Figure Legends:
Fig 1 Bias plot showing the recruitment of β-arrestin-2 (Tango assay) as the abscissae and activation of a genetically engineered firefly luciferase cAMP biosensor (GloSensor; Promega, Madison, WI) as the ordinates. Four agonists for the Κ-opioid receptor were compared:GR89696 , 4-([3,4-dichlorophenyl]acetyl)-3-(1-pyrrolidinylmethyl)-1-piperazinecarboxylic acid methyl ester fumarate salt, ICI 199,441 , 2-(3,4-dichlorophenyl)-N-methyl-N-([1S]-1-phenyl-2-[1-pyrrolidinyl]ethyl)acetamide hydrochloride; RB 48, 22-thiocyanatosalvinorin A and RB 64 , 22-bromosalvinorin A. Plot shows that the salvinorin analogues RB64 and RB48 are biased toward G protein while ICI199441 and GR89696 are biased toward β-arrestin. Data from White et al, 2014.
Fig 2 A. Angiotensin receptor II type 1 receptor downstream signaling pathways and BRET sensors for G protein and β-arrestin activation, specifically heterotrimeric G proteins and activation of βarr2. cAMP, adenosine 3′,5′-cyclic monophosphate. B. Heatmap of relative activity (ΔLog(τ/KA) (Kenakin et al, 2012) values for angiotensin analogs showing activation of various G proteins and β-arrestin. Data from Namkung et al (2018)
Fig 3. Theoretical scheme for bias testing showing how secondary bias assays may further differentiate and better characterize an initial bias estimate.
Fig 4: Concentration-response (CR) curves for two agonists (denoted by black and red curves) A. Theoretical model showing receptor R interacting with an agonist A and two G proteins G1 and G2 (Kenakin, 2003). B. Non biased agonist parameters: Agonist1 (black curve): α = 10, γ1 = γ2 =2, KG1= KG2 = 3: Agonist2(red curve): α = 10, γ1 = γ2 =10, KG1= KG2 = 1. Composition of Cell Types: CellA = [G1]= 1, [G2]= 10: CellB = [G1]= 10, [G2] = 1. C. Biased agonist parameters: Agonist1 (black curve): α = 10, γ1 =1, γ2 =10, KG1= 3, KG2 = 1: Agonist2 (red curve): α = 10, γ1 = 10, γ2 =1, KG1 = 1, KG2 = 3. Cell type composition as in Panel B.
Fig 5. Bias at different levels within the cell. A. Schematic diagram showing three general zones where assays may access bias. Usually it is detected at the level of the receptor. As the receptor-mediated signals spread through the cell various other components become involved which can modify bias in different areas. Finally, whole cell response integrates these signals to yield a quantity and quality of efficacy. B. Data for seven dopamine agonists producing six responses in the cell. Radar plot shows the relative bias of the agonists for each pathway (color coded) through ΔΔLog(max/EC50) values. Note how the bias for the agonists differs depending on which part of the signaling pathway is assessed. Data from Klein Herrenbrink et al, 2016.