30G-Protein Coupled Receptors (GPCRs) transmit signals across the cell membrane via 31 an allosteric network from the ligand-binding site to the G-protein binding site via a series of 32 conserved microswitches. Crystal structures of GPCRs provide snapshots of inactive and 33 active states, but poorly describe the conformational dynamics of the allosteric network that 34 underlies GPCR activation. Here we analyse the correlation between ligand binding and 35 receptor conformation of the α 1A -adrenoceptor, known for stimulating smooth muscle 36 contraction in response to binding noradrenaline. NMR of 13 C ε H 3 -methionine labelled α 1A -37 adrenoreceptor mutants, each exhibiting differing signalling capacities, revealed how 38 different classes of ligands modulate receptor conformational equilibria. 13 C ε H 3 -methionine 39 residues near the microswitches revealed distinct states that correlated with ligand efficacies, 40 supporting a conformational selection mechanism. We propose that allosteric coupling 41 between the microswitches controls receptor conformation and underlies the mechanism of 42 ligand modulation of GPCR signalling in cells. 43 44 45 46 47 3 93 al., 2017)), the NPxxY switch, and the intracellular G protein binding site, characterized by 94 the DRY motif (or switch). How these microswitches coordinate the transmission of the 95 extracellular signal is not clear, but molecular dynamics (MD) simulations and NMR data 96 have led to a mechanistic description of "loose allosteric coupling" (Latorraca et al., 2017).97 5 98 99 Figure 1. Methionine residues in α 1A -AR. (a) The location of six methionines on a cartoon 100 representation of α 1A -AR. Methionine sidechains are highlighted as red sticks. Bound adrenaline and 101 G protein are coloured in green and purple respectively. (b-d) Homology models of α 1A -AR-A4 in the 102 inactive state (blue; modeled on the X-ray crystal structure of inactive β 2 -AR, pdb id: 5jqh) and 103 active state (pink; modeled on the X-ray crystal structure of active β 2 -AR, pdb id: 3sn6) are 104 superimposed showing inferred conformational changes that occur in the ligand binding pocket (b), 105 transmission switch (c) and G protein binding site (d). 106 107This mechanism refers to each microswitch as conformationally independent from the others, 108 that is an active DRY motif state is not significantly dependent on an active state in the 109 transmission switch. That said, an active state in the transmission switch does increase the 110 probability of the DRY motif (and thus the receptor) to sample active states (thus, loose 111 allosteric coupling) (Latorraca et al., 2017). Put simply, the conformational changes that 112 occur in the microswitches are thought to drive the overall equilibrium state of the receptor 113 6 system. Despite recent work, it is not well understood how the binding of ligands such as 114 inverse agonists influence the microswitch state equilibria to decrease basal receptor activity. 115 α 1 -adrenoceptors (α 1 -ARs) comprise three G q -co...