InteractiveFly: GeneBrief

Sensory neuron membrane protein 1: Biological Overview | References

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, antennal protein Sensory neuron membrane protein 1 (SNMP1), was previously shown to facilitate detection of lipid-derived pheromones by their cognate receptors in olfactory cilia. This study investigated how SNMP1 functions in vivo. Structure-activity dissection demonstrates that SNMP1's 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. A model is proposed in which SNMP1 funnels hydrophobic pheromones from the extracellular fluid to integral membrane receptors (Gomez-Diaz, 2016).

The CD36 (cluster of differentiation 36) family of transmembrane proteins is broadly conserved in animals but displays remarkable functional versatility. The three mammalian CD36 proteins (CD36, SR-BI and LIMP-2) are implicated in lipoprotein scavenging, fatty acid transport, innate immune signalling, cell adhesion, lysosomal protein sorting and gustatory fat detection<. Consistently, mutation or misregulation of CD36 proteins in humans has been linked to several diseases, including arterial hypertension, diabetes, cardiomyopathy and epilepsy (Gomez-Diaz, 2016).

Despite the importance of these proteins, the precise mechanism(s) by which they function is enigmatic. Many molecular studies on mammalian CD36 family members have exploited in vitro biochemical assays, which have identified a large number of lipidic and protein ligands (for example, fatty acids, oxidized low-density lipoproteins, thrombospondin 1 and hexarelin) and correspondingly diverse ligand-binding regions in the large ectodomain of these proteins. The downstream consequences of ligand/CD36 protein interactions have mostly been analysed in heterologous cell culture expression systems. Such studies have revealed potential roles for these proteins in mediating ligand translocation across the membrane, receptor-mediated ligand internalization, transfer of ligands to other cell surface signalling receptors or direct activation of intracellular signalling cascades (for example, via Lyn and Yes tyrosine kinases. However, demonstration of the relevance of many of these biochemical and cellular properties of CD36 proteins in their native environment has rarely been tested. In part, this reflects the challenge of molecular genetic analysis of CD36 family members that have broad tissue expression and multiple, essential functions (Gomez-Diaz, 2016).

The genetic model, Drosophila melanogaster, offers a powerful system to investigate CD36 protein function in vivo. Drosophila possesses a repertoire of 14 CD36-like proteins (Nichols, 2008), most of which have tissue-specific expression patterns, suggesting that they have distinct roles. Two family members, NINAD (neither inactivation nor afterpotential D) and Santa Maria, are important for transport of dietary carotenoids from the gut to the photoreceptors (Voolstra, 2006; Wang, 2007). Others, such as Croquemort and Peste, have been implicated in immune recognition, as they are required for the uptake of Staphylococcus aureus and Mycobacteria, respectively, at least in Drosophila cell lines (Stuart, 2005; Philips, 2005). In flies, Croquemort and another CD36 homologue, Debris Buster, act in phagosome maturation in epidermal cells during clearance of degenerating neural processes (Han, 2014; Gomez-Diaz, 2016 and references therein).

Previous studies have characterized a Drosophila CD36 family member called sensory neuron membrane protein 1 (SNMP1; originally named SNMP) (Benton, 2007; Jin, 2008; Li, 2014). Drosophila SNMP1, as well as its orthologues in other insects, is expressed in olfactory sensory neurons (OSNs) that detect lipid-derived pheromones (Rogers, 2001a; Rogers, 2001b; Rogers, 1997; Forstner, 2008; Pregitzer, 2014). SNMP1 is targeted to the dendritic cilia that are exposed to external chemical signals, where odorant receptors (ORs) are located. The best-characterized function of Drosophila SNMP1 is in OSNs expressing OR67d that, together with the obligate OR co-receptor ORCO, detects the male sex pheromone (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA). Loss of SNMP1 drastically reduces the sensitivity of these neurons to cVA stimulation, but does not affect OR67d/ORCO expression or cilia localization. Although these data implicated SNMP1 as a key component of the pheromone signal transduction pathway, its mechanism of action is unknown (Gomez-Diaz, 2016).

Several non-mutually exclusive models can be envisaged for the function of SNMP1 in pheromone signalling. SNMP1 could bind free pheromone molecules and/or complexes of pheromones with secreted odorant binding proteins (OBPs) in the extracellular space. These interactions could simply concentrate pheromone molecules in the vicinity of pheromone receptors in the cilia membranes or directly facilitate transfer of these ligands to their cognate OR. SNMP1 could also participate in pheromone transduction by coupling to an intracellular signalling cascade, for example, by recruiting proteins to the cilia membranes via its cytosolic tails. Regardless of the exact mechanism, the genetic requirement for SNMP1 in coupling the presence of extracellular lipidic ligands to downstream cellular responses (that is, neuronal firing) was reminiscent of the function of mammalian CD36 in gustatory sensing of fats and immune recognition of pathogenic bacterial lipids and lipoproteins. These parallels indicate that SNMP1 might be a relevant model for understanding CD36 proteins in vivo (Gomez-Diaz, 2016).

This study describes a structure–function dissection of SNMP1, using molecular genetic, cellular, biochemical, electrophysiological and homology modelling approaches. The data demonstrate that the SNMP1 ectodomain is essential for its function, but the intracellular domains are dispensable. SNMP1 can be substituted by mammalian CD36, whose ectodomain can interact with insect pheromones. In a structural model of the SNMP1 ectodomain, a tunnel was identified that may funnel pheromone molecules to their cognate receptors. This work provides novel insights into insect pheromone transduction and highlight both conserved and divergent molecular mechanisms of CD36 protein function (Gomez-Diaz, 2016).

SNMP1 was shown to be expressed in pheromone-sensing neurons nearly two decades ago (Rogers, 1997) and genetically implicated in pheromone transduction 10 years later (Benton, 2007; Jin, 2008), but understanding how this CD36 protein acts has been largely elusive. This study considers evidence both for and against potential mechanisms of action of SNMP1 to propose a new model for its function in pheromone signalling (Gomez-Diaz, 2016).

One model suggested that SNMP1 acts as an inhibitory subunit of OR/ORCO complexes, whose influence is released in the presence of pheromone, thereby leading to an increase in neuronal firing (Jin, 2008). This proposition was based on the prominent elevated firing of OR67d neurons in the apparent absence of cVA stimulation, which was previously interpreted as spontaneous activity. Recent evidence (2014), however, indicates that this elevated firing reflects instead a highly prolonged, low-level, ligand-evoked activity due to the exposure of flies to environmental sources of cVA (for example, male flies in culture tubes), indicating a role for SNMP1 in controlling pheromone signalling kinetics rather than inhibiting ORs. Moreover, a purely inhibitory function of SNMP1 is incompatible with observations that expression of SNMP1 is required to enhance sensitivity of responses of ORs to pheromone when expressed in ectopic cells (Gomez-Diaz, 2016).

A second model is that SNMP1 transduces signals across the cell membrane to regulate intracellular signalling cascades. Although this mechanism is a prominent feature of mammalian CD36, the current demonstration that neither cytosolic tail is required for SNMP1 localization or function argues that SNMP1 is not likely to transmit signals intracellularly, at least for its role in pheromone signalling that was assayed. These observations also imply the existence of a novel type of cilia-targeting signal in SNMP1, as all cilia localization signals identified in other membrane proteins are found in cytoplasmic domains. Moreover, the lack of distinguishing primary structural features of the SNMP1 transmembrane helices compared with other CD36 proteins and the exchangeability of these sequences by those of NINAD suggest that these regions simply fulfil a structural role in membrane anchoring, rather than contributing a specific function in pheromone transduction. Finally, SNMP1's inability to assemble into homomeric complexes suggests it is unlikely to form, for example, membrane-spanning channels in cilia or rely on receptor clustering for its function (Gomez-Diaz, 2016).

