Constitutive and Conditional Epitope Tagging of Endogenous G-Protein–Coupled Receptors inDrosophila

Abstract

To visualize the cellular and subcellular localization of neuromodulatory G-protein–coupled receptors inDrosophila, 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 forDrosophila5-HT1A,5-HT2A,5-HT2B,Octβ1R,Octβ2R, two isoforms ofOAMB, andmGluR. 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 expression patterns for these receptors in female brains and that 5-HT1A and 5-HT2B localize to the mushroom bodies (MBs) and central complex, respectively, as predicted by their roles in sleep. By contrast, the unexpected enrichment of Octβ1R 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 functions 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 of5-HT1Ato show that it localizes to distinct sites within the MBs as both a postsynaptic receptor in Kenyon cells and a presynaptic autoreceptor.