The Third Extracellular Loop of Mammalian Odorant Receptors Is Involved in Ligand Binding

Abstract

Mammals recognize chemicals in the air via G protein-coupled odorant receptors (ORs). In addition to their orthosteric binding site, other segments of these receptors modulate ligand recognition. Focusing on human hOR1A1, which is considered prototypical of class II ORs, we used a combination of molecular modeling, site-directed mutagenesis, and in vitro functional assays. We showed that the third extracellular loop of ORs (ECL3) contributes to ligand recognition and receptor activation. Indeed, site-directed mutations in ECL3 showed differential effects on the potency and efficacy of both carvones, citronellol, and 2-nonanone.

Keywords: ECL3; functional assays; ligand selectivity; molecular modeling; odorant receptors.

Figure 1

(a) Structure of hOR1A1 from homology modeling (Modeller) compared to that obtained from AlphaFold2 (AlphaFold). In both structures, the third extracellular loop (ECL3) (shown in red) was predicted to be close to the orthosteric binding cavity, shown as a cyan surface. (b) Conservation analysis of ECL3 sequences of both classes of human odorant receptors and the highlight of hOR1A1 specific ECL3 sequence.

Figure 2

Entry of (−)-carvone inside receptor hOR1A1. The ligand is initially located outside the receptor (1). It then migrates to the cradle of the orthosteric binding cavity (2,3), as indicated by Y251^6.48. During this process, the ligand interacts with several residues from ECL3 (indicated in red). Contour map of (−)-carvone migration as the minimum distance from S266 (taken as the distance from ECL3) and minimum distance from Y251^6.48 (taken as the distance from the cradle of the cavity). All replicas were considered. The three highlighted basins show the ligand’s largest density.

Figure 3

Chemical structure of four agonists of hOR1A1.

Figure 4

In vitro data of hOR1A1 and mutant ORs. (a) In vitro dose–response curves of four ligands (−)-carvone, (+)-carvone, citronellol, and 2-nonanone towards wt hOR1A1 and mutant ORs at positions P261, T263, S266, and D269. (*) indicates the response value is significantly different compared to wt hOR1A1 (one-way ANOVA, followed by a Dunnett test; * p < 0.05). (RLU = relative luminescence unit) (b) The fluorescence intensity of the R-phycoerythrin (R-PE) signal of wt hOR1A1 and mutant ORs at positions P261, T263, S266, and D269. (c) Normalized graph of cell-surface expression of ECL3 mutant ORs against wt hOR1A1 (one-way ANOVA, followed by a Dunnett test; * p < 0.05, ** p < 0.01, and *** p < 0.001).