TY - JOUR

T1 - Structural motifs of importance for the constitutive activity of the orphan 7TM receptor EBI2: analysis of receptor activation in the absence of an agonist

AU - Benned-Jensen, Tau

AU - Rosenkilde, Mette M

N1 - Keywords: Alanine; Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Amino Acids, Aromatic; Cell Line; Cell Membrane; Conserved Sequence; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Fluorescent Antibody Technique, Indirect; Forskolin; Genes, Reporter; Green Fluorescent Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Herpesvirus 4, Human; Humans; Hydrogen Bonding; Kidney; Luciferases; Lymphocytes; Models, Molecular; Molecular Sequence Data; Protein Structure, Secondary; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Signal Transduction; Static Electricity; Transfection

PY - 2008

Y1 - 2008

N2 - The Epstein-Barr induced receptor 2 (EBI2) is a lymphocyte-expressed orphan seven transmembrane-spanning (7TM) receptor that signals constitutively through Galphai, 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 A 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.

AB - The Epstein-Barr induced receptor 2 (EBI2) is a lymphocyte-expressed orphan seven transmembrane-spanning (7TM) receptor that signals constitutively through Galphai, 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 A 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.

U2 - 10.1124/mol.108.049676

DO - 10.1124/mol.108.049676

M3 - Journal article

C2 - 18628402

VL - 74

SP - 1008

EP - 1021

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 4

ER -