Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

• Published in: The FASEB journal Vol. 26; no. 12; pp. 4951 - 4965
• Main Authors: Hudson, Brian D., Christiansen, Elisabeth, Tikhonova, Irina G., Grundmann, Manuel, Kostenis, Evi, Adams, David R., Ulven, Trond, Milligan, Graeme
• Format: Journal Article
• Language: English
• Published: United States The Federation of American Societies for Experimental Biology 01.12.2012
• Subjects: Amino Acid Sequence; Animals; Arrestins - genetics; Arrestins - metabolism; beta-Arrestins; Binding Sites - genetics; Cattle; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases - metabolism; Fatty Acids - chemistry; Fatty Acids - metabolism; Fatty Acids - pharmacology; GPCR; HEK293 Cells; Humans; Ligands; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutation; Phosphorylation - drug effects; Protein Binding - drug effects; Protein Structure, Tertiary; RASSL; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Sequence Homology, Amino Acid; Species Specificity; Structure-Activity Relationship
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fj.12-213314

When it is difficult to develop selective ligands within a family of related G‐protein‐coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling and mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2‐RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2‐RASSL displayed a >100‐fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pEC50 values for inhibition of cAMP, 5.83 ± 0.11; Ca2+ mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 ± 0.06. This FFA2‐RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.—Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs. FASEB J. 26, 4951–4965 (2012). www.fasebj.org