A third model posits that SNMP1 forms an extracellular platform for capture of pheromones or pheromone/OBP complexes near the sensory cilia membrane. The sensitivity of the entire ectodomain to small deletions underlies its importance in SNMP1 function. Moreover, although a subregion of the ectodomain encompassing the disulfide bonds appears at least partially dispensable for cilia localization, the complete loss of signalling function of all deletion mutants suggests that this ectodomain acts as a single structural entity, rather than functionally separable subdomains. In support of this model, this study has shown that the ectodomain of mammalian CD36, which can partially substitute for SNMP1 in vivo, is able to interact with a variety of insect pheromones. Although it is not yet possible to assess direct interactions between Drosophila SNMP1 and pheromones, pioneering studies in A. polyphemus using a radiolabelled photoaffinity pheromone analogue identified a single labelled ∼70-kDa, antennal-specific membrane protein. Subsequent biochemical purification from olfactory cilia of a protein with these properties led to the initial identification of SNMP1 (Rogers, 1997), suggesting that it is a prominent pheromone-interacting membrane protein in olfactory cilia (Gomez-Diaz, 2016).

Although SNMP1 is very important for cVA detection, pheromones can directly induce OR-dependent responses in heterologous neurons or other cells, at least when applied at high concentration. These data imply that pheromones must ultimately interact with ORs, and that SNMP1 is not an integral part of the molecular machinery required for OSN firing. In the context of the third model, what, then, is the role of the SNMP1 ectodomain? The sequence of this region lacks obvious homology to other proteins. Consistently, the three-dimensional structure of the LIMP-2 ectodomain exhibits a novel global protein fold. The presence of a central cavity in LIMP-2 (also preserved in homology models of CD36, SR-BI (Neculai, 2013) and SNMP1) provides an intriguing new hypothesis for the ectodomain in acting as a tunnel for transport of small molecules from the extracellular/extraluminal space to or into the membrane. While direct visualization of movement of molecules through such a tunnel awaits development of appropriate assays, steric blockage of the predicted tunnel in SR-BI by pharmacological or genetic manipulations decreases (by about twofold) cholesterol uptake in cultured cells. Similarly, this study found introduction of larger amino-acid side chains within the presumed SNMP1 tunnel diminishes pheromone sensitivity (Gomez-Diaz, 2016).

Together these data lead to a model in which SNMP1 acts by transporting pheromone ligands in the extracellular lymph via an ectodomain tunnel to the cognate pheromone detecting OR in the cilia membrane. Why should pheromone sensing require such a mechanism? After entering the lymph, pheromones are thought to be encapsulated by OBPs (such as Lush for cVA), which induces a conformational change in these proteins. Subsequent release of pheromone molecules might therefore require energetic input to reverse this conformational change. It is hypothesized that pheromone release is triggered by transient interaction of OBP/pheromone complexes with SNMP1. Pheromone molecules must ultimately end up in the ligand-binding pocket of a cognate OR. Although very little is known about the biochemistry of OR/ligand interactions, available data suggest that the binding site lies within the transmembrane regions. The tunnel of SNMP1 might therefore facilitate direct delivery of hydrophobic pheromone molecules to this pocket, thereby protecting them from exposure to the aqueous lymph fluid or odorant degrading enzymes that are abundant in this compartment. Alternatively, and akin to the lipid transport function of CD36 and SR-BI (Ehehalt, 2006), the tunnel might direct pheromone molecules into the lipid bilayer, from where they move laterally into the OR ligand-binding pocket. This latter possibility would be analogous to the mechanism by which ligands enter the binding site in the free fatty acid receptor GPR40. Recent analysis of the kinetics of the low-frequency pheromone-evoked responses to high stimulus concentrations in the absence of SNMP1 indicated that this protein is important for both rapid activation and termination. It is possible, therefore, that SNMP1 serves to funnel pheromone molecules both to and from the OR ligand-binding pocket (Gomez-Diaz, 2016).

Experimental testing of this model is technically challenging, as it demands the functional reconstitution of three transmembrane proteins (SNMP1, OR67d and ORCO), a secreted protein (LUSH) and a radioactively or fluorescently labelled pheromone ligand in an assay system that permits biochemical assessment of dynamic interactions between these components. Moreover, the inability to recapitulate high-sensitivity responses to cVA by misexpression of OR67d, SNMP1 and LUSH in non-pheromone sensing olfactory sensilla, also hints that other signalling components are involved. Nevertheless, the available data do support the insect pheromone detection system as an elegant signalling mechanism that couples low-specificity/high-sensitivity components (such as OBPs and SNMP1, with high-specificity/low-sensitivity components (that is, pheromone-detecting ORs, which typically recognize a single ligand). This mechanism might underlie the widely documented detection of these important intraspecific signals with both high sensitivity and specificity (Gomez-Diaz, 2016).

CD36-related genes have been identified across animals, as well as in unicellular eukaryotes, indicating an ancient origin of this superfamily. How functionally distinct CD36 proteins have evolved is poorly understood. Comparison of the properties of SNMP1 with long-studied mammalian homologues provides initial insight into this question. Most strikingly, the observation that mammalian CD36 can compensate for loss of SNMP1 implies the existence of a conserved, ancestral mechanism of action across this functionally diverse protein family (Gomez-Diaz, 2016).

Unexpectedly, another insect CD36 protein, NINAD, is much less effective in replacing SNMP1 function than the mammalian homologue. It is suggested that this reflects a distinction in the evolution of the CD36 repertoires in mammals and insects. While mice and humans have only three, broadly expressed CD36 family members, insect species have evolved a dozen or more proteins. In Drosophila, at least, individual genes exhibit distinct tissue-specific expression patterns. These expression properties might reflect their different roles in, for example, the digestive, sensory or immune system, where they recognize different ligands and might couple to other types of transmembrane receptors. It is suggested that, in parallel with their acquisition of unique expression patterns, the insect proteins have become structurally and functionally specialized and hence are unable to effectively substitute for each other. For example, SNMP1's lack of dependence on intracellular domains might reflect its exclusive requirement to transfer pheromone molecules from the lymph to the ORs, without transducing signals intracellularly. By contrast, mammalian CD36 appears to have retained functional versatility, reflecting its implication in multiple distinct signalling roles in different tissues. Future study of chimeric versions of SNMP1 and other insect CD36 proteins might help uncover the conserved and divergent molecular mechanisms by which this protein family recognizes and transduces external signals in vivo (Gomez-Diaz, 2016).

Requirement for Drosophila SNMP1 for rapid activation and termination of pheromone-induced activity

Pheromones are used for conspecific communication by many animals. In Drosophila, the volatile male-specific pheromone 11-cis vaccenyl acetate (cVA) supplies an important signal for gender recognition. Sensing of cVA by the olfactory system depends on multiple components, including an olfactory receptor (OR67d), the co-receptor ORCO, and an odorant binding protein (LUSH). In addition, a CD36 related protein, sensory neuron membrane protein 1 (SNMP1) is also involved in cVA detection. Loss of SNMP1 has been reported to eliminate cVA responsiveness, and to greatly increase spontaneous activity of OR67d-expressing olfactory receptor neurons (ORNs). This study found the snmp1¹ mutation did not abolish cVA responsiveness or cause high spontaneous activity. The cVA responses in snmp1 mutants displayed a delayed onset, and took longer to reach peak activity than wild-type. Most strikingly, loss of SNMP1 caused a dramatic delay in signal termination. The profound impairment in signal inactivation accounted for the previously reported 'spontaneous activity,' which represented continuous activation following transient exposure to environmental cVA. This study introduced the silk moth receptor (BmOR1) in OR67d ORNs of snmp1¹ flies and found that the ORNs showed slow activation and deactivation kinetics in response to the BmOR1 ligand (bombykol). The bombykol receptor complex was expressed in Xenopus oocytes in the presence or absence of the silk moth SNMP1 (BmSNMP), and addition of BmSNMP was found to accelerate receptor activation and deactivation. The results thus clarify SNMP1 as an important player required for the rapid kinetics of the pheromone response in insects (Li, 2014).

