e to structure via the formation of intra-molecular disulphide bonds, although this remains to be demonstrated experimentally. They also contain an arginine at the bottom of TMD III within the highly conserved Asp–Arg-Tyr RY) domain of class A GPCRs. FFA2 contains a Glu-Arg-Tyr motif in this location, whereas in FFA3 it is a Glu-Arg-Phe motif. The orthosteric binding pocket Both FFA2 and FFA3 respond to short-chain free fatty acids with carbon chain length C1 to C5 . However, the potency of these ligands is low, within the high mM to low mM range. Because of the lack of high affinity ligands that interact with FFA2 and/or FFA3, identification of residues that contribute to the binding of orthosteric ligands in these two GPCRs has so far been inferred PF-562271 price simply from loss of function or impairment of signalling of point mutants of these receptors or from modelling studies. In initial studies designed to explore the contributions of the six amino acids predicted to differ between human FFA3 and the non-functional GPR42 sequence, Brown et al. systematically inter-converted each variant residue. Alteration of Arg174 of FFA3, that is predicted to be located in extracellular loop 2, to 
the corresponding Trp residue of GPR42 generated an FFA3 mutant that was no longer able to respond to the C3 fatty acid propionate. Conversely, introduction of Arg174 into GPR42 rendered the previously unresponsive protein able to generate signals to propionate. Alteration of the residue equivalent to Leu227 of human FFA2 to Val at the bottom of TMD VI in the rat orthologue of FFA3 resulted in constitutive signalling. This region is often implicated in GPCR activation of G proteins. Based on both the lack of function of fatty acid amides at each of FFA1-3 and the loss of function of longer-chain fatty acids at PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19809470 forms of FFA1 in which the Arg residues at 5.39 and 7.35 were mutated, Stoddart et al., aligned human FFA1, FFA2 and FFA3 and noted the conservation of these two residues. Mutation of Arg 5.39 in FFA2 to a range of amino acids, including Lys, resulted in loss of capacity of the C2 and C3 fatty acids acetate and propionate to cause elevation of intracellular Ca2+ in HEK293 cells transfected to express these variants transiently. Because it was unclear in such studies if the mutated forms of FFA2 were able to reach the cell surface efficiently, Stoddart et al. established stable HEK293 cell lines able to express C-terminally enhanced yellow fluorescent protein tagged forms of either wild-type FFA2 or Arg 5.39 Ala FFA2, only upon addition of the antibiotic doxycycline. Imaging of such induced cells demonstrated similar subcellular distribution of the two forms and confirmed that Arg 5.39 Ala FFA2 was unable to respond to either acetate or propionate. These cells had the additional benefit of providing proof that induction of expression of FFA2 was required for the short-chain free fatty acids to elevate intracellular Ca2+ and that this was not a non-specific effect of the ligands. Construction of HEK293 cells able to express Arg 7.35 Ala FFA2-eYFP in an inducible fashion also demonstrated the importance of this residue for the function of acetate and propionate. As noted earlier, FFA3 couples predominantly to pertussis toxin-sensitive G proteins of the Gi/Go subfamily. Generation and inducible expression of Arg 5.39 Ala FFA3-eYFP and Arg 7.35 Ala FFA3-eYFP also demonstrated the importance of these residues in FFA3 as propionate was unable to increase binding of
