QUANTIFICATION OF SIGNALING COMPONENTS AND AMPLIFICATION IN THE BETA-ADRENERGIC-RECEPTOR-ADENYLATE CYCLASE PATHWAY IN ISOLATED ADULT-RAT VENTRICULAR MYOCYTES

We have investigated the stoichiometric relationship of proteins invol ved in beta-adrenergic-receptor-mediated signal transduction in isolat ed rat cardiac myocytes. These cells contain about 2.1 x 10(5) beta-ad renergic receptors per cell, as determined by radioligand-binding assa ys. We have assessed the amount of G(s) alpha present in myocyte membr anes by immunoblotting using a purified glutathione S-transferase-G(s) alpha fusion protein as a standard for quantification. By this method , we determined that cardiac myocytes contain about 35 x 10(6) and 12 x 10(6) molecules per cell of the 45 and 52 kDa forms of G(s) alpha, r espectively. [H-3]Forskolin binding assays were used to assess the for mation of high-affinity forskolin binding sites representing G(s) alph a-adenylate cyclase complexes occurring in response to G(s) alpha acti vation. Quantification of the adenylate cyclase complexes was facilita ted by the permeabilization of cells with saponin. The addition of iso prenaline (isoproterenol) and guanosine 5'-[gamma-thio]trisphosphate t o saponin-permeabilized myocytes results in the formation of 6 x 10(5) G(s) alpha-adenylate cyclase complexes. Taken together, the data pres ented here demonstrate that, in a physiologically relevant setting, G- protein is present in large stoichiometric excess relative to both rec eptor and effector. In addition, we show that, overall, only modest si gnal amplification occurs between receptor and adenylate cyclase. Thus adenylate cyclase (rather than G(s)) is the component distal to recep tor that limits agonist-mediated increases in cyclic AMP production. A lthough limited data are as yet available for other G-protein-regulate d effecters, we hypothesize that the stoichiometry of signalling compo nents and the extent of signal amplification described for the beta-ad renergic response pathway will be applicable to other G-protein-couple d hormone receptor systems.