ABSTRACTThe β1-adrenoceptor (β1AR) is a G protein-coupled receptor (GPCR) activated by the hormone noradrenaline, resulting in the coupling of the heterotrimeric G protein Gs1. 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 Gsand induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins (biased agonism)3is important in drug development, as the therapeutic effect may arise from only one signalling cascade, whilst the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, we determined the electron cryo-microscopy (cryo-EM) structure of the β1AR-βarr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound β1AR coupled to the G protein mimetic nanobody Nb806. βarr1 couples to β1AR in a distinct manner to how Gscouples to β2AR7, 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 Gs. The conformation of the finger loop in βarr1 is different from that adopted by the finger loop in visual arrestin when it couples to rhodopsin8, and its β-turn configuration is reminiscent of the loop in Nb80 that inserts at the same position. β1AR coupled to βarr1 showed significant differences in structure compared to β1AR 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 Ser2115.42and Ser2155.46, and a reduction in affinity of formoterol for the β1AR-βarr1 complex compared to β1AR coupled to mini-Gs. These differences provide a foundation for the development of small molecules that could bias signalling in the β-adrenoceptors.