Differential conservation analysis identifies residues defining constitutive internalization in beta-adrenergic receptors

G protein-coupled receptors (GPCRs) are major drug targets and key regulators of cell signaling. The basis of functional diversification between individual GPCRs and families of GPCRs can be revealed by investigating evolutionary conservation patterns. In this study, we investigated the functional role of specifically conserved residues in the TM1/TM7/H8 dimerization interface of beta-adrenergic receptors (BARs). Residues specifically conserved for B2AR compared to B1AR and B3AR subtypes were identified via phylogenetic analysis. The significance of residues differentially conserved between receptor subtypes at the TM1/TM1 interface was investigated using molecular dynamics (MD) simulations in combination with biophysical and functional studies. Our findings suggest that differentially conserved residues within TM1 of BARs modulate receptor conformation without disrupting dimerization to impact cell surface expression, basal activity, and endocytosis. This highlights the importance of TM1 in modulating receptor function and provides new insights into the evolutionary and functional differences among beta-adrenergic receptor subtypes.