All possible pairs of IRs resulted in either no or very weak localization of IR25a to cilia and basal phenylethyl amine responses (Figures 8A–8C). By contrast, upon coexpression of all three IRs, we observed consistent localization of IR25a to sensory cilia and robust concentration-dependent odor-evoked responses (Figures 8A–8C). Moreover, the magnitude of these responses was highly comparable to phenylethyl amine-evoked activity in endogenous ac4 sensilla neurons (Figure 8D). These results ATM/ATR assay reveal a thus far unique case, where three distinct subunits form a functional olfactory receptor. Chemosensory synapses” between

the environment and sensory neurons have been proposed as novel models to characterize mechanisms of neuronal activation and regulation by external stimuli (Shaham, 2010). The IRs provide an intriguing example of molecular homology between peripheral sensory and postsynaptic receptors, and motivated us to define the conserved and divergent properties of these olfactory receptors compared with their iGluR

ancestors. Cross-species analyses have demonstrated that IR25a is the “ancestral” INCB018424 mouse IR, as orthologs of this gene are expressed in chemosensory neurons in insects, nematode worms, and mollusks (Croset et al., 2010). By contrast, IR8a is a recently evolved, insect-specific duplicate of IR25a, although it retains a similar domain organization and sequence identity to iGluRs (Croset

et al., 2010). The chemosensory role of IR25a in the common protostome ancestor is unknown, but it is attractive to suggest that it initially retained function as a glutamate-sensing receptor in the distal dendritic membranes of peripheral sensory neurons, analogous to the role of iGluRs in postsynaptic membranes of interneurons. Subsequent expansion of the IR repertoire may have allowed specialization of IR8a and IR25a as coreceptors acting in conjunction with more divergent odor-specific IRs. The dedication of these relatively slowly evolving members of the IR repertoire as a structural core of heteromeric IR complexes may help maintain the central function of these receptors as ligand-gated cation channels. Our analysis of IR8a suggests that one specific function of the coreceptors Amine dehydrogenase may be to link IR complexes to the cilia transport pathway through their intracellular cytoplasmic tail, similar to the role of this region in coupling iGluRs to the postsynaptic transport machinery (Groc and Choquet, 2006). Conserved motifs for subcellular targeting are not apparent between iGluRs and IR8a or IR25a (data not shown), perhaps reflecting the novel signals required to localize IRs to specialized sensory cilia membranes. The maintenance of LBDs in coreceptor IRs raises the possibility that these proteins still bind ligands.