In vivo assembly and trafficking of olfactory Ionotropic Receptors

Abstract

Background Ionotropic receptors (IRs) are a large, divergent subfamily of ionotropic glutamate receptors (iGluRs) that are expressed in diverse peripheral sensory neurons and function in olfaction, taste, hygrosensation and thermosensation. Analogous to the cell biological properties of their synaptic iGluR ancestors, IRs are thought to form heteromeric complexes that localise to the ciliated dendrites of sensory neurons. IR complexes are composed of selectively expressed ‘tuning’ receptors and one of two broadly expressed co-receptors (IR8a or IR25a). While the extracellular ligand-binding domain (LBD) of tuning IRs is likely to define the stimulus specificity of the complex, the role of this domain in co-receptors is unclear. Results We identify a sequence in the co-receptor LBD, the ‘co-receptor extra loop’ (CREL), which is conserved across IR8a and IR25a orthologues but not present in either tuning IRs or iGluRs. The CREL contains a single predicted N -glycosylation site, which we show bears a sugar modification in recombinantly expressed IR8a. Using the Drosophila olfactory system as an in vivo model, we find that a transgenically encoded IR8a mutant in which the CREL cannot be N -glycosylated is impaired in localisation to cilia in some, though not all, populations of sensory neurons expressing different tuning IRs. This defect can be complemented by the presence of endogenous wild-type IR8a, indicating that IR complexes contain at least two IR8a subunits and that this post-translational modification is dispensable for protein folding or complex assembly. Analysis of the subcellular distribution of the mutant protein suggests that its absence from sensory cilia is due to a failure in exit from the endoplasmic reticulum. Protein modelling and in vivo analysis of tuning IR and co-receptor subunit interactions by a fluorescent protein fragment complementation assay reveal that the CREL N -glycosylation site is likely to be located on the external face of a heterotetrameric IR complex. Conclusions Our data reveal an important role for the IR co-receptor LBD in control of intracellular transport, provide novel insights into the stoichiometry and assembly of IR complexes and uncover an unexpected heterogeneity in the trafficking regulation of this sensory receptor family. Electronic supplementary material The online version of this article (10.1186/s12915-019-0651-7) contains supplementary material, which is available to authorized users.

Comparison of transcriptomes from two chemosensory organs in four decapod crustaceans reveals hundreds of candidate chemoreceptor proteins

“…Since IRs, like the iGluRs from which they evolved, are heterotetramers, these IRs exist in combinations to form functional channels. One or two of the constituent subunits are co-receptor IRs and the other two to three subunits are tuning IRs [16].…”

“…The ORs likely evolved in insects from GRs, and so far there is no evidence of their existence in non-insect arthropods [4,6,[10][11][12][13]. The IRs evolved from ionotropic glutamate receptors (iGluRs), and they are organized as heterotetramers [4][5][6] containing two classes of IRs that are different in structure and function: co-receptor IRs, for which there are four (IR25a, IR8a, IR93a, and IR76b); and tuning IRs, for which there are many more [3,8,[14][15][16]. The co-receptors IR25a and IR8a differ from co-receptors IR93a and IR76b as they have high sequence identity to each other and to iGluRs.…”

“…The co-receptors IR25a and IR8a differ from co-receptors IR93a and IR76b as they have high sequence identity to each other and to iGluRs. IR25a and IR8a retain the amino-terminal domain (ATD) of iGluRs that is also largely absent in most tuning IRs and have a distinctive co-receptor extra loop (CREL) region in their ligand binding domain (LBD) that is lacking in all other IRs [16]. IR25a and IR8a are also necessary for targeting the IRs to the dendritic membrane to make a functional receptor-channel and may also contribute to the tuning specificity of the cell [14].…”

“…Tuning IRs, on the other hand, only confer specific sensitivity to the IRs heterotetramer by virtue of the particular combination of types. There is evidence in Drosophila that the heterotetrameric IR in some olfactory sensory neurons (OSNs) is composed of two co-receptor IRs and two tuning IRs [16].…”

“…"arthropod-conserved" [10,18]. The tuning IRs have evolved more recently [10,19] and are more specific to phyla or individual species [16]. In fact, IRs are not just chemoreceptors but can participate as "environmental sensors," detecting temperature and humidity [20][21][22][23].…”