Decoding functional specialization in G protein‐coupled receptors (GPCRs) through evolution‐guided residue profiling

BACKGROUND AND PURPOSE: G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate physiological processes by enabling cells to detect and respond to diverse stimuli. Although many subfamily-specific functional hotspots have been described, the family-wide determinants of common and subfamily-specific functions remain incomplete. EXPERIMENTAL APPROACH: Here, we developed an evolutionary framework utilizing conservation within orthologs and variation across paralogs to classify positions as common residues (CRs) or selective residues (SRs). KEY RESULTS: Common residues (CRs) cluster in sites linked to structural stability and activation, whereas SRs concentrate at selective interfaces involved in ligand and transducer binding. SR distributions across families revealed that some classes mainly diversify through changes in ligand-recognition sites, whereas others through changes at transducer-binding interfaces. We also uncovered CRs involved in family-specific and cross-family motifs, including conserved disulfide bridges and cholesterol-contact sites. CONCLUSIONS AND IMPLICATIONS: Together, these findings provide an evolutionary blueprint of family-wide features, reinforce known associations and deliver testable hypotheses that are especially valuable for understudied families.