpressing GFP alone, isoform-1-GFP, and isoform-2-GFP, respectively. Our results showed that both isoforms significantly inhibited vascular epidermal growth factor (VEGF)-induced cell migration. Taken together, we have provided several lines of experimental evidence that two mouse ECSCR splicing variants/isoform precursors exist. They are differentially expressed in a variety of tissue types and likely involved in modulation of vascular EC migration. We have also defined the gene structure of mouse ECSCR using bioinformatics tools, which provides new information towards a better understanding of alternative splicing of ECSCR.
authors.affiliation: Department of Obstetrics and Gynecology, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85004, USA.|Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85013, USA.|Department of Pathogen Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Wu
authors.affiliation: Department of Obstetrics and Gynecology, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85004, USA.
authors.forename: Chunwei
authors.initials: C
authors.lastname: Shi
authors.affiliation: Department of Obstetrics and Gynecology, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85004, USA.
authors.forename: Fanxin
authors.initials: F
authors.lastname: Ma
authors.affiliation: Department of Obstetrics and Gynecology, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85004, USA.
authors.forename: James
authors.initials: J
authors.lastname: Balducci
authors.affiliation: Department of Pathogen Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.
authors.forename: Hanju
authors.initials: H
authors.lastname: Huang
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at Tyler, Tyler, TX 75708, USA.
authors.forename: Hong-Long
authors.initials: HL
authors.lastname: Ji
authors.affiliation: Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85013, USA.
authors.forename: Yongchang
authors.initials: Y
authors.lastname: Chang
authors.affiliation: Department of Obstetrics and Gynecology, St. Joseph's Hospital and Medical Center, Phoenix, AZ 85004, USA.
authors.forename: Yao
authors.initials: Y
authors.lastname: Huang
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D002465:Cell Movement / Q000235:genetics*; D003001:Cloning, Molecular; D042783:Endothelial Cells / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D018919:Neovascularization, Physiologic / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics; D016415:Sequence Alignment; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C587669:Ecscr protein, mouse; D008565:Membrane Proteins; D020033:Protein Isoforms; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D049452:Green Fluorescent Proteins