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Structural Motifs of Importance for the Constitutive Activity of the Orphan 7TM Receptor EBI2: Analysis of Receptor Activation in the Absence of an Agonist

Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, the Panum Institute, Copenhagen University, Copenhagen, Denmark.

The Epstein-Barr induced receptor 2 (EBI2) is a lymphocyte-expressed orphan seven transmembrane-spanning (7TM) receptor that signals constitutively through G_i, as shown, for instance by guanosine 5'-O-(3-thio)triphosphate incorporation. Two regions of importance for the constitutive activity were identified by a systematic mutational analysis of 29 residues in EBI2. The cAMP response element-binding protein transcription factor was used as a measure of receptor activity and was correlated to the receptor surface expression. PheVI:13 (Phe²⁵⁷), and the neighboring 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 with EBI2 wild type (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 with wt EBI2. IleIII:03 (Ile¹⁰⁶) is located only 4 Å from ArgII:20, and a favorable electrostatic interaction with ArgII:20 was created by introduction of Glu in III:03, given that the activity increased to 4.4-fold of that wt EBI2. It is noteworthy that swapping these charges by introduction of Glu in II:20 and Arg in III:03 resulted in a 2.7-fold increase compared with wt EBI2, thereby rescuing the two signaling-deficient single mutations, which 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, in that it may facilitate the rational design and development of small-molecule inverse agonists against EBI2 of putative importance as antiviral- or immune modulatory therapy.

Address correspondence to: Mette M. Rosenkilde, Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, The Panum Institute, Copenhagen University, Blegdamsvej 2, 2200 Copenhagen, Denmark. E-mail: rosenkilde{at}sund.ku.dk