Tissue-Level Dynamics and Molecular Regulation of the Glucagon-like Hormone/GPCR signaling Pathway

Abstract

In Drosophila melanogaster, adipokinetic hormone (AKH), a functional homolog of mammalian glucagon, acts on its receptor (AkhR) in the fat body to mobilize energy/lipid stores during increased energy demand. Despite the physiological importance of AKH/AkhR signaling, the systemic cues that trigger AKH release, the tissue level AkhR signaling dynamics in the fat body, and the intracellular regulators that determine AkhR pathway competence remain incompletely defined. This dissertation aims to defines multi layer regulation of AKH signaling across organismal, tissue, and molecular scales. First, I investigate how AKH release is regulated and how AkhR signaling coordinates fat body inter-cellular calcium dynamics in vivo. I show that specific dietary amino acids stimulate AKH producing neurosecretory cells, resulting in promoting AKH hormone release into the hemolymph. Circulating AKH subsequently drives robust, global intercellular calcium waves in the larval fat body through a gap junction–independent mechanism that consequently promotes lipolysis of stored fat. In contrast, in the adult fly fat body, AKH triggers intercellular calcium waves by a previously unknown gap junction–dependent mechanism, indicating a developmental shift in how AkhR/GPCR signaling is coordinated across adipose tissue. Second, to identify intracellular modulators of the AkhR–Gαq signaling axis, I conducted a genome wide pooled CRISPR/Cas9 knockout screen in Drosophila S2R+ cells. Phenotype based selection readout for this screen is GPCR-Gaq driven cytoskeletal remodeling and cell death. This screen identified the fly homolog of palmitoyl acyltransferase ZDHHC8 and its cofactor CG5447 (a mammalian GOLGA7 ortholog) as key regulators of pathway output. Building on the established requirement for N terminal palmitoylation in Gαq signaling, I show that the ZDHHC8–CG5447 complex is required for Gαq palmitoylation and robust Gαq coupled signaling, and that disruption of this module also reduces AkhR stability. Together, these studies connect nutrient sensing and endocrine hormone release to tissue scale calcium wave dynamics to molecular regulation of AkhR in a palmitoylation dependent control of intracellular signal competence. By dissecting a glucagon like GPCR pathway in Drosophila, this work provides a framework for understanding how Gαq coupled metabolic receptors can be tuned to generate coordinated physiological responses, these principles are likely to be relevant across conserved signaling networks.