Abstract

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 design 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 light sensitivity by maximizing light-driven responses while minimizing thermally driven noise. To investigate this, we integrated our assay with deep mutational scanning of relevant rhodopsin domains to test the impacts of about 2,000 single-residue substitutions in rhodopsin across all three molecular phenotypes (over 6,000 measurements). By analyzing these data in the context of rhodopsin protein structure, we discovered complex pleiotropic effects that act asymmetrically to impose joint constraints, restricting mutational tolerance in the retinal binding pocket and G protein interaction interface of rhodopsin. 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 sensory capabilities of receptor proteins.