Splicing variants of the orphan G-protein-coupled receptor GPR56 regulate the activity of transcription factors associated with tumorigenesis

Abstract

GPR56 is an orphan G-protein-coupled receptor of the adhesion family involved in brain development. In some cancer cells and tissues, GPR56 is highly expressed and may contribute to tumorigenesis phenotypes such as cell adhesion and metastasis. Although the ligand for GPR56 is unknown, the overexpression of the receptor induces the activity of several transcription factors. We identified five splicing forms of GPR56 by searching the genome database. In this study, we tried to assess the properties of the splicing variants on the activation of the transcription factors. Genome structure of human GPR56 genes was analyzed using the Ensembl genome browser. All splicing variants were constructed using PCR with the GPR56 wild-type gene as template and the appropriate primers and their expression was verified by western blotting. We examined the effect of GPR56 splicing forms on the cellular responses through reporter gene assay with various promoters. We also confirmed the GPR56-mediated transcriptional activity by silencing GPR56 expression through shRNA-mediated RNA interference. We found that the coding sequence of GPR56 consist of 13 exons and alternative splicing occurs in the second and tenth exons. In reporter gene assays, GPR56 overexpression increased the activity of the serum-response element, NFAT, and E2F response elements, whereas this overexpression downregulated c-myc and p53 response element activity. Furthermore, increased promoter activity of the COX2, iNOS, and VEGF genes was observed. Variants 1 and 2 potently enhanced SRE-mediated transcription compared with wild-type GPR56. Variants 3 and 4 hardly affect the activity of the promoters. These results suggested that the splicing of GPR56 may induce differential tumorigenic responses owing to their varied ability to activate transcription factors.