Two Arginine-Glutamate Ionic Locks Near the Extracellular Surface of FFAR1 Gate Receptor Activation

Activation of a number of class A G protein-coupled receptors (GPCRs) is thought to involve two molecular switches, a rotamer toggle switch within the transmembrane domain and an ionic lock at the cytoplasmic surface of the receptor; however, the mechanism by which agonist binding changes these molecular interactions is not understood. Importantly, 80% of GPCRs including free fatty acid receptor 1 (FFAR1) lack the complement of amino acid residues implicated in either or both of these two switches; the mechanism of activation of these GPCRs is therefore less clear. By homology modeling, we identified two Glu residues (Glu-145 and Glu-172) in the second extracellular loop of FFAR1 that form putative interactions individually with two transmembrane Arg residues (Arg-183(5.39) and Arg-258(7.35)) to create two ionic locks. Molecular dynamics simulations showed that binding of agonists to FFAR1 leads to breakage of these Glu-Arg interactions. In mutagenesis experiments, breakage of these two putative interactions by substituting Ala for Glu-145 and Glu-172 caused constitutive receptor activation. Our results therefore reveal a molecular switch for receptor activation present on the extracellular surface of FFAR1 that is broken by agonist binding. Similar ionic locks between the transmembrane domains and the extracellular loops may constitute a mechanism common to other class A GPCRs also.G protein-coupled receptors (GPCRs)³ are important components of signal transduction machineries that regulate many physiological processes. They are also important as targets for therapeutic agents; a large percentage of drugs in the marketplace are GPCR ligands or modulators. Knowledge of structure-function relationships of GPCRs has been gained through many pharmacological, biochemical, and biophysical studies, and has been used extensively to enhance the discovery of GPCR ligands that have been developed into therapeutically useful agents (1–3). Knowledge of the molecular details of ligand-receptor interaction and of the mechanism of receptor activation will also likely improve efforts to identify agonists with better potency and efficacy. Tan et al. (3) have recently reported their design of agonists with higher potency and efficacy for the trace amine receptor 1 based on the rotamer toggle switch model of receptor activation that is thought to operate in a number of class A GPCRs. The rotamer toggle switch typically involves the aromatic residues Trp and Phe within transmembrane helix 6 (TMH6) of GPCRs. During agonist-mediated receptor activation or in constitutively active receptors, the dihedral angle on the side chain of these residues is predicted to be rotated compared with the inactive state and thereby triggers a movement of TMH6 away from TMH3 (e.g. Ref. 4). It is also thought that an ionic lock between an Arg residue in TMH3 and a Glu in TMH6 near the cytoplasmic surface of some GPCRs holds the receptor in the inactive conformation and that receptor activation is accompanied by breakage of the ionic bond when agonist binds; the ionic lock may also be broken by receptor mutation (e.g. Ref. 5). Although these models of receptor activation have been proposed for a number of class A GPCRs, it is not certain how generally this hypothesis can be applied across all members of this GPCR class. From the alignment of 372 sequences of human GPCRs, we noted that about 80% of GPCRs do not have the putative residues that play a role in either the rotamer toggle switch, the ionic lock, or both. For these receptors, the interaction responsible for regulating interconversion between inactive and active receptor conformations therefore remains unknown.The free fatty acid receptor 1 (FFAR1) is a G_q-coupled, class A GPCR-activated endogenously by free fatty acids, with a preference for medium-to-long chain fatty acids (C8–12) (reviewed in Ref. 6). The receptor has been suggested to be a potential target for treatment of type 2 diabetes, as offered by the action of agonists to potentiate glucose-stimulated insulin release (reviewed in Refs. 7, 8). Several groups, including ours, have reported the discovery of novel small molecule ligands for FFAR1 (9–13). Most of these compounds were identified by high-throughput screening followed by chemical optimization (10–12). Our group has delineated the ligand-binding pocket of FFAR1 (14, 15) and used the information as a rational approach to ligand discovery by means of virtual screening (13). The mechanism of FFAR1 activation; however, remains unknown especially because this receptor does not contain either the rotamer toggle switch or the ionic lock between TMHs 3 and 6.We have previously identified nine residues in the ligand-binding pocket of FFAR1 that are important for ligand recognition and/or receptor activation (14). In particular, two Arg residues (Arg-183(5.39)⁴ and Arg-258(7.35)) and an Asn residue (Asn-244(6.55)) in the TMHs coordinate the carboxylate head group of the naturally occurring agonist linoleate and the synthetic agonist GW9508. In the present study, by a collaborative effort using computational modeling and receptor mutagenesis, we report the identification of Glu-172 in the second extracellular loop (ECL2) of FFAR1 that may function together with Arg-183(5.39) and Arg-258(7.35) as locks to control activation of the receptor. Our results suggest that these ionic locks at the extracellular surface hold the receptor in an inactive state. Agonists, through interaction with the arginine residues, may break the arginine-glutamate interactions thereby allowing the receptor to adopt an active conformation. Therefore, our results have provided insights into the mechanism of activation of class A GPCRs that function in a manner not explicable by the more well-studied models.