Mutations that disrupt SNMP1 are reported to cause two impairments in OR67d ORNs in Drosophila (Benton, 2007; Jin, 2008). The first is insensitivity to cVA, and the second is increased spontaneous activity of OR67d ORNs in the absence of cVA stimulation. This latter phenotype motivated the proposal that the presence of SNMP1 somehow suppressed the spontaneous activity of OR67d (Li, 2014).

Rapid termination is critical for an appropriate pheromone response, particularly for insects that use pheromones as tracking cues such as the silk moth, which relies on pheromone trails that are composed of intermittent odor pockets separated by clean air spaces. Thus, to follow this trail, the pheromone-sensitive ORNs must quickly terminate their responses. It has been suggested that rapid inactivation of the pheromone response is due to degradation mediated by pheromone-degrading enzymes. However, a mathematical model proposed that a soluble scavenger is required for the fast clearance of bombykol in the sensilla lymph, as enzymatic degradation may not be fast enough (Li, 2014).

The current work found that in contrast to previous studies, loss of SNMP1 neither eliminated cVA responsiveness nor caused high spontaneous activity. In support of these conclusions, snmp1¹ mutant females raised in isolation from males did not display elevated spontaneous activity. However, the snmp1¹ females exhibited high frequencies of action potentials if they were raised along with males, or if the isolated females were exposed to cVA prior to performing the recordings. The snmp1¹ mutation also did not eliminate cVA responsiveness, since the Or67d ORNs produced cVA-induced action potentials when the pheromone was puffed in close range to the mutant females. Thus, SNMP1 was not absolutely essential for OR67d ORN activation. This conclusion is supported by the finding that when OR67d is ectopically expressed in basiconic ORNs, which lack SNMP, the ORNs can be activated by cVA, if it is applied directly to the sensilla (Li, 2014).

Of primary importance here, SNMP1 was required for rapid kinetic responses to cVA-both for rapid activation and termination of the responses. The pheromone-induced action potentials were dramatically delayed as they persisted for longer than 10 minutes, as opposed to ∼1 second for wild-type. Slow termination of cVA-induced responses also occurs upon introduction of SNMP1 antibodies to the recording pipet in wild-type flies (Jin, 2008). It is proposed that the so-called spontaneous activity displayed by snmp1¹ null flies, was a consequence of extremely long-lived activity of OR67 ORNs following exposure to environmental cVA (Li, 2014).

In addition to OR67d, ORCO and SNMP, a phospholipid flippase (dATP8B) and an OBP referred to as LUSH contribute to the sensitivity of ORNs to cVA. Loss of dATP8B affects the function of odorant receptors, at least in part by decreasing the concentration of OR67d in the ORN dendrites. However, the role of LUSH is controversial. While OBPs are typically thought to be carriers that transport hydrophobic odorants through the aqueous endolymph to the receptors, an in vitro study indicates that the cVA-LUSH complex is the activating ligand for OR67d. This conclusion has recently been questioned, in part because OR67d neurons devoid of LUSH are activated by strong cVA stimulation in vivo. Consistent with this latter report, this study also found that cVA evoked responses in the lush¹ mutants and lush¹,snmp1¹ double mutants if the pheromone was applied using the close-range application assay. Therefore, the proposal is favored that OR67d ORNs are activated directly by the pheromone (Li, 2014).

SNMP1 function does not appear to be specific to cVA since the initiation and termination of the bombykol responses were also delayed in transgenic flies expressing the silk pheromone receptor, BmOR1. However, the delayed termination in the absence of SNMP1 was not as dramatic in response to bombykol as compared to cVA. The ORNs in T3 sensilla also express SNMP1 and respond to odors from fly bodies (Benton, 2007; van der Goes van Naters, 2007). However, the T3 ORNs from wild-type or snmp1¹ males or females raised in groups or in isolation displayed similar basal activities. Thus, loss of SNMP1 does not always result in extremely prolonged activities in trichoid ORNs that are exposed to their ligands (Li, 2014).

A key question is whether SNMP1 regulates the pheromone response at the level of the receptors, or whether it modulates ORN activity downstream of receptor activation. To address whether SNMP1 activity modulated the response at the level of the receptors, the bombykol receptor complex was expressed in Xenopus oocytes, since this in vitro expression system was not likely to express other downstream signaling proteins that functioned in insect ORNs. Introduction of SNMP1 accelerated receptor activation by bombykol, and promoted rapid inactivation during wash out of the pheromone. A simple explanation for this result is that the pheromone binds to and dissociates from the receptor faster in the presence of SNMP1. It is proposed that SNMP1 facilitates the association and dissociation between ligands and receptors so that the receptor activation and inactivation are accelerated. On the surface, such a dual function might seem surprising, as association and dissociation are opposing processes. In this context it is noteworthy that an enzyme can increase both the forward and reverse reaction rates by lowering the activation energy of a reversible reaction (Li, 2014).

Farnesol-detecting olfactory neurons in Drosophila

This study set out to deorphanize a subset of putative Drosophila odorant receptors expressed in trichoid sensilla using a transgenic in vivo misexpression approach. Farnesol was identified as a potent and specific activator for the orphan odorant receptor Or83c. Farnesol is an intermediate in juvenile hormone biosynthesis, but is also produced by ripe citrus fruit peels. This study shows that farnesol stimulates robust activation of Or83c-expressing olfactory neurons, even at high dilutions. The CD36 homolog Snmp1 is required for normal farnesol response kinetics. The neurons expressing Or83c are found in a subset of poorly characterized intermediate sensilla. These neurons mediate attraction behavior to low concentrations of farnesol, and tOr83c receptor mutants are defective for this behavior. Or83c neurons innervate the DC3 glomerulus in the antennal lobe and projection neurons relaying information from this glomerulus to higher brain centers target a region of the lateral horn previously implicated in pheromone perception. These findings identify a sensitive, narrowly tuned receptor that mediates attraction behavior to farnesol and demonstrates an effective approach to deorphanizing odorant receptors expressed in neurons located in intermediate and trichoid sensilla that may not function in the classical 'empty basiconic neuron' system (Ronderos, 2014).

Basiconic sensilla typically contain neurons expressing broadly tuned odorant receptors that are activated directly by food odors, although a few receptors are narrowly tuned. Coeloconic neurons typically express ionotropic glutamate receptors that detect humidity and a variety of volatile compounds including acids, ammonia, and the insect repellant DEET. Trichoid sensilla contain olfactory neurons specialized for pheromone detection in most insects. Or83c-expressing neurons were previously categorized as trichoid neurons based on in situ experiments, but this study showed that these neurons are actually located in intermediate sensilla. The intermediate and trichoid sensilla were previously characterized and named based on morphology and the number of neurons contained. Accordingly, the intermediate sensillum containing two neurons are referred to as 'ai2' (previously called 'at2') and this study refers to the intermediate sensillum with three neurons as 'ai3'. A recent study refers to the intermediate sensillum containing three neurons as 'ai2' (Ronderos, 2014).

This study found that Or83c is a narrowly tuned odorant receptor selectively activated by farnesol and expressed in neurons located in intermediate sensilla. Flies are attracted to farnesol and this attraction is defective in flies lacking Or83c expression. Farnesol emitted by Drosophila flies, pupae, or larvae, were not found suggesting that farnesol does not act as a pheromone cue. However, volatiles from citrus fruit peels known to contain farnesol could reliably activate Or83c-expressing neurons. Given the known presence of farnesol in these rinds and the farnesol-selective activation of Or83c, it is very likely that the odorant emitted by the fruit rinds is farnesol. Drosophila encounter farnesol in citrus fruit peels and consume the yeast that grows on them. In addition, it has been shown recently that Drosophila females prefer to lay their eggs in citrus fruit peels. This preference is mediated by Or19a neurons expressed in ai3 intermediate sensilla that are tuned to detect valencene and other citrus volatiles. Nonetheless, the citrus volatiles that activate Or19a neurons do not promote olfactory attraction. Because farnesol is attractive even at very low concentrations, the results suggests that Drosophila might use farnesol and Or83c activation to guide long-range attraction to citrus fruit peels followed by activation of Or19a neurons by other citrus volatiles to guide egg-laying decisions. If farnesol detection is conserved in other, more destructive insect species, then farnesol may be a useful component of lures and traps to protect citrus crops (Ronderos, 2014).

