Liliane Abuin

Complementary Function and Integrated Wiring of the Evolutionarily Distinct Drosophila Olfactory Subsystems

To sense myriad environmental odors, animals have evolved multiple, large families of divergent olfactory receptors. How and why distinct receptor repertoires and their associated circuits are functionally and anatomically integrated is essentially unknown. We have addressed these questions through comprehensive comparative analysis of the Drosophila olfactory subsystems that express the ionotropic receptors (IRs) and odorant receptors (ORs). We identify ligands for most IR neuron classes, revealing their specificity for select amines and acids, which complements the broader tuning of ORs for esters and alcohols. IR and OR sensory neurons exhibit glomerular convergence in segregated, although interconnected, zones of the primary olfactory center, but these circuits are extensively interdigitated in higher brain regions. Consistently, behavioral responses to odors arise from an interplay between IR- and OR-dependent pathways. We integrate knowledge on the different phylogenetic and developmental properties of these receptors and circuits to propose models for the functional contributions and evolution of these distinct olfactory subsystems.

An olfactory receptor for food-derived odours promotes male courtship in Drosophila

Many animals attract mating partners through the release of volatile sex pheromones, which can convey information on the species, gender and receptivity of the sender to induce innate courtship and mating behaviours by the receiver. Male Drosophila melanogaster fruitflies display stereotyped reproductive behaviours towards females, and these behaviours are controlled by the neural circuitry expressing male-specific isoforms of the transcription factor Fruitless (FRUM). However, the volatile pheromone ligands, receptors and olfactory sensory neurons (OSNs) that promote male courtship have not been identified in this important model organism. Here we describe a novel courtship function of Ionotropic receptor 84a (IR84a), which is a member of the chemosensory ionotropic glutamate receptor family, in a previously uncharacterized population of FRUM-positive OSNs. IR84a-expressing neurons are activated not by fly-derived chemicals but by the aromatic odours phenylacetic acid and phenylacetaldehyde, which are widely found in fruit and other plant tissues that serve as food sources and oviposition sites for drosophilid flies. Mutation of Ir84a abolishes both odour-evoked and spontaneous electrophysiological activity in these neurons and markedly reduces male courtship behaviour. Conversely, male courtship is increased—in an IR84a-dependent manner—in the presence of phenylacetic acid but not in the presence of another fruit odour that does not activate IR84a. Interneurons downstream of IR84a-expressing OSNs innervate a pheromone-processing centre in the brain. Whereas IR84a orthologues and phenylacetic-acid-responsive neurons are present in diverse drosophilid species, IR84a is absent from insects that rely on long-range sex pheromones. Our results suggest a model in which IR84a couples food presence to the activation of the fruM courtship circuitry in fruitflies. These findings reveal an unusual but effective evolutionary solution to coordinate feeding and oviposition site selection with reproductive behaviours through a specific sensory pathway.

Anchoring of both PKA and 14‐3‐3 inhibits the Rho‐GEF activity of the AKAP‐Lbc signaling complex

A‐kinase anchoring proteins (AKAPs) target the cAMP‐regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP‐Lbc, which functions as a PKA‐targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP‐Lbc Rho‐GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14‐3‐3 as a novel regulatory protein interacting with AKAP‐Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP‐Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14‐3‐3, which inhibits the Rho‐GEF activity of AKAP‐Lbc. AKAP‐Lbc mutants that fail to interact with PKA or with 14‐3‐3 show a higher basal Rho‐GEF activity as compared to the wild‐type protein. This suggests that, under basal conditions, 14‐3‐3 maintains AKAP‐Lbc in an inactive state. Therefore, while it is known that AKAP‐Lbc activity can be stimulated by Gα12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14‐3‐3.

Constitutively active mutants of the β1-adrenergic receptor

We provide the first evidence that point mutations can constitutively activate the β1‐adrenergic receptor (AR). Leucine 322 of the β1‐AR in the C‐terminal portion of its third intracellular loop was replaced with seven amino acids (I, T, E, F, C, A and K) differing in their physico‐chemical properties. The β1‐AR mutants expressed in HEK‐293 cells displayed various levels of constitutive activity which could be partially inhibited by some beta‐blockers. The results of this study might have interesting implications for future studies aiming at elucidating the activation process of the β1‐AR as well as the mechanism of action of beta‐blockers.

Ezrin Directly Interacts with the α1b-Adrenergic Receptor and Plays a Role in Receptor Recycling

Using the yeast two-hybrid system, we identified ezrin as a protein interacting with the C-tail of the α1b-adrenergic receptor (AR). The interaction was shown to occur in vitro between the receptor C-tail and the N-terminal portion of ezrin, or Four-point-one ERM (FERM) domain. The α1b-AR/ezrin interaction occurred inside the cells as shown by the finding that the transfected α1b-AR and FERM domain or ezrin could be coimmunoprecipitated from human embryonic kidney 293 cell extracts. Mutational analysis of the α1b-AR revealed that the binding site for ezrin involves a stretch of at least four arginines on the receptor C-tail. The results from both receptor biotinylation and immunofluorescence experiments indicated that the FERM domain impaired α1b-AR recycling to the plasma membrane without affecting receptor internalization. The dominant negative effect of the FERM domain, which relies on its ability to mask the ezrin binding site for actin, was mimicked by treatment of cells with cytochalasin D, an actin depolymerizing agent. A receptor mutant (ΔR8) lacking its binding site in the C-tail for ezrin displayed delayed receptor recycling. These findings identify ezrin as a new protein directly interacting with a G protein-coupled receptor and demonstrate the direct implication of ezrin in GPCR trafficking via an actin-dependent mechanism.

