Chemical Synthesis of a Full-Length G-Protein-Coupled Receptor β₂-Adrenergic Receptor with Defined Modification...

The β₂-adrenergic receptor (β₂AR) is a G-protein-coupled receptor (GPCR) that responds to the hormone adrenaline and is an important drug target in the context of respiratory diseases, including asthma. β₂AR function can be regulated by post-translational modifications such as phosphorylation and ubiquitination at the C-terminus, but access to the full-length β₂AR with well-defined and homogeneous modification patterns critical for biochemical and biophysical studies remains challenging. Here, we report a practical synthesis of differentially modified, full-length β₂AR based on a combined native chemical ligation (NCL) and sortase ligation strategy. An array of homogeneous samples of full-length β₂ARs with distinct modification patterns, including a full-length β₂AR bearing both monoubiquitination and octaphosphorylation modifications, were successfully prepared for the first time. Using these homogeneously modified full-length β₂AR receptors, we found that different phosphorylation patterns mediate different interactions with β-arrestin1 as reflected in different agonist binding affinities. Our experiments also indicated that ubiquitination can further modulate interactions between β₂AR and β-arrestin1. Access to full-length β₂AR with well-defined and homogeneous modification patterns at the C-terminus opens a door to further in-depth mechanistic studies into the structure and dynamics of β₂AR complexes with downstream transducer proteins, including G proteins, arrestins, and GPCR kinases.