Misexpression of Or83c in basiconic neurons fails to confer farnesol sensitivity. However, misexpression in at1 neurons confers farnesol sensitivity, as does misexpression in Or23a-expressing neurons located in intermediate sensilla neurons that are normally insensitive to this odorant. This indicates that there are farnesol sensitivity factors expressed in intermediate and trichoid sensilla that are lacking in basiconic sensilla. Snmp1 was shown to be one factor lacking in most basiconic sensilla that is important for normal responses to farnesol in trichoid and intermediate sensilla. However, when Or83c is expressed in intermediate or trichoid sensilla lacking Snmp1 (in Snmp1^Z0249) it can still respond to farnesol, but when Or83c is expressed in basiconic sensilla neurons, the responses are completely abolished. This suggests that there are likely additional, as yet unknown factors in intermediate and trichoid sensilla that are required for Or83c function. Interestingly, ab4b is the only basiconic neuron expressing Snmp1 (Benton, 2007) and these neurons are narrowly tuned to detect geosmin, a microbial-produced volatile that alerts flies to the presence of potentially toxic molds and bacteria. The Or19a neurons shown recently to mediate oviposition toward citrus substrates also express Snmp1 (Benton, 2007; Dweck, 2013), but it is not known whether Snmp1 plays any role in the detection of either valencene or geosmin. However, it is interesting that all of the Snmp1-expressing olfactory neurons characterized to date appear to have relatively specialized functions (Ronderos, 2014).

Neurons located in trichoid sensilla typically mediate responses to pheromones and basiconic neurons respond primarily to food odorants. The Or83c DC3 PNs innervate higher brain centers also targeted by PNs from VA1lm (Or47b) and VL2a (Ir84a) glomeruli. This region of the lateral horn is associated with mating behaviors, but this study shows that farnesol signaling leads to food-seeking behaviors despite innervating this region of the lateral horn. What might account for this discrepancy in wiring and function? One possibility is that the farnesol-activated projection neurons synapse with different populations of lateral horn neurons than those associated with mating, thus activating distinct circuits. VA1lm and VL2a are two of three projection neuron populations (the third being DA1) that express the sexually dimorphic fruitless (Fru^M) transcription factor, which is required for establishing the entire olfactory neural circuit from olfactory neurons to motor neurons that regulate mating behaviors. DC3 PNs, which do not express Fru^M, might not integrate into this courtship circuit, perhaps targeting interneurons that regulate other olfactory behaviors. Another possibility is that organization of the lateral horn might not be a simple pheromone versus food division, as postulated previously, but instead may predominately reflect continuation of a sensillar organization (basiconic vs intermediate vs trichoid vs coeloconic) into higher brain centers. Nonetheless, the surprising overlap of courtship and food attraction in the lateral orn requires future investigation on how these different olfactory signals are further processed to mediate distinct behaviors (Ronderos, 2014).

Finally, this study shows that Or83c is a selective farnesol receptor that can confer farnesol sensitivity on intermediate and trichoid olfactory neurons in a cell autonomous fashion. Therefore, misexpression of Or83c in other trichoid or intermediate olfactory neurons in the Or83c^MB11142 mutant background may provide an odorant-driven strategy to gain insights into the behavioral outputs of these circuits (Ronderos, 2014).

Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein

Detection of volatile odorants by olfactory neurons is thought to result from direct activation of seven-transmembrane odorant receptors by odor molecules. This study shows that detection of the Drosophila pheromone, 11-cis vaccenyl acetate (cVA), is instead mediated by pheromone-induced conformational shifts in the extracellular pheromone-binding protein, Lush. Lush undergoes a pheromone-specific conformational change that triggers the firing of pheromone-sensitive neurons. Amino acid substitutions in Lush that are predicted to reduce or enhance the conformational shift alter sensitivity to cVA as predicted in vivo. One substitution, Lush^D118A, produces a dominant-active Lush protein that stimulates T1 neurons through the neuronal receptor components Or67d and SNMP in the complete absence of pheromone. Structural analysis of Lush^D118A reveals that it closely resembles cVA-bound Lush. Therefore, the pheromone-binding protein is an inactive, extracellular ligand converted by pheromone molecules into an activator of pheromone-sensitive neurons and reveals a distinct paradigm for detection of odorants (Laughlin, 2008).

This study has shown that cVA binds to the pheromone-binding protein Lush and induces conformational changes. Mutations predicted to reduce or enhance the conformational changes also reduce or enhance cVA sensitivity in vivo. One Lush mutant, Lush^D118A, is dominantly active, triggering robust action potentials in T1 neurons in the absence of pheromone. This effect is specific to T1 neurons, as basiconic and other trichoid olfactory neurons are unaffected by this protein. Lush^D118A activates T1 neurons through the putative cVA-activated neuronal receptor components, Or67d and SNMP, accounting for the specificity of the dominant Lush. The data reveal that pheromone molecules are not required for activation of T1 neurons and define a novel olfactory signaling paradigm in which the pheromone-induced conformational change in Lush mediates activation of T1 neurons (Laughlin, 2008).

cVA can trigger weak responses in T1 neurons in the absence of Lush when applied at high concentrations. Direct effects of cVA on Or67d/SNMP receptor complexes may mediate these Lush-independent responses, as these two components confer marginal cVA sensitivity to the empty neuron preparation (Benton, 2007). Alternatively, activated Lush may normally dimerize with an unknown cofactor that alone can weakly activate T1 receptors in the presence of cVA. However, the sensitivity for cVA in the absence of Lush is so poor that lush¹ mutants are blind to the pheromone in aggregation assays. In proximity experiments, cVA levels emanating from single male flies are below detection limits in the absence of Lush. Therefore, the Lush-independent activation of T1 neurons is unlikely to play a role in cVA responses in vivo (Laughlin, 2008).

Olfactory neurons are thought to be tuned to odorants exclusively by the odorant receptors they express. Indeed, in Drosophila melanogaster, activation of many odorant receptors results from direct binding of food odorants. Why does cVA reception require a binding protein intermediate? It is suggested that the binding protein may enhance sensitivity and specificity in the pheromone detection process. If a pheromone induces a stable, ligand-specific conformational change in a binding protein, single pheromone molecules could be detectable if the neuronal receptor complex is specifically tuned to that conformation. Further, if the conformation of the binding protein that activates the receptors is specific to the pheromone-bound state, other environmental stimuli are less likely to activate the neurons, even if they interact with the binding protein. Consistent with this idea, Lush increases the sensitivity of T1 neurons to cVA over 500-fold, but, remarkably, does not sensitize the neurons to structurally similar chemicals, such as vaccenyl alcohol or vaccenic acid. Indeed, Lush can bind a large array of chemicals, but only cVA activates T1 neurons. Other OBPs have been shown to bind to a wide range of unnatural compounds, including plasticizers and dyes, and the electrophysiological or behavioral responses to a specific ligand do not correlate with the binding affinity of the OBP for that ligand. Therefore, binding is clearly not sufficient for sensitization. However, by utilizing a ligand-specific conformational shift in a binding protein, detection of rare pheromone molecules is possible with high fidelity and sensitivity by creating an active binding protein species that diffuses within the sensillum lymph until it contacts and activates a receptor on the dendrites (Laughlin, 2008).

