Abstract

The human μ-opioid receptor (μOR) mediates the therapeutic and adverse effects of opioids, yet its structural determinants of function remain poorly understood. This study employed Scanning Site Saturation Mutagenesis (SSSM) to systematically analyze the role of 288 transmembrane core residues, generating a comprehensive library of 5,472 receptor variants. Using a yeast-based biosensor platform, we screened the library against the μOR agonist DAMGO to identify functional variants. Our results revealed critical insights into receptor-ligand interactions, including the identification of L178R, a variant exhibiting nearly double the signaling sensitivity of the wild-type receptor. This substitution, localized to intracellular loop 2, highlights the role of conserved residues in stabilizing receptor-G protein interactions. Additionally, we characterized non signaling variants to identify residues essential for receptor function. By integrating high-throughput sequencing and variant characterization, this platform provides a robust tool for probing receptor functionality and offers a foundation for refining opioid receptor-based drug discovery.