title: Novel splice variants of CXCR4 identified by transcriptome sequencing.
abstract: Chemokine receptor CXCR4 is involved in tumor growth, angiogenesis and metastasis. Its function is regulated in many ways and one of them is alternative splicing. We identified two novel coding splice variants (CXCR4-3 and CXCR4-4) of CXCR4 in Ewing sarcoma (EWS) cell lines by whole transcriptome sequencing and validated these with reverse transcriptase- PCR and Sanger sequencing. The novel splice variants were expressed at RNA level in Ewing sarcoma samples and in other tumor cell lines and placenta, but not in lung. Due to inclusion of an additional exon the new isoforms have a 70 and 33 amino acid elongation of the N-terminal end of CXCR4. For validation at protein and functional level, the identified isoforms and normal CXCR4 were cloned into an EYFP tagged vector and ectopically expressed in HEK293T cell line and EWS cell line A673. Of the novel isoforms CXCR4-3 showed cell membrane localization and a functional response after addition of CXCR4 ligand CXCL12a. CXCR4-4 showed strong cytoplasmic accumulation and no response to ligand treatment. The role of the newly discovered isoforms in CXCR4 signaling is likely to be limited. Our data stresses the importance of functional validation of newly identified isoforms.
authors: Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
authors: L G L
authors: LG
authors: Sand
authors: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors: A G
authors: AG
authors: Jochemsen
authors: Huygens-Kamerlingh Onnes Laboratory, Leiden Institute of Physics, Leiden University, Leiden, The Netherlands.
authors: E
authors: E
authors: Beletkaia
authors: Huygens-Kamerlingh Onnes Laboratory, Leiden Institute of Physics, Leiden University, Leiden, The Netherlands.
authors: T
authors: T
authors: Schmidt
authors: Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
authors: P C W
authors: PC
authors: Hogendoorn
authors: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: k.szuhai@lumc.nl.
authors: K
authors: K
authors: Szuhai
mesh_terms: D001859:Bone Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling; D057809:HEK293 Cells; D006801:Humans; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D019718:Receptors, CXCR4 / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D012512:Sarcoma, Ewing / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D059467:Transcriptome
chemical_list: D020033:Protein Isoforms; D019718:Receptors, CXCR4; D002118:Calcium
keywords: Bone tumors; CXCR4; Ewing sarcoma; Next generation sequencing; Soft tissue tumor; Splicing