Phosphorylation via PKC Regulates the Function of the Drosophila Odorant Co-Receptor

Bill S Hansson,Dieter Wicher,Merid Negash Getahun,Shannon B Olsson,Sofía Lavista Llanos,Vardanush Sargsyan

doi:10.3389/fncel.2011.00005

Abstract

Insect odorant receptors (ORs) have a unique design of heterodimers formed by an olfactory receptor protein and the ion channel Orco. Heterologously expressed insect ORs are activated via an ionotropic and a metabotropic pathway that leads to cAMP production and activates the Orco channel. The contribution of metabotropic signaling to the insect odor response remains to be elucidated. Disruption of the Gq protein signaling cascade reduces the odor response (Kain et al., 2008). We investigated this phenomenon in HEK293 cells expressing Drosophila Orco and found that phospholipase C (PLC) inhibition reduced the sensitivity of Orco to cAMP. A similar effect was seen upon inhibition of protein kinase C (PKC), whereas PKC stimulation activated Orco even in the absence of cAMP. Mutation of the five PKC phosphorylation sites in Orco almost completely eliminated sensitivity to cAMP. To test the impact of PKC activity in vivo we combined single sensillum electrophysiological recordings with microinjection of agents affecting PLC and PKC function and observed an altered response of olfactory sensory neurons (OSNs) to odorant stimulation. Injection of the PLC inhibitor U73122 or the PKC inhibitor Gö6976 into sensilla reduced the OSN response to odor pulses. Conversely, injection of the PKC activators OAG, a diacylglycerol analog, or phorbol myristate acetate (PMA) enhanced the odor response. We conclude that metabotropic pathways affecting the phosphorylation state of Orco regulate OR function and thereby shape the OSN odor response.

Highlights

Olfaction in nematodes and vertebrates utilizes G protein-coupled receptor (GPCR) signaling
We investigate the effect of Gq protein downstream signaling on heterologously expressed Orco proteins and demonstrate the relevance of these results using single sensillum electrophysiology in Drosophila olfactory sensory neurons (OSNs) combined with microinjection of compounds affecting the Gq protein signaling cascade
Mutant flies with disrupted Gq protein/phospholipase C (PLC) signaling cascade show reduced odor responses (Kain et al, 2008)

Summary

Introduction

Olfaction in nematodes and vertebrates utilizes G protein-coupled receptor (GPCR) signaling. Odorant receptor (OR) proteins share the seven-transmembrane topology of GPCRs but retain no sequence-similarity (Benton et al, 2006). They form heterodimers of a ligand-binding OR and an ubiquitous co-receptor such as Dmel/Orco (previously Or83b in Drosophila; Vosshall and Hansson, 2011), both of which are inversely oriented in the membrane compared to GPCRs (Benton et al, 2006; Lundin et al, 2007). We concentrate on OR22a as ligandbinding receptor. This is probably the most well-investigated OR of D. melanogaster. It is expressed in large basiconic sensilla and is tuned to fruit volatiles (ethyl butyrate, ethyl hexanoate) emitted by, e.g., pineapple (Stensmyr et al, 2003; Hallem and Carlson, 2006)

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