[Functional analysis of G protein-coupled receptor kinase (beta ARK1) by intracellular immunization].
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 beta ARK1) involves desensitization of the beta 1-adrenergic receptor-mediated response in heart, we tried to apply monoclonal antibody which could recognize only beta ARK1 and inhibit its phosphorylating activity to the heart cells. Monoclonal antibody was obtained by immunization of carboxyl terminus of beta ARK1 as fusion protein of glutathione-S-transferase (GST). The resulting monoclonal antibody specifically reacted with beta ARK1, and inhibited the binding of purified beta gamma 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 beta 1-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.
Kurose
H
H
Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan.
Takikawa
E
E
Akahane
S
S
Tanaka
R
R
Nagao
T
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000276:immunology / Q000494:pharmacology; D000939:Epitopes; D006168:Guinea Pigs; D006321:Heart / Q000187:drug effects; D006801:Humans; D010766:Phosphorylation; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000187:drug effects; D051552:beta-Adrenergic Receptor Kinases
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011943:Receptors, Adrenergic, beta; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases