Analysis of the signaling pathway of the adhesion GPCR homolog CG15744/Remoulade in Drosophila

• Applicant: Beatriz Blanco Redondo, Ph.D.
• Subject Area: Pharmacology
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 574945774

The Drosophila genome includes five loci coding for adhesion Gprotein coupled receptors (aGPCRs). Phylogenetic analysis indicates that the remoulade (remo) gene is a homolog of the vertebrate ADGRA family, sharing a similar receptor domain structure. In-vivo expression shows Remo's presence in the central nervous system (CNS) and peripheral nervous system (PNS) larvae (L3) and adult flies. In L3 PNS, Remo is expressed in pickpocket (PPK) neurons, which are involved in sensory information transduction, including nociception. Larvae with remo knocked out (remoKO) and those with remo knocked down in PPK-neurons via RNA interference show increased nocifensive response, suggesting that Remo is necessary for this behavior in PPK-neurons. To investigate Remo's biochemical properties, we conducted immunoprecipitation analysis. Our findings indicate that the receptor is cleaved even without a consensus GPS sequence, undergoing proteolytic processing likely by the GAIN domain or an alternative protease. The mammalian Remo homolog, ADGRA2/Gpr124, cooperates with GPCRs from the Frizzled family and transmembrane proteins RECK and Lrp5/6. These proteins form a cell surface complex acting as a recognition platform for Wnt ligands. The structural dynamics of this complex are not well understood, with limited pharmacological and in vivo systems available for its characterization. Remo may play a role in this specific signaling pathway, warranting further investigation. This project leverages our experience with orphan aGPCRs like Cirl, flamingo, ketchup, and mayo in Drosophila, along with advanced techniques to achieve the following objectives. The first work package focuses on determining Remo proteolytic fragments and extra- and intracellular interactors through immunoprecipitation, mass spectrometry, Western blot, or Edman degradation sequencing. The second work package involves reconstituting Remo signaling to establish and analyze its signaling cascade in comparison to its vertebrate homolog A2/Gpr124 using cloning, ELISA, or luciferase assays. The third work package examines the effects of Remo signaling in vivo, validating in vitro Remo signaling data through phenotypic analysis of neuronal functions using nocifensive response assay (NRA) and axon guidance assay (AGA) with the Drosophila genomic engineering platform. Each work package is divided into specific aims to analyze the similarities between Remo and A2, establish a Drosophila model for ADGRAsubfamily functions, and identify ways to modulate this receptor group's biological activity. By achieving these objectives, the project aims to enhance the understanding of Remo receptor functions and signaling mechanisms, providing insights into their biological activity and potential modulation strategies. Our long-term objective is to identify nervous system disorders resulting from misregulation of aGPCR signaling pathways and develop strategies to target these pathways effectively with drugs.

• DFG Programme: Research Grants