Distinct combinations of variant ionotropic glutamate receptors mediate thermosensation and hygrosensation in Drosophila

Ionotropic Receptors (IRs) are a large subfamily of variant ionotropic glutamate receptors present across Protostomia. While these receptors are most extensively studied for their roles in chemosensory detection, recent work has implicated two family members, IR21a and IR25a, in thermosensation in Drosophila. Here we characterize one of the most evolutionarily deeply conserved receptors, IR93a, and show that it is co-expressed and functions with IR21a and IR25a to mediate physiological and behavioral responses to cool temperatures. IR93a is also co-expressed with IR25a and a distinct receptor, IR40a, in a discrete population of sensory neurons in the sacculus, a multi-chambered pocket within the antenna. We demonstrate that this combination of receptors is required for neuronal responses to dry air and behavioral discrimination of humidity differences. Our results identify IR93a as a common component of molecularly and cellularly distinct IR pathways important for thermosensation and hygrosensation in insects. DOI: http://dx.doi.org/10.7554/eLife.17879.001

Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50.

The internalization properties of the α1a- and α1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the α1b-AR displayed robust agonist-induced endocytosis, the α1a-AR did not. Constitutive internalization of the α1a-AR was negligible, whereas the α1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the α1-AR subtypes with β-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and β-arrestin translocation assays indicated that the agonistinduced interaction of the α1a-AR with β-arrestins was much weaker than that of the α1b-AR. In addition, the α1a-AR did not bind AP50. The α1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding β-arrestins despite its binding to AP50. In contrast, the α1b-AR mutant ΔR8, lacking AP50 binding, bound β-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that β-arrestin 2 plays a prominent role in α1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the α1a- and α1b-AR and provide evidence that the lack of significant endocytosis of the α1a-AR is linked to its poor interaction with β-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the α1b-AR is important for receptor/β-arrestin interaction and that this interaction is the main event triggering receptor internalization.

A CD36 ectodomain mediates insect pheromone detection via a putative tunnelling mechanism.

CD36 transmembrane proteins have diverse roles in lipid uptake, cell adhesion and pathogen sensing. Despite numerous in vitro studies, how they act in native cellular contexts is poorly understood. A Drosophila CD36 homologue, sensory neuron membrane protein 1 (SNMP1), was previously shown to facilitate detection of lipid-derived pheromones by their cognate receptors in olfactory cilia. Here we investigate how SNMP1 functions in vivo. Structure–activity dissection demonstrates that SNMP1s ectodomain is essential, but intracellular and transmembrane domains dispensable, for cilia localization and pheromone-evoked responses. SNMP1 can be substituted by mammalian CD36, whose ectodomain can interact with insect pheromones. Homology modelling, using the mammalian LIMP-2 structure as template, reveals a putative tunnel in the SNMP1 ectodomain that is sufficiently large to accommodate pheromone molecules. Amino-acid substitutions predicted to block this tunnel diminish pheromone sensitivity. We propose a model in which SNMP1 funnels hydrophobic pheromones from the extracellular fluid to integral membrane receptors.

Olfactory receptor pseudo-pseudogenes

Pseudogenes are generally considered to be non-functional DNA sequences that arise through nonsense or frame-shift mutations of protein-coding genes. Although certain pseudogene-derived RNAs have regulatory roles, and some pseudogene fragments are translated, no clear functions for pseudogene-derived proteins are known. Olfactory receptor families contain many pseudogenes, which reflect low selection pressures on loci no longer relevant to the fitness of a species. Here we report the characterization of a pseudogene in the chemosensory variant ionotropic glutamate receptor repertoire of Drosophila sechellia, an insect endemic to the Seychelles that feeds almost exclusively on the ripe fruit of Morinda citrifolia. This locus, D. sechellia Ir75a, bears a premature termination codon (PTC) that appears to be fixed in the population. However, D. sechellia Ir75a encodes a functional receptor, owing to efficient translational read-through of the PTC. Read-through is detected only in neurons and is independent of the type of termination codon, but depends on the sequence downstream of the PTC. Furthermore, although the intact Drosophila melanogaster Ir75a orthologue detects acetic acid—a chemical cue important for locating fermenting food found only at trace levels in Morinda fruit—D. sechellia Ir75a has evolved distinct odour-tuning properties through amino-acid changes in its ligand-binding domain. We identify functional PTC-containing loci within different olfactory receptor repertoires and species, suggesting that such ‘pseudo-pseudogenes’ could represent a widespread phenomenon.

An expression atlas of chemosensory ionotropic glutamate receptors identifies a molecular basis of carbonation detection

Taste perception is thought to involve the encoding of appetitive and aversive chemical cues in food through a limited number of sensory pathways. Through expression analysis of the complete repertoire of Drosophila Ionotropic Receptors (IRs), a sensory subfamily of ionotropic glutamate receptors, we reveal that the majority of IRs is expressed in diverse peripheral neuron populations across gustatory organs in both larvae and adults, implying numerous roles in taste-evoked behaviours. We characterise Ir56d, which labels two anatomically-distinct classes of neurons in the proboscis: one represents a subset of sugar- and fatty acid-sensing neurons, while the other responds to carbonated solutions and fatty acids. Mutational analysis shows that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological activation by carbonation and fatty acids, but not sucrose. We further demonstrate that carbonation is behaviourally attractive to flies (in an IR56d-dependent manner), but in a distinct way to other appetitive stimuli. Our work provides a valuable toolkit for investigating the taste functions of IRs, defines a molecular basis of carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory receptors in distinct neuron types to coordinate behaviour.