Fig. 3: TAIII acts as a previously unrecognized allosteric inhibitor of A2AR.

a Photosensitive biotinylated-TAIII (TAIII-P) was generated via click chemistry and used for proximity labeling. b Human T cells isolated from PBMCs were treated with TAIII-P (0–20 μM) for 4 h. Association with A2AR was assessed by avidin-biotin pulldown and western blot (upper), and quantified using ImageJ (lower). Data are presented as mean ± SD from four independent experiments (n = 4 biological replicates); two-way ANOVA with Dunnett’s test. c–d Competition assay in cell lysates pretreated with TAIII, followed by TAIII-P pulldown. Relative A2AR levels were quantified. Data are presented as mean ± SD (n = 3 biological replicates); two-way ANOVA with Dunnett’s test. e 293 T cells were co-transfected with A2AR and a CREB luciferase reporter, followed by treatment with the indicated compounds. CREB transcriptional activity was measured using a luciferase assay. NECA was not included, as A2AR overexpression alone was sufficient to activate the CREB reporter. f 293 T cells transfected with A2AR were incubated with NECA (0.1 μM) and TAIII or AZD4635 for 30 min. cAMP levels were determined by LANCE assay; fluorescence ratios (615/665 nm) were proportional to cAMP. Blank wells were negative controls. Data are presented as mean ± SD from four independent experiments (n = 4 biological replicates); ordinary one-way ANOVA with Dunnett’s test. g 293 T cells transfected with or without A2AR were treated with indicated compounds; cAMP levels were measured by LANCE assay. Data are presented as mean ± SD (n = 3 biological replicates); two-way ANOVA with Dunnett’s test. h Predicted mode of binding of TAIII to A2AR based upon molecular modeling (PDB ID: 4EIY). i–j CREB-Fluc reporters with wild-type or mutant A2AR residues were used to assess the contribution of three residues to TAIII binding (i) and dose-dependent effects (j). Data are presented as mean ± SD (n = 3 biological replicates); two-way ANOVA with Dunnett’s test.