Abstract: | Mammals detect and discriminate numerous odors via a large family of G protein-coupled odorant receptors (ORs). However, little is known about the molecular and structural basis underlying OR response properties. Using site-directed mutagenesis and computational modeling, we studied ORs sharing high sequence homology but with different response properties. When tested in heterologous cells by diverse odorants, MOR256-3 responded broadly to many odorants, whereas MOR256-8 responded weakly to a few odorants. Out of 36 mutant MOR256-3 ORs, the majority altered the responses to different odorants in a similar manner and the overall response of an OR was positively correlated with its basal activity, an indication of ligand-independent receptor activation. Strikingly, a single mutation in MOR256-8 was sufficient to confer both high basal activity and broad responsiveness to this receptor. These results suggest that broad responsiveness of an OR is at least partially attributed to its activation likelihood.G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins which play essential roles in converting extracellular stimuli into intracellular signals in a variety of cell types. Odor detection by olfactory sensory neurons (OSNs) in the mammalian nose depends on a large family of G protein-coupled odorant receptors (ORs) (1), which endows the olfactory system with an extraordinary power of odor detection and discrimination. Although OR-ligand binding is the first step toward smell perception, little is known about the molecular and structural basis underlying odor response properties of individual ORs.Most mammalian ORs respond to a small fraction of all of the tested odorants (2). In contrast, recent studies have identified a small number of ORs that respond to a large set of diverse odorants with comparable potency and efficacy as the former. Curiously, several broadly responsive ORs including MOR256-3 (Olfr124 or SR1), MOR256-31 (Olfr263), and human OR2W1 (ortholog of MOR256-31) belong to the same subfamily, which also contains ORs such as MOR256-8 (Olfr1362) and MOR256-22 (Olfr1387) that respond to a few odorants (3–6). Identification of ORs within the same subfamily (i.e., sharing >50% amino acid identity) but with different response properties offers an opportunity for dissecting out the molecular features that define the tuning properties of these ORs.Mammalian ORs belong to class A (or rhodopsin family) GPCRs. The structure-function relationship of several class A members (e.g., rhodopsin and β2-adrenergic receptor) has been investigated in great details via various approaches including site-directed mutagenesis, X-ray crystallography (7, 8), and molecular modeling (9–11). Although no crystal structure is available for any OR, site-directed mutagenesis and/or computational modeling have shed light on structure-function relationship for a few ORs (12–18).Using a joint approach of site-directed mutagenesis and computational modeling, we investigated the response properties of mutant ORs based on MOR256-3 and MOR256-8, which responded to a large and small set of odorants, respectively. Three-dimensional atomic models of these ORs were built to map locations of the mutated residues. Most mutations in MOR256-3 altered the responses to different odorants in a similar manner. Remarkably, MOR256-8 was converted into a broadly responsive OR by swapping a single or a few residues. More generally, we found that an OR’s total response was positively correlated with its basal activity, an indication of ligand-independent receptor activation. These data suggest that broad responsiveness of an OR is not only determined by ligand binding, but also by activation mechanism. |