They also BIBW2992 nmr produced flies expressing the C-terminal half of YFP fused to the intracellular region of tethered OBP49a in the same neurons. When flies expressing the Gr64a receptor fusion were crossed to the tethered OBP49a-YFP fusion, strong fluorescence was detected in the labellum, suggesting that the intracellular domains of Gr64a and the membrane-tethered OBP49a are in close proximity. These findings are consistent with OBP49a interacting with, and inhibiting, sweet responses through Gr64a (Figure 2D). Surprisingly,

no fluorescence was detected when the tethered OBP49a-YFP flies were crossed to the Gr64f-YFP fusion, indicating there may be a specific interaction between OBP49a and the Gr64a subunit that permits association of the two YFP fragments. Addition of bitter ligands did not alter the fluorescence in either combination. This could mean that OBP49a is always bound to the receptor and only inhibits when bitters are present, or perhaps that adding an

artificial membrane anchor to OBP49a results in some structural configuration that is only able to interact with Gr49a-YFP. Split YFP experiments have to be interpreted with caution, as these findings only demonstrate that the proteins are in INK1197 mw close proximity and do not implicate or rule out any specific protein-protein interactions between OBP49a and membrane receptor subunits. Additional work remains to demonstrate exactly how this inhibition works at the receptor level. Does OBP49a actually bind to tastants? Jeong et al. (2013) purified OBP49a from flies and bound it to sensor chips and used surface

Histamine H2 receptor plasmon resonance to examine what tastants bind to OBP49a. They found that bitter chemicals bound to OBP49a in a dose-dependent manner, but sucrose did not. Together, these data support a model in which OBP49a binds to bitter tastants and inhibits the firing of the sugar-sensing neurons, possibly by direct interactions with the neuronal sweet taste receptors (Figure 2D). OBP49a is expressed in all sensilla on the labellum, so while L-type sensilla were studied by Jeong et al. (2013) to rule out potential crosstalk between the bitter-sensing neurons and sweet-sensing neurons in the same sensilla, it is likely that this mechanism is also present in the S-type and I-type type sensilla as well. This would be consistent with the potent effects observed in the OBP49a mutants on bitter avoidance behavior. These data support the controversial view that members of the odorant-binding protein family can directly interact with membrane receptors in a ligand-dependent manner and influence neuronal activity. To fully understand how OBP49a functions, some structural studies are in order. OBP49a is 30% larger than most mature OBP proteins and contains 12 cysteines instead of 6 (Nagnan-Le Meillour and Jacquin-Joly, 2003).