ABSTRACT Tweety Homologues (TTYH1-3) comprise a unique family of membrane proteins that are highly expressed in the nervous system. Knockout studies in mice demonstrate that these proteins play important roles in neurogenesis, nociception, and brain development. The cryogenic electron microscopy (cryo-EM) structures of mammalian TTYH1-3 have revealed that they form homodimers with a unique fold, distinct from any structures currently available in the database. However, the molecular functions of these membrane proteins remain unclear. Without knowing how TTYHs function, it remains challenging to target this important class of membrane proteins for potential new treatments for various neurological diseases. The long-term goal is to elucidate the mechanism by which TTYH family membrane proteins mediate crucial physiological and pathological events in the nervous system. The specific objectives for this application are to uncover a non- homodimeric configuration of TTYH and to explore the role of this membrane protein in neurodevelopment and regeneration. The central hypothesis is that TTYH forms a complex with another membrane protein to transduce extracellular cues into intracellular signals for neurite outgrowth. The rationale for the proposed research is that by uncovering a new configuration, the mechanistic understanding of how TTYH operates will improve, moving beyond the limitations of the currently available homodimeric structures. In addition, establishing its role in neurodevelopment and regeneration will enable us to fill in the critical gap in understanding how malfunctioning TTYH can cause diverse neurological diseases. The central hypothesis will be tested by performing two specific aims: 1) Uncover the heteromultimeric configuration of TTYH using biochemical and cryo-EM approaches, and 2) Determine whether TTYH mediates neurodevelopment and regeneration using live imaging and laser ablation in transgenic worms. The proposed research is innovative because it intends to provide the first near-atomic resolution structure of TTYH in complex with an interacting partner discovered from endogenously expressed protein. It is also innovative because it will take advantage of C. elegans, a powerful model organism for studying neurodevelopment and neurogenesis, on TTYH for the first time. The contribution of the proposed research is significant because it will provide crucial new insights into the unforeseen heteromultimerization and specific function of this unique class of membrane proteins in the nervous system. These efforts represent an important initial step toward understanding what this family of membrane proteins do, why they are highly expressed in the nervous system, and how they mediate reported pathophysiological activities.

PROJECT NARRATIVE Tweety homologues (TTYHs) are potentially druggable membrane proteins important for brain development and cancer. Despite their clinical relevance, their molecular functions remain unclear. The objective of the proposed research is to provide the missing structural information and clarify their potential roles as signal transducers, which may pave the way for designing novel pharmacological agents to treat neurological diseases.