Developmental Roles of Beta-Arrestin Kurtz Revealed by Structure-Function Analysis

β-arrestins control signaling via the G protein coupled receptors (GPCRs), serving as both signal terminators and transducers. Previous studies identified several structural elements in β-arrestins that contribute to their functions as GPCR regulators. However, the importance of these elements in vivo is unclear, and the developmental roles of β-arrestins are not well understood. I carried out an in vivo structure-function analysis of Kurtz (Krz), the single ortholog of mammalian β-arrestins in the Drosophila genome. A combination of Krz mutations affecting the GPCR-phosphosensing and receptor core-binding (“finger loop”) functions (Krz-KKVL/A) resulted in a complete loss of Krz activity during development. Endosome recruitment and bioluminescence resonance energy transfer (BRET) assays revealed that the KKVL/A mutations abolished the GPCR-binding the ability of Krz. Moreover, the isolated “finger loop” mutation (Krz-VL/A), while having a negligible effect on GPCR internalization, severely affected Krz function, suggesting that tight receptor interactions are necessary for proper termination of signaling in vivo. Genetic analysis, as well as live imaging, demonstrated that mutations in Krz led to hyperactivity of the GPCR Mist (also known as Mthl1), which is activated by its ligand Folded gastrulation (Fog) and is responsible for cellular contractility and epithelial morphogenesis. Another mutation that affects phosphoinositol binding (Krz-KRK/Q), implicated Krz in the regulation of AMP-activated protein kinase (AMPK) signaling. This mutation impaired the ability of Krz to scaffold the complex containing a regulatory phosphatase subunit Fiery mountain (Fmt), phosphatase V (PpV), and AMPK. The formation of the Krz/Fmt/PpV/AMPK complex is required for limiting AMPK activity during development. By identifying key signaling pathways controlled by Krz and the critical molecular determinants required for this activity, my work expands the knowledge of the developmental functions of β-arrestins