Abstract
Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3′-diindolylmethane. 3,3′-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3′-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine172, located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3′-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3′-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [3H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3′-diindolylmethane and was not affected adversely by mutation of arginine172. These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings.
Highlights
GPR84 is designated officially as an ‘orphan’ G protein-coupled receptor (GPCR)[1]
Limited in detailed characterisation, there are studies consistent with the presence of multiple binding pockets in GPR843,24. We address these issues for the purported medium chain fatty acids (MCFAs)-GPR84 pairing and in so doing demonstrate that, as expected for a free fatty acid receptor, an arginine residue, here located within the 2nd extracellular loop (ECL2) of GPR84, acts as the putative charge partner for MCFAs to allow their recognition by the receptor
We show that a series of other GPR84 agonist ligands with long hydrophobic tails and carboxylate bioisostere head groups[4,25,26] bind the receptor in a manner overlapping with the MCFAs and, as such, these each act as ‘orthosteric’ agonists
Summary
GPR84 is designated officially as an ‘orphan’ G protein-coupled receptor (GPCR)[1]. This terminology indicates that the endogenous ligands that activate the receptor remain unidentified or that suggested ligands are not accepted with unanimity by the research community. Limited in detailed characterisation, there are studies consistent with the presence of multiple binding pockets in GPR843,24 We address these issues for the purported MCFA-GPR84 pairing and in so doing demonstrate that, as expected for a free fatty acid receptor, an arginine residue, here located within the 2nd extracellular loop (ECL2) of GPR84, acts as the putative charge partner for MCFAs to allow their recognition by the receptor. DIM, and certain related molecules produce extensive increases in the measured potency of both MCFAs and other orthosteric agonists and this suggests that endogenously produced molecules acting in a similar way may result in significantly lower concentrations of MCFA being required to activate GPR84 than would have previously been predicted. We demonstrate that a group of GPR84 antagonist ligands[28], able to block signalling by both orthosteric and allosteric agonist ligands, do so non-competitively by binding at a location that is distinct from either of these agonist sites
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