An Efficacy-Dependent Effect of Cardiac Overexpression of β₂-Adrenoceptor on Ligand Affinity in Transgenic Mice

Abstract

In previous studies, it was shown that the overexpression of β₂-adrenoceptor (β₂AR) in the hearts of transgenic mice (Tg) leads to agonist-independent activation of adenylate cyclase and enhanced myocardial function. Here, we measured the physical coupling of β₂AR and G_s by evaluating the coimmunoprecipitation of β₂AR and G_s and the ligand binding properties of β₂AR in the hearts of Tg mice to investigate the details of the interaction among ligand, receptor, and G protein. The following results were obtained: (i) coimmunoprecipitation of β₂AR and G_s was increased in the absence of agonist in Tg mice compared with the control animals. This demonstrates directly the increased interaction between unliganded β₂AR and G_s, which is consistent with increased background cAMP production and cardiac function in the hearts of Tg mice. (ii) Guanosine-5′-(β,γ-imido)triphosphate abolished the association of β₂AR/G_s in the immunoprecipitate. (iii) The affinities for ligands that show agonist (isoproterenol, clenbuterol, and dobutamine), neutral antagonist (alprenolol and timolol), and negative antagonist (propranolol and ICI 118551) activities in this experimental system were increased, not changed and decreased, respectively, in Tg mice compared with the controls. (iv) This efficacy-dependent alteration in ligand affinities was still observed in the presence of a guanosine-5′-(β,γ-imido)triphosphate concentration that abolishes β₂AR/G_scoupling. This suggests that the altered β₂AR binding affinities in Tg mice are not due to the increased interaction between β₂AR and G_s. These data cannot be explained by using ternary, quinternary, two-state extended ternary, or cubic ternary complex models. We therefore discuss the results using a “two-state polymerization model” that includes an isomerization step for the conversion of receptor between an inactive and an active form (denoted as R and R*, respectively) and a polymerization of the active state (R*_n). The simplest form of this model (i.e., noncooperative dimerization of the receptor) is found to be consistent with the experimental data.