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bioRxiv · 10.1101/2023.12.27.573472

Constitutive and conditional epitope-tagging of endogenous G protein coupled receptors in Drosophila

Bonanno, S. L.·Sanfilippo, P.·Eamani, A.·Sampson, M.·Binu, K.·Li, K.·Burns, G.·Makar, M. M.·Zipursky, S. L.·Krantz, D. E.
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Abstract

To visualize the cellular and subcellular localization of neuromodulatory G-protein coupled receptors (GPCRs) in Drosophila, we implement a molecular strategy recently used to add epitope tags to ionotropic receptors at their endogenous loci. Leveraging evolutionary conservation to identify sites more likely to permit insertion of a tag, we generated constitutive and conditional tagged alleles for Drosophila 5-HT1A, 5-HT2A, 5-HT2B, Oct{beta}1R, Oct{beta}2R, two isoforms of OAMB, and mGluR. The conditional alleles allow for the restricted expression of tagged receptor in specific cell types, an option not available for any previous reagents to label these proteins. We show that 5-HT1A and 5-HT2B localize to the mushroom bodies and central complex respectively, as predicted by their roles in sleep. By contrast, the unexpected enrichment of Oct{beta}1R in the central complex and of 5-HT1A and 5-HT2A to nerve terminals in lobular columnar cells in the visual system suggest new hypotheses about their function at these sites. Using an additional tagged allele of the serotonin transporter, a marker of serotonergic tracts, we demonstrate diverse spatial relationships between postsynaptic 5-HT receptors and presynaptic 5-HT neurons, consistent with the importance of both synaptic and volume transmission. Finally, we use the conditional allele of 5-HT1A to show that it localizes to distinct sites within the mushroom bodies as both a postsynaptic receptor in Kenyon cells and a presynaptic autoreceptor. Significance StatementIn Drosophila, despite remarkable advances in both connectomic and genomic studies, antibodies to many aminergic GPCRs are not available. We have overcome this obstacle using evolutionary conservation to identify loci in GPCRs amenable to epitope-tagging, and CRISPR/Cas9 genome editing to generated eight novel lines. This method also may be applied to other GPCRs and allows cell-specific expression of the tagged locus. We have used the tagged alleles we generated to address several questions that remain poorly understood. These include the relationship between pre- and post-synaptic sites that express the same receptor, and the use of relatively distant targets by pre-synaptic release sites that may employ volume transmission as well as standard synaptic signaling.

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Bonanno, S. L., Sanfilippo, P., Eamani, A., Sampson, M., Binu, K., Li, K., Burns, G., Makar, M. M., Zipursky, S. L., Krantz, D. E.. 2023-12-27. Constitutive and conditional epitope-tagging of endogenous G protein coupled receptors in Drosophila. https://doi.org/10.1101/2023.12.27.573472

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