Attempted were made to reconstitute the cVA detection pathway in basiconic neurons lacking endogenous receptors. The CD36 homolog SNMP is expressed in most or all trichoid neurons and is required for sensitivity to cVA (Benton, 2007; Jin, 2008). SNMP colocalizes with the odorant receptor complex in T1 neuron dendrites (Benton, 2007), and antiserum to SNMP infused into the lymph of T1 sensilla phenocopies SNMP loss-of-function mutants, suggesting that SNMP directly mediates pheromone sensitivity (Jin, 2008). Expression of SNMP, Or67d, and Lush together in the empty neuron system failed to recapitulate T1 cVA sensitivity. Or67d alone was unresponsive, but adding Lush through the recording pipette did sensitize Or67d receptors slightly to cVA in the absence of SNMP, suggesting that Lush interacts directly with Or67d. Coexpressing SNMP and Or67d enhanced cVA sensitivity, but, surprisingly, adding Lush failed to further enhance sensitivity. These differences between the empty neuron responses and T1 neurons may reflect reduced levels of one or more components when expressed in basiconic sensilla or, more likely, indicate that additional components are missing. Indeed, in a screen for cVA-insensitive mutants, mutations were recovered in the known sensitivity factors as well as three additional unknown genes encoding factors that are essential for cVA sensitivity. It is expected that, when all of these components are identified and expressed in the basiconic neurons, full cVA sensitivity will be conferred (Laughlin, 2008).

OBPs, like Lush, are a large family of soluble proteins secreted into the lymph fluid surrounding the olfactory neurons. Proposed functions for OBPs include transporting ligands to the ORs, protecting the odor from degradation or deactivation by odorant-degrading enzymes (ODEs), and forming a complex with an odor that either directly activates ORs or binds to other accessory proteins, which ultimately results in OR activation. In vitro studies of the pheromone-binding protein (PBP) from Bombyx mori show that the OBP undergoes a conformational change at low pH that prevents ligand binding, suggesting that OBPs may function primarily as passive carriers and changes in the local pH stimulate pheromone release in the vicinity of the neuronal membrane. Furthermore, previous studies reported that high concentrations of moth pheromones can directly activate cognate pheromone receptors expressed in tissue culture and that DMSO is as effective as the pheromone-binding proteins at sensitizing the neurons to pheromone, leading to the conclusion that the binding proteins are pheromone solubilizers/carriers. However, similar studies implicate the binding proteins as factors in receptor specificity. The current data support the latter view. It is noted that Lush homologs in other insects and the 12 Drosophila species have conserved the amino acids predicted to form the salt bridge. Only Drosophila ananassae (D. ana) is predicted to lack the phenylalanine corresponding to F121 in melanogaster (replaced by leucine). A similar activation mechanism, therefore, is likely to occur in these species. Recent work in rodents reveals that vertebrate pheromones can be peptides or protein. It will be interesting to determine whether the conformational activation mechanism identified for Lush is conserved in analogous extracellular binding proteins in other species (Laughlin, 2008).

The SNMP/CD36 gene family in Diptera, Hymenoptera and Coleoptera: Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae, Aedes aegypti, Apis mellifera, and Tribolium castaneum

Sensory neuron membrane proteins (SNMPs) are membrane bound proteins initially identified in olfactory receptor neurons of Lepidoptera and are thought to play a role in odor detection; SNMPs belong to a larger gene family characterized by the human protein CD36. This study has identified 12-14 candidate SNMP/CD36 homologs from each of the genomes of Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae and Aedes aegypti (Diptera), eight candidate homologs from Apis mellifera (Hymenoptera), and 15 from Tribolium castaneum (Coleoptera). Analysis (sequence similarity and intron locations) suggests that the insect SNMP/CD36 genes fall into three major groups. Group 1 includes the previously characterized D. melanogaster emp (epithelial membrane protein). Group 2 includes the previously characterized D. melanogaster croquemort, ninaD, santa maria, and peste. Group 3 genes include the SNMPs, which fall into two subgroups referred to as SNMP1 and SNMP2. D. melanogaster SNMP1 (CG7000) shares both significant sequence similarity and five of its six intron insertion sites with the lepidopteran Bombyx mori SNMP1. The topological conservation of this gene family within the three major holometabolous lineages indicates that it predates the coleopteran and hymenoptera/dipera/lepidoptera split 300+ million years ago (Nichols, 2008).

SNMP is a signaling component required for pheromone sensitivity in Drosophila

The only known volatile pheromone in Drosophila, 11-cis-vaccenyl acetate (cVA), mediates a variety of behaviors including aggregation, mate recognition, and sexual behavior. cVA is detected by a small set of olfactory neurons located in T1 trichoid sensilla on the antennae of males and females. Two components known to be required for cVA reception are the odorant receptor Or67d and the extracellular pheromone-binding protein LUSH. Using a genetic screen for cVA-insensitive mutants, a third component required for cVA reception has been identified, sensory neuron membrane protein (SNMP). SNMP is a homolog of CD36, a scavenger receptor important for lipoprotein binding and uptake of cholesterol and lipids in vertebrates. In humans, loss of CD36 is linked to a wide range of disorders including insulin resistance, dyslipidemia, and atherosclerosis, but how CD36 functions in lipid transport and signal transduction is poorly understood. This study shows that SNMP is required in pheromone-sensitive neurons for cVA sensitivity but is not required for sensitivity to general odorants. Using antiserum to SNMP infused directly into the sensillum lymph, it has been shown that SNMP function is required on the dendrites of cVA-sensitive neurons; this finding is consistent with a direct role in cVA signal transduction. Therefore, pheromone perception in Drosophila should serve as an excellent model to elucidate the role of CD36 members in transmembrane signaling (Jin, 2008).

CVA (11-cis-vaccenyl acetate) mediates social behaviors in Drosophila, and its reception requires the odorant receptor Or67d and the extracellular pheromone-binding protein Lush. Misexpression of Or67d receptors in trichoid neurons that are normally insensitive to pheromone confers cVA sensitivity but only if Lush is present. However, Or67d and Lush are not sufficient to confer cVA sensitivity to basiconic neurons. This finding reveals that there are additional factors required for cVA sensitivity present in trichoid sensilla that are lacking in basiconic sensilla. Using a genetic screen, attempts were made to identify additional components important for cVA sensitivity. ~3,000 mutagenized third-chromosome lines selected for homozygous viability were screened. Each mutant line was screened for T1 electrophysiological responses to cVA using single sensillum electrophysiological recordings. Five complementation groups were identified that were cVA-insensitive yet retained spontaneous activity in the pheromone-sensing neurons (the vains phenotype). The presence of spontaneous activity indicates that the neurons are present, are viable, and can sustain action potentials, thereby eliminating nonspecific mutants affecting development or general neuronal function. Of the five complementation groups recovered, two, Or67d and Or83b, affect genes previously implicated in cVA or general odorant detection, two remain unmapped, and the fifth encodes SNMP, a new cVA detection component (Jin, 2008).

Two alleles of vainsA (vainsA¹) were isolated. Both mutants are defective for cVA sensitivity but also have striking defects in most olfactory responses. Deficiency mapping localized vainsA to the third chromosome at position 83 on the polytene map. A candidate gene in this interval, Or83b, encodes a coreceptor required to deliver odorant receptors to the dendrites. Mutants lacking Or83b are insensitive to most odorants due to lack of functional receptors exposed to the environment. Or83b mutants detect CO₂ normally because this gas is detected by gustatory receptors Gr21a and Gr63a, and gustatory receptors do not require Or83b for function. vainsA mutants, like previously reported Or83b mutants, have normal CO₂ responses but lack responses to general odorants (Jin, 2008).

