Rabbit polyclonal antiserum was raised to bacterially expressed Lush protein for direct examination of the expression of this protein in wild-type and lush flies. Affinity-purified anti-Lush antibodies recognize protein in accessory cells of trichoid sensilla on the ventral-lateral portion of the third-antennal segment in wild-type males and females, in a pattern identical to LacZ expression in ET249. In contrast to the LacZ that is localized to the support cell cytoplasm in ET249 flies, Lush protein is clearly present within the shafts of the trichoid sensilla, confirming that it is secreted into the sensillum lymph. No labeling of olfactory neurons is observed. Western blots of antennal extracts from wild-type and lush mutant flies probed with anti-Lush antiserum reveal that the mutants are completely defective for Lush expression. Southern blot analysis of lush mutant DNA confirms that the 3-kb deletion removes the entire protein-coding region of the lush gene. This suggests that loss of this odorant-binding protein gene is responsible for the chemosensory defects in lush mutants (Kim, 1998).

Effects of Mutation or Deletion

Drosophila OBP LUSH is required for activity of pheromone-sensitive neurons

Odorant binding proteins (OBPs) are extracellular proteins localized to the chemosensory systems of most terrestrial species. OBPs are expressed by nonneuronal cells and secreted into the fluid bathing olfactory neuron dendrites. Several members have been shown to interact directly with odorants, but the significance of this is not clear. Mutants of the Drosophila OBP lush are completely devoid of evoked activity to the pheromone 11-cis vaccenyl acetate (VA), revealing that this binding protein is absolutely required for activation of pheromone-sensitive chemosensory neurons. lush mutants are also defective for pheromone-evoked behavior. Importantly, a genetic effect of lush mutants on spontaneous activity in VA-sensitive neurons in the absence of pheromone is described. The defects in spontaneous activity and VA sensitivity are reversed by germline transformation with a lush transgene or by introducing recombinant Lush protein into mutant sensilla. These studies directly link pheromone-induced behavior with OBP-dependent activation of a subset of olfactory neurons (Xu, 2005).

Mutants lacking Lush are insensitive to 11-cis vaccenyl acetate, both at the level of olfactory neuron activation and at the level of aggregation behavior normally elicited by this pheromone. These defects are due specifically to loss of Lush expression in T1 sensilla, because transgenic expression of Lush protein restores function. Other members of the OBP family (OBP83a, or OS-F and OBP83b, or OS-E and moth APO3, or unfolded Lush) do not functionally compensate for the loss of Lush. Therefore, Lush is absolutely and specifically required in the sensillum lymph for VA pheromone signal transduction by T1 neurons, providing a clear demonstration that an OBP member functions in olfaction and mediates activation of olfactory neurons (Xu, 2005).

lush mutants are also defective for spontaneous activity in T1 neurons. This unexpected pheromone-independent phenotype reveals a genetic interaction between Lush and T1 neuron activation mechanisms. T1 neurons do not produce Lush and do not require it for cell fate determination or general health, since direct introduction of recombinant Lush restores function as fast as the protein can diffuse into the sensillum lymph. This time frame would seem too short for any growth factor-like signal requiring transcription and translation. Individual action potentials have normal shape and kinetics in lush mutants, further suggesting there is no intrinsic defect in the T1 neurons. Even relatively dilute preparations of exogenous Lush restore spontaneous activity and VA sensitivity, while expression of other OBPs at high levels does not. This eliminates any nonspecific osmotic effects resulting from absence of the abundant Lush protein in the sensillum lymph producing the observed defects. Finally, VA activates wild-type T1 but not T2 sensilla, though both sensilla types express Lush. This demonstrates a requirement for both Lush and a T1 neuron-specific factor. The simplest explanation consistent with these findings is that extracellular Lush protein can stimulate, directly or indirectly, T1 neurons to produce action potentials through an unknown T1-specific receptor (Xu, 2005).

Members of the OBP family interact directly with odorant ligands, leading to several proposals for OBP function. For example, OBPs may function by removing or inactivating odorants from the sensillum lymph, by solubilizing hydrophobic pheromone ligands, by concentrating pheromone molecules in the lymph, by acting as filters to screen out subsets of odorants, by functioning as buffers to prevent saturation of the responses during high stimulus intensities, or by transporting odorants to the olfactory neurons or to act as coreceptors with odorants to activate olfactory neurons. Recent structural studies have led to the proposal that local pH changes near dendrites might induce unloading of pheromone that subsequently proceeds to activate neuronal receptors. However, there has been little direct in vivo evidence to support or refute these models. Diffusion of antiserum to an OBP in taste sensilla reduced activity, suggesting that the binding protein might facilitate activation of a chemosensory neuron. A role for binding proteins has been implicated in the specificity of neuronal activation in moths. This work showed that the wrong pheromone could activate a pheromone-sensitive neuron when prebound to an OBP that normally binds the activating pheromone (Pophof, 2002). Lush OBP is absolutely required for activation of T1 neurons by VA. This finding is not consistent with odorant removal as a sole function for Lush. Similarly, a role in activation of pheromone-sensitive neurons indicates that Lush is not a buffer or filter for VA. The data suggest that Lush activates T1 neuronal surface receptors responsible for action potential generation. Therefore, while Lush may bind and transport pheromone, it is not a simple carrier or solubilizing factor for pheromone but instead has a more specific role as a signal transduction component. This model would be consistent with the findings of Pophof in the moth system (Pophof, 2002) and may reflect a general mechanism through which OBPs function in insects (Xu, 2005).

