دورية أكاديمية
Constitutive and conditional epitope-tagging of endogenous G protein coupled receptors in Drosophila .
| العنوان: | Constitutive and conditional epitope-tagging of endogenous G protein coupled receptors in Drosophila . |
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| المؤلفون: | Bonanno SL; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA., Sanfilippo P; Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.; Howard Hughes Medical Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA., Eamani A; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA., Sampson MM; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA., Kandagedon B; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA., Li K; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA., Burns GD; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA., Makar ME; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA., Zipursky SL; Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.; Howard Hughes Medical Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA., Krantz DE; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA. |
| المصدر: | The Journal of neuroscience : the official journal of the Society for Neuroscience [J Neurosci] 2024 Jun 27. Date of Electronic Publication: 2024 Jun 27. |
| Publication Model: | Ahead of Print |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Society for Neuroscience Country of Publication: United States NLM ID: 8102140 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1529-2401 (Electronic) Linking ISSN: 02706474 NLM ISO Abbreviation: J Neurosci Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: Washington, DC : Society for Neuroscience Original Publication: [Baltimore, Md.] : The Society, c1981- |
| مستخلص: | 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, Octβ1R, Octβ2R, 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 expression patterns for these receptors in female brains, and 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 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 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 Statement In 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 generate eight novel lines. This method may also be applied to other GPCRs and allows cell-specific expression of the tagged receptor. We have used the tagged alleles we generated to address several questions that remain poorly understood. These include the relationship between pre- and postsynaptic sites that express the same receptor, and the use of relatively distant targets by presynaptic release sites that may employ volume transmission as well as standard synaptic signaling. (Copyright © 2024 the authors.) |
| التعليقات: | Update of: bioRxiv. 2023 Dec 27:2023.12.27.573472. doi: 10.1101/2023.12.27.573472. (PMID: 38234787) |
| تواريخ الأحداث: | Date Created: 20240627 Latest Revision: 20240708 |
| رمز التحديث: | 20240708 |
| DOI: | 10.1523/JNEUROSCI.2377-23.2024 |
| PMID: | 38937100 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1529-2401 |
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| DOI: | 10.1523/JNEUROSCI.2377-23.2024 |