DNA and RNA were isolated from vainsA¹ and vainsA² mutants and the genomic DNA and cDNAs encoding Or83b were sequenced. Both vainsA alleles were found to contain lesions predicted to disrupt Or83b function. vainsA¹ mutants have a lesion in the splicing donor sequence GTGAGT at the start of intron 3 that is mutated to ATGAGT. Therefore, this intron is not recognized by the splicing machinery and is included in the mature transcript. Inclusion of this intron terminates the Or83b polypeptide prematurely at residue 350. vainsA² mutants also have a single point mutation that produces a splicing defect. In this case, the mutants are defective in the splicing acceptor sequence, CAG, of intron 4, that is mutated from CAGAG to CAAAG. This mutation simultaneously creates a new splicing acceptor, AAG, two base pairs downstream that results in a 2-bp deletion in the mature message. Use of this novel acceptor results in a frame-shift mutation that encodes a polypeptide longer than wild type Or83b, which lacks the putative seventh transmembrane domain of the coreceptor. To reflect the fact that vainsA mutants are new alleles of Or83b, these mutants were renamed Or83b^Z4506 and Or83b^Z0061 (Jin, 2008).

vainsC¹ fails to complement Or67d² null mutants, revealing that vainsC¹ is defective for Or67d function. Indeed, sequence analysis reveals that Or67d has a single-amino-acid substitution in vainsC¹, C23W, which completely disrupts cVA signaling. This mutation, near the N terminus, is predicted to be intracellular, so this mutation could disrupt the structural integrity of the receptor or its ability to activate downstream components. Henceforth, vainsC¹ is referred to as Or67d^Z5499 (Jin, 2008).

vainsB¹, vainsD¹, and vainsE¹ mutants complement lush and Or67d and thus represent previously uncharacterized sensitivity factors for cVA. vainsB and vainsE loci have not been mapped. However, it was possible to map vainsD. vainsD¹ T1 neurons are completely defective for cVA pheromone responses but are unique among the cVA detection mutants with respect to spontaneous activity. The T1 neurons from vainsD¹ display increased basal activity (14-25 spikes per second compared with wild type at ≈1 spike per second). This phenotype is distinct from Or67d mutants and lush mutants which have almost no spontaneous neuronal activity present in the T1 neurons (Jin, 2008).

To determine whether vainsD¹ is required for olfactory responses in general, the odor-evoked electrophysiological responses of large and small basiconic and non-T1 sensilla to a wide range of odorants were surveyed. The results show that the basal activity and olfactory responses of basiconic neurons in vainsD¹ mutants are indistinguishable from wild-type controls. Thus, vainsD¹ is not an olfactory component mediating olfaction in a global manner but instead is selectively required for cVA activation of T1 neurons. Importantly, both Or67d and Lush, the two factors known to be required for cVA detection, appear unaffected in the vainsD¹ mutant background (Jin, 2008).

Seficiency mapping was used to localize the vainsD¹ mutation. One deficiency, Df(3R)93B;93D, failed to complement vainsD¹. The known genes mapping to the 93B-93D interval was surveyed for likely candidates. Notably, a strong candidate gene in this interval, Snmp (or CG7000), encodes a 551-aa homolog of SNMP, a moth protein expressed in pheromone-sensitive olfactory neuron dendrites (Rogers, 1997; Rogers, 2001a; Rogers, 2001b). Moth SNMP is a 67-kDa polypeptide with similarity to members of the CD36 family of lipid binding proteins (Rogers, 1997). In vertebrates, CD36 is an 88-kDa integral membrane protein receptor that mediates internalization of oxidized low-density lipoprotein by macrophages (Collot-Teixeira, 2007), formation of atherosclerotic plaques (Febbraio, 2004), and the import of long-chain fatty acids by adipose, heart, and other tissues (Coburn, 2000; Febbraio, 2007). In humans, loss of CD36 is linked to a wide range of disorders including insulin resistance, dyslipidemia, and atherosclerosis (Coburn, 2000; Febbraio, 2007; Pravenec, 2007; Miyaoka, 2001; Hirano, 2003). CD36 molecules share a common domain structure with short intracellular domains at the N and C termini, two membrane spanning domains, and a large extracellular domain (Febbraio, 2007; Jin, 2008 and references therein).

To examine whether Drosophila Snmp is defective in vainsD¹ mutant animals, its nucleotide sequence was compared with parental controls. Indeed, Snmp harbors a 5-bp deletion not present in parental controls that introduces a frame shift and a concomitant premature termination at residue 204, approximately halfway through the protein. Snmp mRNA was surveyed to check global expression patterns; abundant expression was found in antennae and heads lacking appendages (antennae and maxillary palps) and a lower expression level in the body. As expected, antiserum raised to the extracellular domain of the SNMP protein reveals that it is present in parental control flies and is clearly expressed in trichoid neurons and dendrites but is not detected in vainsD¹ mutants. To confirm that the vainsD¹ (Snmp^Z0429) phenotype results exclusively from the loss of the Snmp gene product, a wild type Snmp cDNA was expressed under control of the Or67d T1 neuron promoter or the lush nonneuronal supporting cell promoter in the Snmp^Z0429 mutant background. Expression of SNMP in the T1 neurons restored cVA sensitivity, but cVA sensitivity was not restored when SNMP was expressed in the support cells with the lush promoter. These findings provide direct evidence that cVA pheromone detection requires SNMP expression in T1 neurons and that this CD36 homolog has a specific role in pheromone detection in the antennae. Consistent with this finding, double mutants defective for both Snmp and lush have high spontaneous activity, indicating that SNMP functions downstream of Lush in cVA signaling (Jin, 2008).

The rescue experiments prove that SNMP functions in T1 neurons but do not reveal whether SNMP directly mediates cVA detection or whether SNMP acts indirectly by mediating the expression or transport of another cVA sensitivity factor. If SNMP is required directly for cVA detection, it is predicted that SNMP function should be required on the surface of the T1 neuron dendrites. Therefore, the antiserum to the extracellular domain of SNMP was infused into the sensillum lymph of T1 sensilla from wild type flies through the recording pipette and spontaneous activity and cVA sensitivity was monitored. Initially the T1 neurons behave normally; but 30 min after immune serum is infused through the recording pipette, striking effects were observed on T1 behavior: (1) spontaneous activity was dramatically increased, similar to what was observed in Snmp^Z0429 mutants; (2) dose-response analysis reveals that the cVA sensitivity is reduced ~10-fold by the antibody treatment. Thus, disruption of SNMP function on the dendrites of T1 neurons phenocopies loss-of-function mutants in SNMP. (3) An unexpected prolongation of cVA responses was also observed following treatment with anti-SNMP antiserum. This finding suggests SNMP is also important for deactivation of cVA responses once initiated. Importantly, infusion of preimmune serum from the same animal at the same concentration had no effect on spontaneous activity, cVA sensitivity, or deactivation kinetics. Essentially identical results were obtained with immune serum from two different animals. These findings reveal that SNMP function is required on the dendritic surface where it is exposed to the sensillum lymph and support the view that SNMP functions directly in cVA signal transduction (Jin, 2008).

The results indicate that cVA perception in Drosophila requires supplemental factors not required for the detection of general food odorants. General food odorants are thought to activate odorant receptors through direct interactions with receptor proteins. It has been shown that misexpression of many Drosophila Ors in 'empty' neurons (neurons lacking a functional odorant receptor) confers the odorant specificity profile of the misexpressed receptor. Thus, receptor expression is necessary and sufficient for neuronal activation by food odors. When Or67d was expressed in the empty neuron system, these workers detected responses to cVA in the absence of Lush but only at concentrations that are orders of magnitude greater than the threshold sensitivity of wild type T1 neurons. Furthermore, these high cVA levels induced submaximal activation in the neurons. Other compounds with no ability to activate T1 neurons in vivo also activated Or67d under these conditions, suggesting that they may be nonspecific. Or67d alone fails to sensitize the empty neuron system to cVA. When Snmp is coexpressed with Or67d, high levels of cVA do elicit responses (Benton, 2007). However, flies with normal expression of Or67d but lacking Lush or SNMP are electrophysiologically and behaviorally insensitive to cVA (Benton, 2007). Thus, in vivo Or67d alone does not recapitulate the sensitivity or specificity to cVA observed in T1 neurons. Lush and SNMP are members of a growing list of components in a unique signaling pathway used for pheromone perception but not for general odorants. It will be interesting to identify the genes affected in vainsB¹ and vainsE¹ mutants, both of which have normal responses to general odorants but are insensitive to cVA (Jin, 2008).

