SummaryG protein-coupled receptors play a major role in transmembrane signalling in higher organisms and many are important drug targets. We report the 2.7 Å resolution crystal structure of a β 1 -adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey receptor had been selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilised by two disulphide bonds and a sodium ion. Cyanopindolol binding to the β 1 -adrenergic receptor and carazolol binding to the β 2 -adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β 2 -adrenergic receptor, directly interacts via a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins that are prevalent in eukaryotes from yeast to man, and which function as key intermediaries in the transduction of signals from outside to inside the cell1. Activating molecules (agonists), such as hormones and neurotransmitters, bind to GPCRs from the extracellular side of the cell membrane and induce a large conformational change which propagates to the cytoplasmic surface2,3, resulting in activation of G proteins and a consequent change in the level of intracellular messengers such as cAMP, Ca 2+ or signalling lipids. There are over 800 different human GPCRs4, all sharing the characteristic arrangement of 7 transmembrane α-helices with the polypeptide N-terminus on the extracellular side of the plasma membrane5. Analysis of their primary amino acid sequences has resulted in the definition of a number of families6, the largest of which, family A, includes the archetypal GPCR, rhodopsin. The three human β-adrenergic receptor (βAR) subtypes, β 1 , β 2 and β 3 , belong to family A and share 51% sequence identity between Trp 1.31 -Asp 5.73 and Glu 6.30 -Cys H8-Cterm i.e. excluding the N-and C-termini and most of cytoplasmic loop 3 ( Supplementary Fig 1; superscripts refer to Ballesteros-Weinstein numbering7). Drugs that * Joint corresponding authors MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK cgt@mrc-lmb.cam.ac.ukgfx@mrc-lmb.cam.ac.uk Telephone +44-(0)1223-402338 +44-(0)1223-402328 Fax +44-(0)1223-213556 . Author contributions. TW devised and carried out receptor expression, purification, crystallisation and cryo-cooling of the crystals. Receptor stabilisation and baculovirus expression were performed by MJSV; both authors were also involved in data collection and preliminary crystallographic analyses of the crystals. PE helped with the crystal cryo-cooling strategy and in diffraction data collection. JGB performed the functional cAMP and reporter gene assays...

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The β 1-adrenoceptor (β 1 AR) is a G-protein-coupled receptor (GPCR) that couples 1 to the heterotrimeric G protein G s. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by 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-known as biased agonism 3-is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects 4. To understand the molecular basis for arrestin coupling, here we ✉