Interactions among key residues regulate mammalian odorant receptor trafficking.

Odorant receptors (ORs) expressed in mammalian olfactory sensory neurons are essential for the sense of smell. However, structure-function studies of many ORs are hampered by unsuccessful heterologous expression. To understand and eventually overcome this bottleneck, we performed heterologous expression and functional assays of over 80 OR variants and chimeras. Combined with literature data and machine learning, we found that the transmembrane domain 4 (TM4) and its interactions with neighbor residues are important for OR functional expression. The data highlight critical roles of T4.62 therein. ORs that fail to reach the cell membrane can be rescued by modifications in TM4. Consequently, such modifications in MOR256-3 (Olfr124) also alter OR responses to odorants. T1614.62 P causes the retention of MOR256-3 in the endoplasmic reticulum (ER), while T1614.62 P/T1484.49 A reverses the retention and makes receptor trafficking to cell membrane. This study offers new clues toward wide-range functional studies of mammalian ORs.

Responsiveness of G protein-coupled odorant receptors is partially attributed to the activation mechanism.

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.

Conserved Residues Control Activation of Mammalian G Protein-Coupled Odorant Receptors.

Odorant receptor (OR) genes and proteins represent more than 2% of our genome and 4% of our proteome and constitute the largest subgroup of G protein-coupled receptors (GPCRs). The mechanism underlying OR activation remains poorly understood, as they do not share some of the highly conserved motifs critical for activation of non-olfactory GPCRs. By combining site-directed mutagenesis, heterologous expression, and molecular dynamics simulations that capture the conformational change of constitutively active mutants, we tentatively identified crucial residues for the function of these receptors using the mouse MOR256-3 (Olfr124) as a model. The toggle switch for sensing agonists involves a highly conserved tyrosine residue in helix VI. The ionic lock is located between the "DRY" motif in helix III and a positively charged "R/K" residue in helix VI. This study provides an unprecedented model that captures the main mechanisms of odorant receptor activation.