Deep mutational scanning of the GPCR rhodopsin reveals pleiotropic mutational effects and their roles in modulating receptor signaling

Biophysical pleiotropy, the phenomenon in which a mutation affects multiple protein properties, underlies many genetic diseases and shapes protein evolution, yet remains poorly understood, limiting synthetic biology and therapeutic development. Although extensively studied in soluble proteins, little is known about how pleiotropic effects shaped the evolution of receptor protein functions. Here, we developed a cell-based assay designed to assess pleiotropic effects in G protein-coupled receptors (GPCRs), the largest receptor class in eukaryotes. Because our assay allows for multiple GPCR molecular phenotypes (basal activity, ligand-dependent signaling, and cellular receptor abundance) to be simultaneously assessed, we applied it to study rhodopsin, a visual GPCR that evolved for high phototransduction capabilities by maximizing light-driven responses while minimizing noise. To investigate this, we integrated our assay with deep mutational scanning to test the impacts of ~2,000 single-residue substitutions in rhodopsin across all three molecular phenotypes (~6,000 measurements). By analyzing these data in the context of rhodopsin's protein structures, we found complex pleiotropic effects that act asymmetrically to impose joint constraints, restricting mutational tolerance in rhodopsin's retinal binding pocket and G protein interaction interface. A dose-response analysis of rhodopsin signaling revealed that these pleiotropic constraints arise from the dual functions of its chromophore, which acts as an agonist upon light-driven isomerization but also an inverse agonist in darkness. Because these constraints reflect mechanistically driven limitations in the evolution of receptor signaling, these findings reveal a role for biophysical pleiotropy in shaping the signaling capabilities of receptor proteins.