Molecular determinants of β-arrestin coupling to formoterol-bound β1-adrenoceptor

ABSTRACT The β 1 -adrenoceptor (β 1 AR) is a G protein-coupled receptor (GPCR) activated by the hormone noradrenaline, resulting in the coupling of the heterotrimeric G protein G s 1 . G protein-mediated signalling is terminated by phosphorylation of the receptor C-terminus and coupling of β-arrestin 1 (βarr1, also known as arrestin-2), which displaces G s and induces signalling through the MAP kinase pathway 2 . The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins (biased agonism) 3 is important in drug development, as the therapeutic effect may arise from only one signalling cascade, whilst the other pathway may mediate undesirable side effects 4 . To understand the molecular basis for arrestin coupling, we determined the electron cryo-microscopy (cryo-EM) structure of the β 1 AR-βarr1 complex in lipid nanodiscs bound to the biased agonist formoterol 5 , and the crystal structure of formoterol-bound β 1 AR coupled to the G protein mimetic nanobody Nb80 6 . βarr1 couples to β 1 AR in a distinct manner to how G s couples to β 2 AR 7 , with the finger loop of βarr1 occupying a narrower cleft on the intracellular surface closer to transmembrane helix H7 than the C-terminal α5 helix of G s . The conformation of the finger loop in βarr1 is different from that adopted by the finger loop in visual arrestin when it couples to rhodopsin 8 , and its β-turn configuration is reminiscent of the loop in Nb80 that inserts at the same position. β 1 AR coupled to βarr1 showed significant differences in structure compared to β 1 AR coupled to Nb80, including an inward movement of extracellular loop 3 (ECL3) and the cytoplasmic ends of H5 and H6. In the orthosteric binding site there was also weakening of interactions between formoterol and the residues Ser211 5.42 and Ser215 5.46 , and a reduction in affinity of formoterol for the β 1 AR-βarr1 complex compared to β 1 AR coupled to mini-G s . These differences provide a foundation for the development of small molecules that could bias signalling in the β-adrenoceptors.