The human sense of smell relies on a vast and chemically diverse array of odor molecules that interact with numerous odorant receptor (ORs) proteins. Despite this complexity, many aspects of olfaction still remain poorly understood. A major challenge in studying these G protein-coupled receptors is their poor functional expression on the cell surface in heterologous or non-olfactory systems, which limits biochemical analysis and insight into their ligand-binding properties. Our goal is to engineer OR constructs with improved surface expression and activation while preserving their native ligand specificity. Here, we build upon previous work which identified N-terminal, transmembrane protein 1 (TM1), and C-terminal modifications that promoted expression but did not contribute to ligand selectivity. We hypothesized that combining these modifications with orthologous OR sequences that respond to the same odorant would further increase receptor expression by enabling substitutions at non-conserved residues between TM2- TM7. We focused on OR5A2, a receptor known for its difficulty to express on the cell surface, and its response to musk, an important fragrance compound. To evaluate our engineered receptors, we used fluorescence activated cell-sorting (FACS) to assess cell surface expression and the GloSensor cAMP assay to assess receptor activation in HEK293T cells transiently expressing native and engineered OR constructs. Our results show that orthologous substitutions to the OR5A2 construct increased cell surface expression, particularly when combined with previously described N- and C-terminal modifications. GloSensor cAMP assays further demonstrated that the engineered receptor responded to the musk odorant in the same manner as the native receptor, suggesting that these modifications improve trafficking and expression without disrupting ligand recognition and downstream signaling. Overall, these findings demonstrate that ortholog-based receptor engineering can enhance odorant receptor expression while preserving native function, providing key insight into structural constraints affecting OR expression in non-olfactory cells.x Symposium Presenter: Emily Zheng

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