Constitutive activity of a spermine receptor is maintained by a single site in the C-terminal

Olfactory receptors are G-protein coupled receptors (GPCRs) that enable olfactory epithelia to detect odorants. These GPCRs may also show constitutive activity, which play important roles in the development and responses of odorant receptor neurons. However, little is known about the molecular characteristics that support the constitutive activities in olfactory receptors. Here, we characterize a pair of olfactory receptor orthologs that show similar ligand-dependent activity but different levels of constitutive activity, and elucidate the molecular characteristics that maintain the constitutive activity. Previously, PmTAAR348, a sea lamprey (Petromyzon marinus) olfactory receptor that is activated by the male sex pheromone spermine has been reported. In this study, we identified LmTAAR348 of Northeast Chinese lamprey (Lethenteron morii) as an ortholog of PmTAAR348. When expressed in HEK293T cell lines, both receptors showed similar levels of activation when exposed to spermine. However, the constitutive activity of LmTAAR348 was 100-fold higher than that of PmTAAR348. Using site-directed mutagenesis, we screened all 13 amino acid residues (aa) that differed between the two orthologs and found that a switch in position 340 reversed the constitutive activity levels between LmTAAR348 and PmTAAR348. Mutating the remaining 12 aa did not affect the ligand-dependent or constitutive activation. Moreover, both the ligand-dependent and constitutive activation of TAAR348 are G_olf (G protein ⍺ subunit olfactory type) independent. We conclude that a single aa in the C-terminal maintains the constitutive activity in a spermine receptor.