A working model is proposed in which Lush functions as an adaptor to bridge the presence of gaseous pheromone molecules to activation of specific neuronal receptors expressed on T1 olfactory neurons. VA may induce a specific conformational change in Lush protein that in turn activates T1 receptors. If such a conformational change occurs spontaneously at low frequency, this would explain the observed loss of spontaneous activity in lush mutants. Ligand-induced conformational changes have been reported previously in pheromone binding proteins from Mamestra brassicae and Bombyx mori upon pheromone binding (Campanacci, 2001; Horst, 2001; Wojtasek, 1999). An important test of this model will be to show that VA pheromone itself is not a direct activator of T1 olfactory neurons, but triggers neuronal activity indirectly through conformational changes in Lush. Consistent with this idea, even 100% VA is incapable of producing activity in T1 neurons in lush mutants. If Lush is the ligand for the T1 receptors, this would refute the pH release model that posits that pheromone release from the OBP mediating activation of neuronal receptors. Alternatively, components of both Lush and an exposed portion of the bound pheromone may activate neuronal receptors (Kaissling, 2001). Lush could also act indirectly by recruiting other factors in the sensillum lymph that ultimately activate T1 neurons. Solving the X-ray crystal structure of the Lush-VA complex and identifying the neuronal receptors that mediate VA sensitivity will allow this model to be corroborated or refuted. Finally, Lush also influences the alcohol responses of T2B neurons. lush mutants are defective for avoidance of concentrated alcohols, and lush mutant T2B neurons fail to show inhibition by concentrated alcohol. Both defects are reversed by expression of a wild-type lush transgene, suggesting the Lush-dependent inhibition of T2B neurons results in behavioral avoidance. This finding would be consistent with the model if a Lush-alcohol complex resulted in activation of T2B receptors and activation of these receptors inhibits these neurons. Inhibition of Drosophila olfactory neurons by odorants has been reported in the literature. Lush binds ethanol (Kruse, 2003) and several phthalate compounds in vitro (Zhou, 2004). Phthalates, even at full strength, do not influence activity in trichoid neurons in the current study. Ethanol influences the responses of T2B neurons but not T2A or T1 neurons, and VA has no effect on T2 neurons. This suggests that ligand binding to a binding protein does not confer universal biological activity in vivo. Instead, different ligands may induce distinct conformations in the binding protein, or perhaps form part of a receptor interaction domain that is discriminated by different neuronal receptors. Structural analysis of these complexes with receptors will allow these interactions to be defined (Xu, 2005).

Social aggregation behavior is induced by activation of T1 neurons, revealing the first stage of a neuronal circuit mediating aggregation in this insect. It is not clear why an aggregation pheromone would be produced only in males. Perhaps this aids roaming flies to identify a safe environment to mate and lay eggs. VA appears to act synergistically with food odorants, consistent with this notion. In Drosophila, at least 35 genes encode OBPs expressed in virtually every Drosophila olfactory and gustatory organ. Recently, a putative taste receptor was implicated in detecting contact pheromone during mating in Drosophila. It will be interesting to determine if OBPs are required for this behavior and if other members of the chemosensory gene family are required for VA sensitivity in T1 neurons. Similarly, it would be of great importance to determine if other OBPs mediate additional behaviors in this animal. In fire ants, the number of egg-laying queens determines the size of the colony. Queen number is determined by workers that kill extra queens depending on the allele of Gp-9 the workers carry. Gp-9 has been identified as a member of the odorant binding protein family (Krieger, 2002). Therefore, it is possible that binding proteins mediate a diverse array of pheromone-mediated social interactions in insects. It should be feasible to design synthetic ligands capable of interacting and inducing appropriate conformational changes with various binding proteins, permitting the manipulation of these signaling pathways. Such compounds could be used to control any number of insect behaviors including aggregation, mating, and colony size (Xu, 2005).

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).


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lush: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

date revised: 10 February 2013

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