Extracellular ionic locks determine variation in constitutive activity and ligand potency between species orthologs of the free fatty acid receptors FFA2 and FFA3.
Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency such that acetate (C2) has been described as FFA2-selective, whereas propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs, it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations, marked variation in ligand-independent constitutive activity was identified using a [(35)S]GTPγS assay. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity in this assay, whereas the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the second extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity and in most cases also yielded corresponding changes in SCFA potency.
Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
Brian D
BD
Hudson
Irina G
IG
Tikhonova
Sunil K
SK
Pandey
Trond
T
Ulven
Graeme
G
Milligan
D000595:Amino Acid Sequence; D000818:Animals; D020148:Butyric Acid / Q000737:chemistry; D005227:Fatty Acids / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007477:Ions; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D020148:Butyric Acid; D005227:Fatty Acids; D019204:GTP-Binding Proteins; D007477:Ions; D008024:Ligands; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C473103:FFAR2 protein, human; C106262:FFAR3 protein, human; C518630:Ffar2 protein, rat; C506482:Gpcr41 protein, mouse; C520305:Gper1 protein, rat; C506481:Ffar2 protein, mouse