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. 2009 Feb 6;284(6):3529-36.
doi: 10.1074/jbc.M806987200. Epub 2008 Dec 8.

Two arginine-glutamate ionic locks near the extracellular surface of FFAR1 gate receptor activation

Chi Shing Sum  1 , Irina G Tikhonova, Stefano Costanzi, Marvin C Gershengorn
Affiliations

Two arginine-glutamate ionic locks near the extracellular surface of FFAR1 gate receptor activation

Chi Shing Sum et al. J Biol Chem. .

Abstract

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.

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Figures

FIGURE 1.
FIGURE 1.
Sequence alignment of FFAR1, rhodopsin, and β2-adrenergic receptor. The most conserved residues in each transmembrane domain are labeled as X.50, according to the Ballesteros and Weinstein nomenclature (17). Symbols indicate positions of residues believed to be involved in the rotamer toggle switch (#) and the ionic lock (*) between the cytosolic ends of TM3 and TM6. Residues forming ionic locks between the TM helical bundle and ECL2 of FFAR1 are colored in red and blue.
FIGURE 2.
FIGURE 2.
Molecular models of the unoccupied FFAR1 and FFAR1 complexed with linoleate or GW9508. The salt bridges between amino acid residues Glu-145(ECL2) and Arg-183(5.39) and Glu-172(ECL2) and Arg-258(7.35) are identified in unoccupied FFAR1 (A) and in FFAR1 complexed with linoleate (B) or GW9508 (C).
FIGURE 3.
FIGURE 3.
Non-bonded interaction energies (van-der-Waals and electrostatic components) between two putative ionic locks. The interaction energies between Glu-145(ECL2) and Arg-183(5.39) (A) and Glu-172 and Arg-258(7.35) (B) for the unoccupied FFAR1 (black) and FFAR1 in complex with linoleate (red) or GW9508 (green) were computed along 3.5-ns trajectories. The changes in energies are expressed as percent deviation from the average energy detected for the unoccupied receptor.
FIGURE 4.
FIGURE 4.
Signaling activities of FFAR1 mutants. A, agonist-stimulated signaling was determined by measuring increases in [Ca2+]i in response to increasing doses of linoleate as described under “Materials and Methods.” RFU represents the relative fluorescence units of the Calcium4 reagent. Data shown are mean ± S.E. of quadruplicate samples in two experiments. B, levels of constitutive activities were determined in CHO-K1 cells by co-transfection of the receptor plasmid and an Elk1-Gal4 trans-activator element and a luciferase reporter as described under “Materials and Methods.” Data shown are mean ± S.E. after normalizing the luminescence levels relative to wild-type receptor. One-way analysis of variance performed indicated significant differences among the groups. Subsequently, Newman-Keuls multiple comparison test was performed to determine whether luciferase activity was statistically different from the lacZ (#) or wild-type group (*). ##, p < 0.001; #, p < 0.01; **, p < 0.001; *, p < 0.05.
FIGURE 5.
FIGURE 5.
Increased constitutive activities of E172A (A) and E145A (B) FFAR1 mutants. The levels of constitutive activity were determined as described under “Materials and Methods.” The amount of receptor plasmid was titrated at a range of 0–48 ng by varying the amount of receptor plasmid while keeping the total amount of DNA transfected constant by supplementing the mixture with lacZ control plasmid. Data shown are means ± S.E. after normalizing the data relative to the value obtained with zero receptor plasmid in the wild-type data set.
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