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Figure 3 Functional Anti-ß₁-Abs Target Distinct Receptor Epitopes

Blocking experiments for different classes of functional anti-ß₁-Abs. (A) The cAMP generation induced by high-activator IgG is attenuated only by specific peptides derived from the second extracellular ß₁-AR loop (ß₁-EC_II). (B) Low-activator signals could be blocked in all instances by peptides corresponding to the first extracellular ß₁-AR loop (ß₁-EC_I) but not by ß₁-EC_II peptides (representative experiments from at least 3 cells per condition are shown). Analysis of the kinetics of the different FRET responses is presented in the lower panels of (A) and (B). Ratio traces were fitted with a mono-exponential function, and the time constant of the decay of the respective FRET signals was determined. In both cases, the effects of EC_I and EC_II peptides differ significantly (*p < 0.05, **p < 0.01; one-way analysis of variance [ANOVA]). (C) Columns represent mean ± SEM (error bars) of cAMP responses induced by (n = 7) representative high-activator IgG, or (D) (n = 10) representative low-activator IgG prepared from DCM patients in the absence (n.b. = not blocked) or presence of peptides corresponding to the first (EC_I) and/or second extracellular ß₁-AR loop (EC_II), or to EC_II of the ß₂-AR. Statistical significance was tested by one-way ANOVA (**p < 0.01 compared with the n.b. group). (E) Dose-response curve showing the effect of increasing concentrations of the ß₁-EC_II peptide on the inhibition of high-activator-induced cAMP responses (representative experiments, n = 5). The curve was fitted to the data using Origin 6.1 (OriginLab Corp., Northampton, Massachusetts) assuming a single binding site. Abbreviations as in Figures 1 and 2.