Beta-adrenergic receptor kinase (betaARK, EC 2.7.1.-) has been implicated in the phosphorylation of G protein-coupled receptors, including opioid receptors. Since delta-opioid receptors of mouse neuroblastoma x rat glioma hybrid cells (NG 108-15) desensitize upon activation, this investigation was designed to find out whether NG 108-15 cells contain betaARK activity. Using the reverse transcription polymerase chain reaction technique, we identified two mRNAs, one coding for rat betaARK1 and the other for rat betaARK2. No hint was found for the presence of mouse betaARK. Examining the cytosolic betaARK activity in these hybrid cells using rhodopsin as substrate, we found a strict functional dependence on the presence of exogenous G protein subunit Gbetagamma. This relationship reflects a characteristic for betaARK1 and 2 out of the known G protein-coupled receptor kinases. Finally, highly purified recombinant betaARK1 proved active to phosphorylate enriched delta-opioid receptor preparations in an opioid agonist-dependent manner. The results reported here provide the basis to study more closely the molecular function of G protein-coupled receptor kinases in a cell line (NG 108-15) most frequently used to investigate acute and chronic opioid actions.

Phosphorylation of G protein‐coupled receptors by β‐adrenergic receptor kinases (βARK) requires the presence of G protein βγ subunits. We have investigated the ability of the two βARK isoforms to distinguish between defined recombinant βγ subunits. βARK2 had an about 25% lower specific activity than βARK1 towards rhodopsin and the β2‐adrenergic receptor but the two kinases shared the selectivity for βγ subunits: βγ complexes consisting of β1 or β2 in combination with γ2, γ5, and γ7 were more efficacious than those with γ3 or β1γ1. Thus, while βARKs differentiate between defined βγ subunits, βγ complexes do not discriminate between βARK isoforms.

Phosphorylation of G protein-coupled receptors by beta-adrenergic receptor kinases (betaARK) requires the presence of G protein betagamma subunits. We have investigated the ability of the two betaARK isoforms to distinguish between defined recombinant betagamma subunits. betaARK2 had an about 25% lower specific activity than betaARK1 towards rhodopsin and the beta2-adrenergic receptor but the two kinases shared the selectivity for betagamma subunits: betagamma complexes consisting of beta1 or beta2 in combination with gamma2, gamma5, and gamma7 were more efficacious than those with gamma3 or beta1 gamma1. Thus, while betaARKs differentiate between defined betagamma subunits, betagamma complexes do not discriminate between betaARK isoforms.

Signal transduction by G-protein-coupled receptors is regulated by various mechanisms acting at the receptor level; those studied most thoroughly are from the beta-adrenergic receptor/Gs/adenylyl cyclase system. We report here a regulatory mechanism occurring at the level of the G proteins themselves. A protein with M(r) 33,000 that inhibits Gs-GTPase activity was purified from bovine brain. This protein is very similar or identical to phosducin, a protein previously thought to be specific for retina and pineal gland. Recombinant phosducin inhibited the GTPase activity of several G proteins, and also inhibited Gs-mediated adenylyl cyclase activation. Blockade of its inhibitory effects by protein kinase A suggests that phosducin may be part of a complex regulatory network controlling G-protein-mediated signalling.