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Article Abstract
In this study, olfactory receptors (ORs) and Golf proteins fused with split luciferase were examined to develop a sensitive assay for identifying OR ligands. Although specific luminescence was observed from HEK293T cells expressing both fusion proteins (OR1A1-LgBiT and SmBiT-Mini-Golf) in response to an OR1A1 ligand, its intensity was low. To improve it, a fusion protein (V1N-G4S3-SmBiT-G4S3-Mini-Golf), was created where V1N contained the palmitoylation site necessary for localizing Mini-Golf to the cell membrane. When OR1A1-LgBiT and V1N-G4S3-SmBiT-G4S3-Mini-Golf were co-expressed in HEK293T cells, specific luminescence by ligand stimulation was considerably increased. The cell surface expression of OR1A1-LgBiT was markedly lower than that of OR1A1. Co-expression of receptor transporting protein 1 short form (RTP1S) promoted the cell surface expression of OR1A1-LgBiT more efficiently than OR1A1. Prediction using AlphaFold3 suggested that the N-terminal domain of RTP1S interacts with LgBiT in OR1A1-LgBiT. Co-expression of OR1A1-LgBiT, V1N-G4S3-SmBiT-G4S3-Mini-Golf, and RTP1S in HEK293T cells enhanced the sensitivity for detecting at least some ligands by 1000- to 3000-fold compared to the conventional cAMP assay. These findings suggest that optimizing the assembly of OR1A1-LgBiT and SmBiT-Mini-Golf at the cell membrane is essential for developing a sensitive assay. The OR response-detection system employing split luciferase holds significant potential for investigating ligands of orphan ORs.
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| http://dx.doi.org/10.1016/j.ab.2025.115954 | DOI Listing |
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