Fig. 3: Identification of an endogenous metal in the tβ1AR–mini-Gs complex and its functional relation to Gs-protein coupling.
a, Endogenous metal adducts were detected in the tβ1AR–mini-Gs complex under stimulation with the agonists isoprenaline (1), norepinephrine (8), carmoterol (9), dobutamine (10) and salbutamol (11). The stoichiometry of metal binding is denoted above individual peaks (1×, one adduct; 2×, two adducts). b, The impact of EDTA on tβ1AR–mini-Gs complex formation and its association with the endogenous metal. The binding stoichiometry of the metal ion is indicated 1×–4×, denoting one to four adducts. A supplement of exogenous ZnCl2 at 25 μM added to EDTA-pretreated receptor and mini-Gs led to the recovery of complex formation of tβ1AR and mini-Gs (bottom spectrum). The peaks assigned to the receptor–mini-Gs complex, receptor monomer and mini-Gs are highlighted in orange, blue and grey, respectively. c, Percentage of endogenous metal adduct with different stoichiometries normalized to the total intensity of the tβ1AR–mini-Gs complex in response to different tβ1AR agonists. The dots refer to the individual data points and the bars represent the mean ± s.d. from a variable number of independent experiments (n = 5 for norepinephrine (8) and dobutamine (10); n = 7 for isoprenaline (1); n = 3 for carmoterol (9) and salbutamol (11)). d, Quantification of tβ1AR–mini-Gs complex formation and its association with metal ion in the presence of EDTA at different concentrations. The relative percentage was calculated by normalizing to the condition without EDTA. The data points represent the mean ± s.d. from three independent experiments.
