Received for publication June 13, 2008.
Revised July 14, 2008.
Accepted for publication July 15, 2008.

Structural motifs of importance for the constitutive activity of the orphan 7TM receptor EBI2 - Analysis of receptor activation in the absence of an agonist

Abstract

The Epstein-Barr induced receptor 2 (EBI2) is a lymphocyte-expressed orphan 7TM receptor, that signals constitutively through Gi as shown for instance by GTPS incorporation. Two regions of importance for the constitutive activity were identified by a systematic mutational analysis of 29 residues in EBI2. The CREB-transcription factor was used as a measure of receptor activity, and was correlated to the receptor surface expression. PheVI:13 (Phe²⁵⁷), and the neighbouring CysVI:12 (Cys²⁵⁶), in the conserved CW/FxP motif in TM-6, acted as negative regulators as Ala substitutions at these positions increased the constitutive activity 5.7 and 2.3 fold, respectively, compared to EBI2 wildtype (wt). In contrast, ArgII:20 (Arg⁸⁷) in TM-2 acted as a positive regulator, as substitution to Ala, but not to Lys, decreased the constitutive activity more than 7 fold compared to wt EBI2. IleIII:03 (Ile¹⁰⁶) is located only 4Å from ArgII:20, and a favourable electrostatic interaction with ArgII:20 was created by introduction of Glu in III:03 as the activity increased to 4.4 fold of wt EBI2. Importantly, swapping these charges by introduction of Glu in II:20 and Arg in III:03, resulted in a 2.7-fold increase compared to wt EBI2, and thereby rescued the two signaling-deficient single-mutations, that exhibited a 3.8- to 4.5-fold decrease in constitutive activity. The uncovering of these molecular mechanisms for EBI2 activation is important from a drug development point-of-view, as it may facilitate the rational design and development of small-molecule inverse agonists against EBI2 of putative importance as antiviral- or immune modulatory therapy.

Key words: Orphan, Gi family, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, Mutagenesis/Chimeric approaches