β₂-Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop

Fluorine-19 nuclear magnetic resonance (NMR) was used to study conformational equilibria at the intracellular tips of helices VI and VII in a variant β₂-adrenergic receptor (β₂AR) containing T4-lysozyme fused into the third intracellular loop (β₂AR-T4L), a G protein-coupled receptor (GPCR) modification widely used in crystal structure determination. G-protein signaling at helix VI showed nearly complete population of an active-like state for all ligand efficacies in the absence of an intracellular protein. For arrestin signaling at helix VII, a native-like equilibrium was observed, except for complexes with ligands devoid of a hydrophobic moiety at the ethanolamine end. These data confirm that response of G-protein and arrestin signaling to ligand efficacy is not coupled, and presents evidence for long-range effects between fusion protein and orthosteric binding cavity, which are suppressed by voluminous bound ligands. Solution NMR thus provides complementary information, which should be considered in functional interpretations of GPCR crystal structures obtained with ICL3 fusions.