Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs.
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 pEC(50) values for inhibition of cAMP, 5.83 ± 0.11; Ca(2+) 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.
Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, UK.
Brian D
BD
Hudson
Elisabeth
E
Christiansen
Irina G
IG
Tikhonova
Manuel
M
Grundmann
Evi
E
Kostenis
David R
DR
Adams
Trond
T
Ulven
Graeme
G
Milligan
D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship; D000071557:beta-Arrestins
D019390:Arrestins; D048049:Extracellular Signal-Regulated MAP Kinases; D005227:Fatty Acids; D008024:Ligands; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins; C473103:FFAR2 protein, human