Amyloid Precursor Protein (APP) was originally identified as the source of beta-amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD) and Down Syndrome. However, APP is normally upregulated in both the developing and injured nervous system, functioning as a guidance receptor that regulates multiple aspects of neuronal motility, including migration and outgrowth, synaptic remodeling, and retraction/regrowth responses. However, the mechanisms by which APP regulates these responses remain poorly understood. Early studies showed that APP could function as an unconventional G protein-coupled receptor (GPCR), interacting with the heterotrimeric G protein Goα to control neuronal responses. Mutations in APP linked with early onset AD also hyperactivated Goα and induced cell death, suggesting that the misregulation of normal APP-Go signaling might provoke neurodegenerative responses. However, subsequent studies produced contradictory results, and until recently, proof that APP acts as a Goα-coupled receptor in vivo remained lacking. Defining the normal functions of APP has been complicated by two close orthologs in mammals (APLP1 and APLP2) with overlapping activities. As an alternative, we adapted a well-characterized assay of neuronal migration in the moth Manduca. Notably, Manduca only contains a single APP ortholog (APP-like; or APPL), which also functionally interacts with Goα in migrating neurons. Moreover, inhibiting APPL-Goα signaling in cultured embryos resulted in ectopic migration and outgrowth, similar to the ectopic migration caused by deleting all APP family members in mice. Using genetic manipulations and split-GFP assays in Drosophila, we also showed that APPL directly binds Goα via a conserved Go-binding motif, and that this interaction is regulated by Goα activation. Using affinity screening methods, we found that Manduca Contactin functions as an authentic ligand for APPL, paralleling recent evidence that mammalian Contactins also bind APP family proteins. These results demonstrate that Manduca provides a powerful discovery system for defining the signaling mechanisms that underlie this evolutionarily conserved pathway. Using our protocols for manipulating protein and gene expression in cultured embryos, we will investigate two novel Goα effectors that may regulate different aspects of APPL-Goα signaling. Specifically, we will test whether APPL-Goα signaling restricts ectopic outgrowth via RhoGEF2, an ortholog of mammalian PDZ-RhoGEFs that activate the small GTPase RhoA (a regulator of actin remodeling). Likewise, we will test whether APP-Goα signaling also induces APPL cleavage by the α-secretase ADAM10/Kuzbanian, providing a novel mechanism for terminating signaling. Lastly, to confirm the relevance of these studies in mammalian neurons, we will test the role of each effector in regulating the APP-Goα signaling using cultured mouse hippocampal neurons.