Abstract
G protein-coupled receptor kinases (GRKs) are believed to involve in desensitization of the G protein-coupled receptors. So far, cDNAs of six GRKs were cloned from several species including human and rat. However, it is unknown whether single GRK phosphorylates various receptors and desensitizes them in the cells. To determine whether GRK2 (also called βARK1) involves desensitization of the β1-adrenergic receptor-mediated response in heart, we tried to apply monoclonal antibody which could recognize only βARK1 and inhibit its phosphorylating activity to the heart cells. Monoclonal antibody was obtained by immunization of carboxyl terminus of βARK1 as fusion protein of glutathione-S-transferase (GST). The resulting monoclonal antibody specifically reacted with βARK1, and inhibited the binding of purified βγ subunit to the carboxyl terminus. Monoclonal antibody completely inhibited phosphorylation of the m2 muscarinic acetylcholine receptor as well as phosphorylation of GST intracellular third loop fusion protein of the m2 receptor. When monoclonal antibody was applied to myocyte prepared from guinea pig heart, the desensitization of the β1-adrenergic receptor was partially inhibited as measured by Ca2+ channel activation. Thus intracellular application of monoclonal antibody is promising approach to analyze function of GRKs.