SNMP is a member of the CD36 family of lipoprotein binding proteins. CD36 knockout mice are defective for uptake of fatty acids into muscle and heart, and macrophages from these lines fail to take up oxidized cholesterol. In Drosophila, other CD36 homologs are important for recognition and removal of dead cells (Franc, 1996) and bacteria (Philips, 2005), and absorption of vitamin A from the gut (Kiefer, 2002; Gu, 2004) and transfer into the retina (Wang, 2007). In vertebrates, CD36 proteins function as receptors and signal transduction molecules. Binding to oxidized sterols triggers CD36 to interact with the nonreceptor tyrosine kinase lyn and MEKK2 which activate c-jun N-terminal kinase to mediate foam cell formation (Rahaman, 2006). SNMP clearly is required for pheromone signaling in Drosophila, and the signaling mechanisms downstream of Or67d are unknown. Whether SNMP signals through a tyrosine kinase pathway remains to be determined (Jin, 2008).

How does SNMP function in cVA signal transduction? lush¹, Snmp^Z0429 double mutants have high spontaneous activity as observed in Snmp^Z0429 mutants, demonstrating that Lush is upstream of SNMP in the cVA reception pathway. These genetic data are consistent with the finding that SNMP function is required in the T1 neurons, whereas Lush is present outside the neurons. Based on the impaired cVA signaling and the increased spontaneous activity after treatment with antiserum to SNMP, it is concluded that SNMP functions on the T1 neuron dendrites, consistent with a direct role in cVA signaling. Disruption of SNMP function, either genetically or with antiserum, results in increased spontaneous activity in T1 neurons. Thus, SNMP normally exerts an inhibitory influence on T1 activity in the absence of cVA. One model consistent with these data is that SNMP is an inhibitory subunit in a complex with Or67d. Such a role could also explain the abnormal deactivation kinetics observed in the antibody experiments (Jin, 2008).

Detection of volatile pheromones is a specialized form of olfaction dedicated to perception of chemical cues with high biological information content delivered from other individuals of the same species. As such, pheromone detection is expected to be highly specific so that spurious environmental stimuli are not mistaken for biologically relevant pheromone cues. The data support the idea that pheromone signaling is more specialized compared with general odor detection and requires additional factors including SNMP and Lush. Future experiments will be required to elucidate the precise functional relationships among these factors (Jin, 2008).

An essential role for a CD36-related receptor in pheromone detection in Drosophila

The CD36 family of transmembrane receptors is present across metazoans and has been implicated biochemically in lipid binding and transport (Ge, 2005). Several CD36 proteins function in the immune system as scavenger receptors for bacterial pathogens and seem to act as cofactors for Toll-like receptors by facilitating recognition of bacterially derived lipids (Hoebe, 2005; Philips, 2004; Stuart, 2005). This study shows that a Drosophila CD36 homologue, Sensory neuron membrane protein (SNMP), is expressed in a population of olfactory sensory neurons (OSNs) implicated in pheromone detection. SNMP is essential for the electrophysiological responses of OSNs expressing the receptor OR67d to (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA), a volatile male-specific fatty-acid-derived pheromone that regulates sexual and social aggregation behaviours (Bartelt, 1985; Xu, 2005; Ejima, 2007; Kurtovic, 2007). SNMP is also required for the activation of the moth pheromone receptor HR13 by its lipid-derived pheromone ligand (Z)-11-hexadecenal (Grosse-Wilde, 2007) but is dispensable for the responses of the conventional odorant receptor OR22a to its short hydrocarbon fruit ester ligands. Finally, this study shows that SNMP is required for responses of OR67d to cVA when ectopically expressed in OSNs not normally activated by pheromones. Because mammalian CD36 binds fatty acids (Bonen, 2004), it is suggested that SNMP acts in concert with odorant receptors to capture pheromone molecules on the surface of olfactory dendrites. This work identifies an unanticipated cofactor for odorant receptors that is likely to have a widespread role in insect pheromone detection. Moreover, these results define a unifying model for CD36 function, coupling recognition of lipid-based extracellular ligands to signalling receptors in both pheromonal communication and pathogen recognition through the innate immune system (Benton, 2007).

Insect odorant receptors represent a novel class of polytopic membrane proteins unrelated to vertebrate G-protein-coupled chemosensory receptors. The functional insect odorant receptor is a heteromer of a ligand-binding subunit and the highly conserved OR83b co-receptor, which mediates transport to sensory cilia. Little is known about how this complex recognizes odours and evokes neuronal depolarization. To isolate novel components involved in insect olfactory detection, a bioinformatic approach was used to identify molecules that exhibit the same insect-specific orthology and olfactory-specific tissue expression as these receptors. Two-thousand one-hundred and thirty-five Drosophila genes with insect-specific orthologues were identified by comparing the fruit fly (Drosophila melanogaster), mosquito (Anopheles gambiae) and eight non-insect genomes using the OrthoMCL algorithm (Li, 2003). Broadly expressed genes were excluded by selecting only the 616 genes with fewer than two expressed sequence tags. All classes of known insect chemosensory genes were recovered, including odorant receptors, gustatory receptors, odorant and other chemosensory binding proteins, and putative odour-degrading enzymes. The remaining genes were classified on the basis of predicted protein domains and included many implicated in immunity and defence (Benton, 2007).

Three-hundred and thirty-nine uncharacterized genes were screened for selective expression in the antenna (the major olfactory organ of Drosophila) by reverse transcriptase-polymerase chain reaction (RT-PCR). Of these, focus was placed on Snmp, an antennal-enriched gene related to the CD36 receptor family. The Anopheles homologue of Snmp was also antennal-specific, consistent with the previously described olfactory-specific expression pattern of the silk moth (Antheraea polyphemus) homologue Snmp-1. SNMPs form an insect-specific sub-group of the CD36 family, explaining how Drosophila Snmp emerged from the bioinformatic screen (Benton, 2007).

In the antenna, Snmp was found prominently expressed in a lateral-distal population of OSNs that co-express Or83b, in non-neuronal support cells that surround these OSNs, and in support cells elsewhere in the antenna and chemosensory organs on the proboscis. Genetic labelling of SNMP-expressing OSNs with mouse CD8 fused to green fluorescent protein (CD8-GFP) revealed that these neurons target nine glomeruli in the antennal lobe -- DA1, VA1d, VA1l/m, DL3, DA4m, DA4l, DA2, DC3 and DC1 -- corresponding to those innervated by OSNs of the trichoid sensilla, which are involved in pheromone detection (Benton, 2007).

Using a peptide antibody, SNMP was found concentrated in trichoid sensory cilia, where it co-localized with OR83b, but only at very low levels in the cell bodies and axons, similar to moth SNMP-1. No SNMP was observed in non-trichoid OSNs, but it was expressed in support cells throughout the antenna. All anti-SNMP immunoreactivity was abolished in an snmp-null mutant, confirming antibody specificity. Although the localization of SNMP in OSN cilia was similar to that of odorant receptors, it did not depend on OR83b when a functional SNMP-GFP fusion protein was expressed in OSNs innervating basiconic sensilla. Therefore, SNMP ciliary trafficking is independent of both specific ligand-binding odorant receptors and OR83b. Whether SNMP might still contact odorant receptors in trichoid cilia was examined by using the fluorescent protein fragment complementation assay. SNMP and OR83b bearing complementary fragments of a yellow fluorescent protein (YFP) reporter were generated and functionally verified. Reconstitution of the fluorescent YFP signal in sensory cilia was observed only when both fusion proteins were expressed. As the YFP fragments do not self-associate, this reconstitution could only result if SNMP and OR83b were brought into close proximity (<80 Å), providing evidence that SNMP is closely apposed to, although not necessarily directly interacting with, odorant receptors in the sensory compartment (Benton, 2007).

