Structural motifs of importance for the constitutive activity of the orphan 7TM receptor EBI2: analysis of receptor activation in the absence of an agonist.
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.
Benned-Jensen
Tau
T
Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, The Panum Institute, Copenhagen University, Blegdamsvej 2, 2200 Copenhagen, Denmark.
Rosenkilde
Mette M
MM
D000409:Alanine / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D024322:Amino Acids, Aromatic / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000032:analysis / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007668:Kidney / Q000166:cytology; D008156:Luciferases / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction; D055672:Static Electricity; D014162:Transfection
D024322:Amino Acids, Aromatic; D017362:Cyclic AMP Response Element-Binding Protein; C080105:GPR183 protein, human; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D008156:Luciferases; D000409:Alanine