Lush is a soluble odorant-binding protein of the fruit fly Drosophila. Mutants not expressing this protein have been reported to lack the avoidance behaviour, exhibited by wild type flies, to high concentrations of ethanol. Very recently, the three-dimensional structure of Lush complexed with short-chain alcohols has been resolved, supporting a role for this protein in binding and detecting small alcohols. Lush does not bind ethanol and wild type flies are in fact attracted by high concentrations of ethanol. Lush binds some phthalates and flies are repelled by such compounds. Finally, fluorescence data, interpreted in the light of the three-dimensional structure of Lush, indicate that the protein undergoes a major conformational change, similar to that reported for the pheromone-binding protein of Bombyx mori, but triggered, in the case of Lush, by ligand (Zhou, 2004).

The behaviour was examined of wild type D. melanogaster to different concentrations of ethanol as well as to dibutyl phthalate, which proved to be the best ligand of Lush. Initially the flies were tested with traps containing food (mashed rotten apple) mixed with ethanol at final concentrations of 1% and 50%. The flies clearly preferred the higher ethanol concentration. To avoid effects of possible contaminants, the absence of organic compounds particularly of phthalates, was verified in the sample of ethanol by GC/MS. Since in these conditions no peak of contaminants was detectable, it is estimated that the concentration of any of such compounds, if present, would be lower than 10 ppm (Zhou, 2004).

Similar experiments were performed with pairs of traps containing dibutyl phthalate at the final concentration of 1 mM, diluted in mashed apples or in 50% ethanol in agarose. In each case the same diluent (mashed apple or 50% ethanol in agarose) was used in the control trap. The flies showed a statistically significant (P<0.001) behaviour of avoidance to dibutyl phthalate, regardless of the medium used (Zhou, 2004).

These behaviour results and the binding affinity of Lush to some phthalates provide a new interpretation of the phenomenon previously observed in previous studies. The original hypothesis stated that Lush could modify the perception of ethanol at high concentrations. This requires some specific binding of ethanol to Lush, which in the current study was not detected. It can be argued that a second binding site could be present on the protein, where ethanol could bind, without affecting the binding of 1-NPN in the other pocket. This hypothesis, however, is not supported by the recently published X-ray structure, which shows the presence of a single binding site in Lush, in agreement with previous reports for other insect OBPs. Moreover, the behaviour experiments performed with pure ethanol clearly show that this compound is a potent attractant for wild type flies at high concentrations (Zhou, 2004 and references therein).

A reinterpretation of previous results, assuming that Lush is specific for aromatic compounds structurally similar to phthalates, rather than for ethanol, provides the first and only evidence that insects use OBPs as tools to distinguish different odorants (Zhou, 2004).

This finding also suggests that binding experiments could be used as a first screening to indicate which OBP gene should be deleted or inactivated in order to get mutants with desired types of anosmia (Zhou, 2004).

In the light of these results, it is concluded that the avoidance previously reported for wild type flies could be due to impurities, such as phthalates or structurally related compounds, present as contaminants in the ethanol used. Furthermore, the loss of avoidance in the mutants would result from the inability to detect large aromatic compounds. This conclusion provides new and increased interest for the previously published data, since Lush would be directly and strictly required for the perception of an odorant, rather than being involved only in modulating the response to ethanol. The recently published structure of Lush with a molecule of butanol in its binding pocket does not, in light of the current study, show a true binding role of this protein for small alcohols (Zhou, 2004).

The C-terminus of Lush, which contains the only tryptophan residue of the protein, is located inside the core of the protein. When the effect of ligands on the tryptophan fluorescence was investigated, it was not possible to measure any quenching up to ligand concentrations of 16 microM. This could indicate that when the ligands (1-NPN or phthalate) enter the binding site, the C-terminus of the protein is displaced and the tryptophan residue moves outside the core of the protein. Such a major conformational change has been observed with the pheromone-binding protein of Bombyx mori, as an effect of a pH increase from 4.5 to 6.5. In the current case, the effect would occur at pH 7 and, most interestingly, as a consequence of ligand binding. This hypothesis, if verified in the structure of Lush complexed with a ligand, would represent the first example of a major conformational change of an OBP related to substrate binding and have interesting implications for the mode of action of these proteins and their interactions with membrane-bound receptorswe investigated (Zhou, 2004).

Insect pheromones elicit stereotypic behaviors that are critical for survival and reproduction. Defining the relevant molecular mechanisms mediating pheromone signaling is an important step to manipulate pheromone-induced behaviors in pathogenic or agriculturally important pests. The only volatile pheromone identified in Drosophila is 11-cis-vaccenyl acetate (VA), a male-specific lipid that mediates aggregation behavior. VA activates a few dozen olfactory neurons located in T1 sensilla on the antenna of both male and female flies. This study identified a neuronal receptor required for VA sensitivity. Two mutants were identified lacking functional T1 sensilla; the expression of the VA receptor is dramatically reduced or eliminated. Importantly, misexpression of this receptor in non-T1 neurons, normally insensitive to VA, confers pheromone sensitivity at physiologic concentrations. Sensitivity of T1 neurons to VA requires Lush, an extracellular odorant-binding protein (OBP76a) present in the sensillum lymph bathing trichoid olfactory neuron dendrites. This study shows that Lush is also required in non-T1 neurons misexpressing the receptor to respond to VA. These data provide new insight into the molecular components and neuronal basis of volatile pheromone perception (Ha, 2006).