Snmp null mutants were generated by gene targeting. snmp mutants are viable and fertile with no gross morphological or locomotor defects. The function of SNMP was examined in the sub-population of trichoid sensilla innervated by neurons expressing OR67d -- the best-characterized Drosophila pheromone receptor that recognizes cVA. In snmp mutants, neither the expression of Or67d nor the ciliary localization of GFP-OR67d or OR83b was affected and axonal projections of snmp mutant OR67d-expressing neurons to the antennal lobe were wild type. The expression of Lush, an odorant-binding protein secreted by trichoid sensilla support cells into the lymph was normal. Thus Snmp is dispensable for the development of trichoid OSNs and support cells (Benton, 2007).

Whether the responses of OR67d neurons to cVA stimulation were altered in snmp mutants was tested. The relatively low spontaneous activity of the OR67d neuron was observable as a sparse distribution of action potentials of uniform amplitude. On stimulation with cVA, wild-type neurons responded with a robust train of action potentials in a dose-dependent manner. snmp mutant neurons displayed no cVA-evoked electrophysiological responses at any concentration tested, but showed an increase in spontaneous activity. Both spontaneous and stimulus-evoked responses were fully restored by expression of the Snmp rescuing transgene in OR67d-expressing neurons, but not by expression in support cells surrounding these neurons. Expression of a distinct Drosophila CD36-related protein, NINAD, in OR67d-expressing neurons did not rescue electrophysiological defects of snmp mutants. Thus, SNMP has an essential, cell-autonomous and specific function in OR67d-expressing neurons in mediating responses to cVA (Benton, 2007).

cVA detection is also dependent on Lush and the OR67d/OR83b heteromeric receptor complex, suggesting that SNMP acts with these proteins in a signalling pathway. In contrast to snmp mutants, however, loss of lush, Or67d or Or83b severely decreased spontaneous activity of these neurons. Double-mutant analysis of this spontaneous activity phenotype revealed that Snmp is epistatic to lush, because OR67d-expressing neurons retained high levels of spontaneous activity in animals lacking both SNMP and Lush. In contrast, snmp Or83b double mutants were, like Or83b, electrically silent. Although the mechanism by which spontaneous activity is regulated in Drosophila OSNs is unknown, genetic analysis indicates that SNMP may act downstream of Lush and upstream of, or in parallel with, odorant receptors in the generation of action potentials (Benton, 2007).

To investigate the specificity of SNMP function, a second receptor, OR22a, which is responsive to fruit esters, such as ethyl butyrate and pentyl acetate, was ectopically expressed in OR67d neurons. Although chemically related to cVA, OR22a ligands lack the long hydrophobic tail of this fatty-acid-derived pheromone. Ectopic expression of OR22a in wild-type OR67d-expressing neurons conferred responses to a panel of known OR22a ligands in addition to the endogenous cVA response, but not to a control odour, geranyl acetate, which activates neither OR67d nor OR22a. In snmp mutants, ectopic OR22a-dependent responses were unaffected, but all cVA responses were lost. The broad expression of SNMP in trichoid OSNs indicates that it might have a general function in pheromone detection. Because no other volatile pheromones have been identified in Drosophila, whether SNMP is required for the activation of the moth (Heliothis virescens) pheromone receptor HR13 by (Z)-11-hexadecenal, a component of the sex pheromone blend of this species, was tested. As previously observed, expression of HR13 in OR67d-expressing neurons conferred responsiveness to this pheromone (Kurtovic, 2007). This response was almost completely abolished in snmp mutants and restored by transgenic rescue of Snmp. Together, these experiments reveal a specific and conserved function for SNMP in mediating pheromone-evoked neuronal activity. OR67d and HR13 share <15% amino acid identity and their ligands have chemically distinct head groups, suggesting that it is the fatty-acid-derived hydrocarbon tail common to these pheromones that necessitates SNMP (Benton, 2007).

Finally, it was asked whether SNMP is required for the activation of OR67d by cVA in neurons not normally responsive to pheromones. OR67d was ectopically expressed in basiconic OSNs that lack the endogenous OR22a ligand-binding odorant receptor, but retain OR83b. All action potentials in these neurons can therefore be ascribed to OR67d/OR83b activity. Or22a mutant neurons expressing OR67d without SNMP exhibited spontaneous firing, but did not respond to cVA. In contrast, when OR67d was co-expressed with SNMP, significant responses to this pheromone were observed; compared to the responses of native OR67d neurons, the frequency of action potentials was lower and exhibited slower rise and decay rates. Such differences may be due to the absence in basiconic sensilla of Lush or odour-degrading enzymes specialized to inactivate pheromone molecules (Benton, 2007).

In summary, through a bioinformatic screen for insect olfactory transduction molecules, Drosophila SNMP was identified as a CD36-related receptor broadly expressed in pheromone-sensing neurons: SNMP is an essential co-factor for detection of the fatty-acid-derived pheromone cVA. Since mammalian CD36 has an important biochemical function in binding and membrane translocation of fatty acids it is suggested that SNMP directly captures pheromone molecules on the surface of OSN cilia -- possibly retrieving them from odorant-binding proteins in the extracellular milieu -- and facilitates their transfer to the odorant-receptor-OR83b complex. OR67d ectopically expressed without SNMP can be activated by cVA when the pheromone was directly applied to the sensillar cuticle overlying the OSN, indicating that pheromone receptors can be directly stimulated by ligand. When pheromones are presented in an air stream to the receptor in its native environment, however, SNMP (and odorant-binding proteins) are essential. It is suggested that the combination of molecular specializations of pheromone-sensing trichoid neurons together contribute to the sensitivity of these cells and that SNMP-related proteins function in the detection of many insect pheromones (Benton, 2007).

The mechanistic basis of CD36 ligand interactions and signalling is still poorly understood in any biological system. These results have three important general implications: (1) SNMP has a specific role in the detection of fatty-acid-derived odour ligands. Because other CD36-related receptors are involved in binding and transport of lipid-based molecules, for example in the mammalian intestine (Ge, 2005), this protein family may represent specialized receptors for extracellular fatty ligands of diverse biological origin and function. (2) It was shown that SNMP acts in concert with other transmembrane odorant receptors in OSN cilia in mediating pheromone-evoked activity. Because CD36 was previously shown to act as a co-receptor for Toll-like receptors, it is suggested that CD36-related proteins have obligate transmembrane partners in all their cellular roles (Benton, 2007).

(3) These results reveal a molecular parallel in the mechanisms of intraspecific recognition through pheromone detection and pathogen recognition through the innate immune system. CD36 proteins in both invertebrates and vertebrates have been implicated in the recognition of specific lipid-derived products from bacterial cell walls, and coupling of this recognition through Toll-like receptors to initiate the innate immune response. Notably, mammalian CD36 has been proposed as a candidate fat taste receptor (Laugerette, 2005). Common molecular recognition mechanisms in immune and chemosensory systems may therefore be widespread (Benton, 2007).

Search PubMed for articles about Drosophila Snmp1

Bartelt, R. J., Schaner, A. M. and Jackson, L. L. (1985). cis-Vaccenyl acetate as an aggregation pheromone in Drosophila melanogaster. J. Chem. Ecol. 11: 1747-1756. FlyBase Citation: FBrf0043078

Benton, R., Vannice, K. S. and Vosshall, L. B. (2007). An essential role for a CD36-related receptor in pheromone detection in Drosophila. Nature 450(7167): 289-93. PubMed ID: 17943085

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date revised: 9 July 2016

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