InteractiveFly: GeneBrief

Sensory neuron membrane protein: Biological Overview | References

Gene name - Sensory neuron membrane protein

Synonyms -

Cytological map position - 93C2-93C2

Function - transmembrane receptor

Keywords - pheromone response, olfactory sensory neurons, cofactor for capture of pheromone molecules

Symbol - Snmp

FlyBase ID: FBgn0260004

Genetic map position - 3R:16,991,220..16,993,927 [-]

Classification - CD36 family

Cellular location - surface transmembrane

NCBI links: | EntrezGene

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).

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 Or83bZ4506 and Or83bZ0061 (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 Or67dZ5499 (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 (SnmpZ0429) 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 SnmpZ0429 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 SnmpZ0429 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, SnmpZ0429 double mutants have high spontaneous activity as observed in SnmpZ0429 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).

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, LushD118A, 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 LushD118A 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, LushD118A, 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. LushD118A 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).


Search PubMed for articles about Drosophila Snmp

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

Bonen, A. et al. (2004). Regulation of fatty acid transport by fatty acid translocase/CD36. Proc. Nutr. Soc. 63: 245-249. PubMed ID: 15294038

Coburn, C. T., et al. (2000). Defective uptake and utilization of long chain fatty acids in muscle and adipose tissues of CD36 knockout mice. J. Biol. Chem. 275: 32523-32529. PubMed ID: 10913136

Collot-Teixeira, S., et al. (2007). CD36 and macrophages in atherosclerosis. Cardiovasc. Res. 75: 468-77. PubMed ID: 17442283

Ejima, A., et al. (2007). Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate. Curr. Biol. 17(7): 599-605. PubMed ID: 17363250

Febbraio, M., Guy, E. and Silverstein, R. L. (2004). Stem cell transplantation reveals that absence of macrophage CD36 is protective against atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 24: 2333-2338. PubMed ID: 15486305

Febbraio, M. and Silverstein, R. L. (2007). CD36: Implications in cardiovascular disease. Int. J. Biochem. Cell Biol. 39: 2012-2030. PubMed ID: 17466567

Franc, N. C., et al. (1996). Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. Immunity 4: 431-443. PubMed ID: 8630729

Ge, Y. and Elghetany, M. T. (2005). CD36: a multiligand molecule. Lab. Hematol. 11: 31-37. PubMed ID: 15790550

Grosse-Wilde, E., Gohl, T., Bouche, E., Breer, H. and Krieger, J. (2007). Candidate pheromone receptors provide the basis for the response of distinct antennal neurons to pheromonal compounds. Eur. J. Neurosci. 25: 2364-2373. PubMed ID: 17445234

Gu, G., Yang, J., Mitchell, K. A. and O'Tousa, J. E. (2004). Drosophila ninaB and ninaD act outside of retina to produce rhodopsin chromophore. J. Biol. Chem. 279: 18608-18613. PubMed ID: 14982930

Hirano, K, et al. (2003). Pathophysiology of human genetic CD36 deficiency. Trends Cardiovasc. Med. 13: 136-141. PubMed ID: 12732446

Hoebe, K. et al. (2005). CD36 is a sensor of diacylglycerides. Nature 433: 523-527. PubMed ID: 15690042

Jin, X., Ha, T. S. and Smith, D. P. (2008). SNMP is a signaling component required for pheromone sensitivity in Drosophila. Proc. Natl. Acad. Sci. 105(31): 10996-1001. PubMed ID: 18653762

Kiefer, C., Sumser, E., Wernet, M. F. and Von Lintig, J. A. (2002). Class B scavenger receptor mediates the cellular uptake of carotenoids in Drosophila. Proc. Natl. Acad. Sci. 99: 10581-10586. PubMed ID: 12136129

Kurtovic, A., Widmer, A. and Dickson, B. J. (2007). A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. Nature 446(7135): 542-6. PubMed ID: 17392786

Laugerette, F., et al. (2005). CD36 involvement in orosensory detection of dietary lipids, spontaneous fat preference, and digestive secretions. J. Clin. Invest. 115(11): 3177-84. PubMed ID: 16276419

Laughlin, J. D., Ha, T. S., Jones, D. N. and Smith, D. P. (2008). Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein. Cell 133: 1255-1265. PubMed ID: 18585358

Li, L., Stoeckert, C. J. and Roos, D. S. (2003). OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 13: 2178-2189. PubMed ID: 12952885

Miyaoka, K, et al. (2001). CD36 deficiency associated with insulin resistance. Lancet. 357: 686-687. PubMed ID: 11247555

Philips, J. A., Rubin, E. J. and Perrimon, N. (2005). Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection. Science 309: 1251-1253. PubMed ID: 16020694

Pravenec, M. and Kurtz, T. W. (2007). Molecular genetics of experimental hypertension and the metabolic syndrome: From gene pathways to new therapies. Hypertension 49: 941-952. PubMed ID: 17339535

Rahaman, S. O., et al. (2006). A CD36-dependent signaling cascade is necessary for macrophage foam cell formation. Cell Metab. 4: 211-221. PubMed ID: 16950138

Rogers, M. E., Steinbrecht, R. A., and Vogt, R. G. (2001a). Expression of SNMP-1 in olfactory neurons and sensilla of male and female antennae of the silkmoth Antheraea polyphemus. Cell Tissue Res. 303: 433-446. PubMed ID: 11320659

Rogers, M, E., Krieger, J., and Vogt, R. G. (2001b). Antennal SNMPs (sensory neuron membrane proteins) of Lepidoptera define a unique family of invertebrate CD36-like proteins. J. Neurobiol. 49: 47-61. PubMed ID: 11536197

Rogers, M. E., Sun, M., Lerner, M. and Vogt, R. G. (1997). Snmp-1, a novel membrane protein of olfactory neurons of the silk moth Antherea polyphemus with homology to the CD36 family of membrane proteins. J. Biol. Chem. 272: 14792-14799. PubMed ID: 9169446

Stuart, L. M., et al. (2005). Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain. J. Cell Biol. 170: 477-485. PubMed ID: 16061696

Wang, T., Jiao, Y. and Montell, C. (2007). Dissection of the pathway required for generation of vitamin A and for Drosophila phototransduction. J. Cell Biol. 177: 305-316. PubMed ID: 17452532

Xu, P., Atkinson, R., Jones, D. N. and Smith, D. P. (2005). Drosophila OBP Lush is required for activity of pheromone-sensitive neurons. Neuron 45: 193-200. PubMed ID: 15664171

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date revised: 30 June 2009

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