With the goal of identifying all genetic loci required for VA pheromone detection, a screen was undertaken to recover mutants with abnormal electrophysiological responses to VA pheromone. Two mutants were identified out of 1200 lines that lack functional T1 sensilla. tod¹ and tot¹ have normal basiconic and non-T1 sensilla, but no T1 sensilla. tod¹ and tot¹ are both recessive and fail to complement one another, revealing these mutants lack T1 sensilla as a result of lesions in independent genes. In normal flies, there is a mixture of T1 and non-T1 subtypes in the trichoid zone, but the proximal part of this zone is enriched in T1 sensilla. Morphologically, all trichoid sensilla are indistinguishable. However, T1 and non-T1 sensilla are clearly distinguishable by electrophysiology. In a survey of over 2000 animals, recordings from random trichoid sensilla in the proximal zone identify VA-sensitive T1 sensilla 89% of the time and non-T1 11% of the time based on spontaneous activity rate and sensitivity to VA. VA could evoke activity in T1 neurons from 0.35 ± 0.14 spikes per second before stimulation to 36.89 ± 3.43 after stimulation. VA did not induce activity in non-T1 neurons. Recordings from either tod¹ mutants or tot¹ mutants from the T1 zone always identified non-T1 sensilla. These sensilla have the classic characteristics of the non-T1 type, including lack of response to VA, more than one neuron present in the sensillum, and a high rate of spontaneous activity. Trichoid and large basiconic sensilla from across the antenna were surveyed in tod¹ mutants and tot¹ mutants, and no VA-responsive neurons could be identified. Therefore, there does not appear to be a simple mislocalization of T1 sensilla to a different part of the antenna, but a complete loss of the T1 functional type (Ha, 2006).

tod¹ and tot¹ mutants lack functional T1 sensilla. Therefore, T1-neuron specific gene products should be absent in these mutants. It was reasoned that a neuronal receptor mediating VA responses might be a member of the odorant receptor family expressed specifically by T1 neurons. Indeed, Ors have been shown to specify odor specificity to olfactory neurons in a number of systems, including Drosophila. Therefore, candidate members of the Drosophila Or gene family were screened for reduced expression in tod¹ and tot¹ mutants. An Or67d spliced transcript was found to be clearly present in wild-type antennas, but is reduced or absent in tod¹ and tot¹ mutants, even after 40 cycles of amplification. Or83b, expressed in most olfactory receptor neurons was present in all three samples, as were all other Or genes tested. These results suggest Or67d expression is specifically reduced or eliminated in tod¹ and tot¹ mutants, and correlates with the loss of the T1 functional class in these genetically distinct mutants (Ha, 2006).

Having identified a candidate receptor correlating with the presence of T1 neurons, attempts were made to establish whether the expression pattern of Or67d in the antenna was consistent with the known T1 neuron distribution. in situ hybridization was performed using fluorescently labeled antisense RNA probes to Or67d to characterize expression of this putative receptor. Antisense probes to Or67d specifically label cells on the ventral–lateral surface of the third antennal segment. Serial sections reveal the labeled cells are concentrated in the proximal T1 zone. These probes failed to identify similar positive cells in antenna tod¹ or tot¹ (3C) consistent with the functional loss of T1 sensilla. Therefore, Or67d expression correlates well with the known distribution of T1 sensilla in wild-type antenna and the absence of T1 sensilla in the mutants (Ha, 2006).

Expression of Or67d in the T1 zone and its absence in tod¹ and tot¹ mutants is consistent with Or67d being the T1 VA receptor, but does not prove this receptor is responsible for VA sensitivity. For example, Or67d may have some functional role specific to T1 neurons that is unrelated to VA sensitivity. Alternatively, a subset of non-T1 class olfactory neurons may also be absent in tod¹ and tot¹ that specifically express Or67d. Therefore, to definitively prove Or67d mediates VA sensitivity, Or67d was misexpressed in olfactory neurons that normally do not express this receptor and are VA insensitive. Previous work has shown that coexpression of an extra odorant receptor in a Drosophila olfactory neuron results in an odor sensitivity profile that is the combination of the sensitivity of the individual receptors. Therefore, Or67d was misexpressed in all neurons by driving Or67d expression with the pan-neuron promoter, ELAV. VA sensitivity of wild-type animals and those misexpressing Or67d in non-T1 sensilla was examined. Neurons in wild-type non-T1 sensilla are insensitive to VA. However, animals misexpressing Or67d in all neurons have non-T1 neurons that are highly responsive to VA. Indeed, dose–response analysis reveals these neurons are nearly as sensitive to VA as wild-type T1 neurons. By all other criteria, these neurons are non-T1 and not T1 neurons. They display high spontaneous activity and contain multiple neurons, and their distribution is typical of the non-T1 functional class. The only difference observed in these neurons compared with wild-type controls was VA sensitivity. Conferring VA sensitivity on non-T1 neurons by expressing Or67d receptors demonstrates that this receptor is both necessary and sufficient to confer VA sensitivity on non-T1 neurons (Ha, 2006).

Lush protein is required in the T1 sensillum lymph for T1 neurons to be sensitive to VA. Non-T1 sensilla also express Lush protein in the sensillum lymph. Is Lush also required for sensitivity of non-T1 neurons misexpressing Or67d? The lush¹ mutation was crossed into the stock misexpressing Or67d in all neurons. Lush protein is critical for non-T1 neurons to respond to VA as well, because when the lush¹ mutation is crossed into the misexpressing flies, VA sensitivity is lost in non-T1 neurons. This clearly demonstrates that Lush is required in the extracellular space in order for non-T1 neurons misexpressing Or67d to be responsive to VA. Therefore, both the receptor Or67d and a specific extracellular binding protein, Lush, are required for VA sensitivity (Ha, 2006).