Insights into the Structure and Function of an Insect Olfactory Receptor Using FRET

Abstract

Insect odorant receptors (ORs) are a novel family of chemosensory receptors that can respond to volatile organic compounds leading to different behaviours such as finding food and location sites to lay eggs, escaping from predators, and finding mates. These receptors form complexes containing at least two subunit members; the noncanonical OR co-receptor (ORCO) and a specificity OR. In addition, the sensory neuron membrane proteins SNMP1 and SNMP2 are also associated with olfactory function, with SNMP1 required for pheromone perception in Drosophila melanogaster. However their exact role and mode of action remain to be elucidated. In this study, fluorescence resonance energy transfer (FRET) was used to gain insights into the structure and function of the specificity OR from D. melanogaster OR22a. In particular, questions regarding protein-protein interactions and the activation mechanism were addressed. First, different heterologous expression systems were tested with the aim to assess which system would be most suitable for FRET studies for the expression of OR22a and ORCO fusion proteins tagged with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). The heterologous expression systems trialled were transient expression in S2 cells, Sf9 cells, and mammalian HEK293 cells, as well as baculovirus-mediated expression in Sf9 and Hi5 cells. Expression was assessed in terms of fluorescence intensity, quality and integrity of expression, and localisation. None of the systems showed good cell surface expression. Due to the higher levels of fluorescence intensity the aculovirus-mediated expression system was selected. In general, baculovirus-mediated expression in Hi5 cells produced full length fusion proteins at levels easily detected by confocal microscopy. Second, FRET experiments were carried out to investigate homomeric and heteromeric interactions involving OR22a, ORCO, SNMP1, and SNMP2 fusion proteins tagged with CFP and YFP. FRET measurements and analysis were optimised allowing the collection and analysis of accurate data. In particular, quantitative FRET analysis looking at the relationship between FRET efficiency of energy transfer (E). and the ratio between YFP and CFP was essential to distinguish specific FRET from random FRET. Inter-molecular FRET evidence indicates that the combinations ORCO + ORCO, ORCO + OR22a, OR22a + OR22a, and SNMP1 + OR22a show FRET E due to specific interactions when expressed in Hi5 cells with baculovirus. This evidence suggests a new model for the formation of the ORCO-OR complex and a possible new role for SNMP1 in general odorant perception. Third, a previous study had suggested that inter-molecular distances and regions of interaction between two subunits could be investigated by inter-molecular FRET measurements with OR22a fusion proteins tagged with CFP and YFP within intracellular loops (ILs; Kiely, 2008). This idea was examined by measuring intermolecular FRET E between different OR22a + OR22a, ORCO + ORCO, and OR22a + ORCO combinations involving OR22a and ORCO fusion proteins tagged with YFP and CFP within ILs. No differences in FRET E were observed for different combinations. Finally, intra-molecular FRET was measured for OR22a constructs labelled with both CFP and YFP. Dynamic FRET experiments were carried out to detect conformational changes upon odorant binding. No changes in FRET E were observed.