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Using BRET-based G protein biosensors to unravel the G protein mediated pathways in insects

Publication date: 2019-11-15

Author:

Lismont, Els
Vanden Broeck, Jozef

Abstract:

G protein-coupled receptors (GPCRs) are membrane-bound receptors which are candidate targets for novel insect pest management strategies. The superfamily of GPCRs is extensively studied in vertebrates since they are pharmacological targets for many important therapeutic compounds. However, in insects, the molecular signaling properties of GPCRs remain poorly understood and studies are usually limited to the detection of fluctuations in the secondary messenger molecules Ca2+ and cyclic adenosine monophosphate (cAMP). In this study we tested if bioluminescence resonance energy transfer (BRET)2-based G protein biosensors, developed to detect a direct activation Gα protein subununits in vertebrates, can also be used to detect Gα protein subununit activation upon insect GPCR activation. We have tested these BRET2-based G protein biosensors, representing members of all four Gα protein subfamilies (Galés et al., 2006), and have shown that they respond to activation of insect GPCRs for three out of four Gα protein subfamilies, namely Gαi/o, Gαs and Gαq/11. In order to test these BRET2-based G protein biosensors, we have characterized three insect receptors from two insect species. The first insect species, the buff-tailed bumblebee, Bombus terrestris, is a beneficial insect that is regularly used as a pollinator of greenhouse crops. In this insect, we have selected the SIFamide receptor (SIFR) which belongs to the Family A GPCRs since almost nothing is known about the downstream signaling pathways of this receptor. We have demonstrated that stimulation of Bomte‑SIFR by Bomte-SIFa activates the Gαi/o and Gαq biosensors. We have also studied the downstream signaling properties of the secondary messenger molecules Ca2+ and cAMP, the agonistic properties of a range of related modified peptides and examined the transcript levels of Bomte-SIFa and Bomte-SIFR in a variety of tissues. The second insect species, the desert locust Schistocerca gregaria, is considered a major pest insect since it can form devastating swarms which have a strong negative impact on agricultural production and human welfare. In this insect we have characterized two receptors from two different GPCR families. The first receptor is the allatotropin receptor (ATR) which belongs to the Family A GPCRs. We have demonstrated that stimulation of Schgr-ATR by Schgr-AT activates the Gαi/o and Gαq/11 biosensors. We have also studied the downstream signaling properties of the secondary messenger molecules Ca2+ and cAMP and found more evidence for the myotropic and allatostimulatory actions of Schgr-AT. In addition, we studied the transcript levels of Schgr-AT and Schgr-ATR in a variety of tissues. The second receptor we characterized in S. gregaria and the third receptor in this study, is the corticotropin-releasing factor (CRF)-related diuretic hormone (DH) receptor (CRF‑DHR1) which belongs to the Family B GPCRs. For this neuropeptide receptor system we have identified three putative receptor sequences (Schgr‑CRF‑DHR1, Schgr-CRF-DHR2 and Schgr-CRF-DHR3) in the (unpublished, in-house) transcriptome database (Verdonck, 2017) and were able to clone the first receptor, namely Schgr‑CRF-DHR1. We have demonstrated that stimulation of Schgr-CRF-DHR1 by Schgr-CRF-DH activates the Gαi/o and Gαs biosensors. We have also studied the downstream signaling properties of the secondary messenger molecules Ca2+ and cAMP and studied the transcript levels of two receptors, namely Schgr‑CRF‑DHR1 and Schgr-CRF-DHR2. If Schgr-CRF-DHR2 and Schgr-CRF-DHR3 exist in vivo and if they are receptors for Schgr-CRF-DH remains a question for future studies. Although we have shown an activation of biosensors from three Gα protein subfamilies, namely Gαi/o, Gαs and Gαq/11, we could not show an activation of any biosensors from the Gα12/13 subfamily. By comparing human Gα sequences with their homologs of B. terrestris and S. gregaria, we found that the Gα12/13 subfamily is less conserved than the three other Gα protein subfamilies.