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Figure 1 Detection of Functional Rat Anti-ß₁-Abs by a Novel FRET-Based Assay

Cell-based approach for the detection of functional activating autoantibodies against the ß₁-adrenergic receptor (anti-ß₁-Abs). (A) Receptor activation induced by binding of antibodies to its accessible extracellular loops leads to an increase in cyclic adenosine monophosphate (cAMP) through sequential activation of G_s proteins and adenylyl cyclase (AC). The cAMP is detected by fluorescence resonance energy transfer (FRET) (grey arrow) between cyan fluorescent proteins (CFP) and yellow fluorescent proteins (YFP) fused to the cAMP-binding domain of Epac1. The sensor (Epac1-based fluorescent cAMP sensor [Epac1-camps]) changes its conformation upon cAMP binding (black arrow), resulting in a decrease in FRET. (B) Measuring cAMP levels in isoproterenol (Iso)-stimulated human embryonic kidney HEK293 cells stably expressing human ß₁-AR by conventional radioimmunoassay (RIA) (Amersham, Freiburg, Germany) or Epac-FRET. One of 3 independent RIA concentration-response curves (EC₅₀ = 0.53 ± 0.19 nmol/l) is shown. The cAMP range that can be monitored by Epac-FRET until it gets saturated is presented by 2 horizontal dotted lines. At 0.05 nmol/l isoproterenol, Epac1-camps becomes saturated, indicating an intracellular cAMP concentration of approximately 20 µmol/l (according to in vitro measurements in cell lysates with pure cAMP [19]). This extremely sensitive sensor is characterized by a high dynamic range at physiologically relevant cAMP concentrations 0.1 to 20 µmol/l (which are covered by only 15% of the maximal cAMP-RIA signal), making minor cAMP changes previously undetected by conventional assays well detectable by Epac-FRET. (C) The immunoglobulin (Ig)G prepared from rats immunized with glutathione-S-transferase (GST) fusion proteins containing the first (EC_I-immunized, center) or second extracellular loop of the human ß₁-AR (EC_II-immunized, right) were assayed for activity using human embryonic kidney HEK293 cells stably expressing human ß₁-AR transfected with Epac1-camps, and compared with nonimmunized animals (control animals, left) to assess the reliability of the method. Representative FRET ratio traces of 1 of 6 independent experiments are presented (% corresponds to the relative change in YFP/CFP intensity ratio). The decrease in FRET reflects an increase in intracellular cAMP.

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