Functional trade-offs in the GPCR rhodopsin revealed by multiphenotypic deep mutational scanning

The pleiotropic effects of mutations underlie many genetic diseases and shape protein evolution, yet they remain poorly characterized, impeding understanding, protein engineering, and therapeutic development. Although extensively studied in globular enzymes, comparatively little is known about how mutations may affect diverse transmembrane protein functions. Here, we integrate yeast-based biosensors of G protein-coupled receptor (GPCR) function with multiphenotypic deep mutational scanning to investigate the pleiotropic effects of mutations in the RHO gene, which encodes the archetypal GPCR rhodopsin responsible for dim-light vision in vertebrates. By testing the impact of ~2,000 single-residue substitutions on rhodopsin dark activity, light activation, and cellular abundance, we uncovered complex variant effect relationships and trade-offs constraining mutational tolerance in rhodopsin's ligand binding pocket and G protein interaction interface. Together, our study reveals the intricate interplay among higher-order mutational effects in a visual GPCR and provides a scalable framework for exploring these effects across the GPCR superfamily.