Molecular Pharmacological Phenotyping of EBI2: AN ORPHAN SEVEN-TRANSMEMBRANE RECEPTOR WITH CONSTITUTIVE ACTIVITY

Mette M Rosenkilde,Tau Benned-Jensen,Helene Andersen,Peter J Holst,Thomas N Kledal,Hans R Lüttichau,Jørgen K Larsen,Jan P Christensen,Thue W Schwartz

doi:10.1074/jbc.m602245200

Abstract

Epstein-Barr virus (EBV)-induced receptor 2 (EBI2) is an orphan seven-transmembrane (7TM) receptor originally identified as the most up-regulated gene (>200-fold) in EBV-infected cells. Here we show that EBI2 signals with constitutive activity through Galpha(i) as determined by a receptor-mediated inhibition of forskolin-induced cAMP production and an induction of the serum response element-driven transcriptional activity in a pertussis toxin-sensitive manner. Galpha(s) and Galpha(q) were not activated constitutively as determined by the lack of cAMP production, the lack of inositol phosphate turnover, and the lack of activities of the transcription factors: cAMP response element-binding protein and nuclear factor-kappaB. Immunohistochemistry and confocal microscopy of FLAG- and green fluorescent protein-tagged EBI2 revealed cell-surface expression. A putative N-terminal truncated version of EBI2, delta4-EBI2, showed similar expression and signaling through Galpha(i) as full-length EBI2. By using a 32P-labeled EBI2 probe we found a very high expression in lymphoid tissue (spleen and lymph node) and peripheral blood mononuclear cells and a high expression in lung tissue. Real-time PCR of EBV-infected cells showed high expression of EBI2 during latent and lytic infection, in contrast to the EBV-encoded 7TM receptor BILF1, which was induced during lytic infection. EBI2 clustered with the orphan GPR18 by alignment analysis as well as by close proximity in the chromosomal region 13q32.3. Based on the constitutive signaling and cellular expression pattern of EBI2, it is suggested that it may function in conjunction with BILF1 in the reprogramming of the cell during EBV infection.

Highlights

The orphan EBI22 receptor belongs to the superfamily of rhodopsinlike 7TM receptors, known as G-protein-coupled receptors
Analysis of the chromosomal localization of EBI2, GPR18, and the CysL1 and -2 receptors revealed an intimate relationship between EBI2 and GPR18, because both genes were located at chromosome 13 in the region 13q32.3 at the minus strand, with only 32,792 bp separation
EBI2 Is Expressed at the Cell Surface and Signals Constitutively through G␣i in a Pertussis toxin (Ptx)-sensitive Manner—The cellular expression was analyzed in two different cell lines, the laboratory fibroblast HEK293 cell line used for binding and signaling characterization of 7TM receptors in general, and the pre-B-cell lymphocyte cell line L12, chosen due to the original cloning of EBI2 from B-lymphocytes infected with Epstein-Barr virus (EBV)

Summary

Introduction

The orphan EBI22 receptor belongs to the superfamily of rhodopsinlike 7TM receptors (seven-transmembrane segment receptors), known as G-protein-coupled receptors. We show that EBI2 signals with constitutive activity through G␣i as determined by a receptor-mediated inhibition of forskolin-induced cAMP production and an induction of the serum response elementdriven transcriptional activity in a pertussis toxin-sensitive manner.

Results

Conclusion