Publications

• Characterization and inhibition of beta-adrenergic receptor kinase in intact myocytes.

  Laugwitz K.L., Kronsbein K., Schmitt M., Hoffmann K., Seyfarth M., Schomig A., Ungerer M.

  <h4>Objectives</h4>beta-Adrenergic receptor kinase (beta ARK) phosphorylates and thereby inactivates agonist-occupied beta-adrenergic receptors (beta AR). beta ARK is thought to play an important role in the regulation of cardiac function. Therefore, we studied beta ARK activation and its inhibiti ... >> More

  <h4>Objectives</h4>beta-Adrenergic receptor kinase (beta ARK) phosphorylates and thereby inactivates agonist-occupied beta-adrenergic receptors (beta AR). beta ARK is thought to play an important role in the regulation of cardiac function. Therefore, we studied beta ARK activation and its inhibition in intact smooth muscle cells and in cardiomyoblasts.<h4>Methods and results</h4>beta AR agonist-stimulated translocation of beta ARK was monitored by immunofluorescence labelling with specific antibodies and confocal laser scanning microscopy in DDT-MF 2 hamster smooth muscle cells and in H9c2 rat cardiomyoblasts. In unstimulated cells. beta ARK was mainly located in the cytosol. After beta AR agonist stimulation, the beta ARK signal was partially translocated to the membranes. Liposomal gene transfer of the COOH-terminus of beta ARK ('beta ARKmini') as a beta ARK inhibitor led to functional expression of this protein in both cell lines with high efficiency. Western blots with beta ARK antibodies showed a gene concentration-dependent immunoreactivity of the 'beta ARKmini' protein. 'beta ARKmini'-transfected myocytes demonstrated reduced membrane targeting of the beta ARK immuno-fluorescence signal. Additionally, the effect of 'beta ARKmini' on beta AR-induced desensitization of myocytic cAMP accumulation was investigated. In control cells, desensitization with isoproterenol led to a subsequent reduction of beta AR-induced cAMP accumulation. In 'beta ARKmini'-transfected myocytes, this beta AR-induced desensitization was significantly diminished, whereas normal beta AR-induced cAMP accumulation was unaffected. A gene concentration of 2 micrograms 'beta ARKmini' DNA/100,000 cardiomyoblasts, and of 0.7 microgram 'beta ARKmini' DNA/100,000 DDT-MF2 smooth muscle cells led to approximately 5.9- and approximately 5.6-fold overexpressions of 'beta ARKmini' vs. native beta ARK, respectively. These gene doses proved sufficient to attenuate beta-adrenergic desensitization significantly.<h4>Conclusions</h4>(1) beta ARK translocation was evidenced in DDT-MF2 smooth muscle cells and in cardiomyoblasts by confocal laser scanning microscopy. (2) Feasibility of 'beta ARKmini' gene transfer to myocytes was demonstrated, and necessary gene doses for beta ARK inhibition were titered. (3) Overexpression of 'beta ARKmini' functionally interacted with endogenous beta-adrenergic signal transduction, leading to sustained cAMP accumulation after prolonged beta-adrenergic stimulation. << Less

  Cardiovasc Res 35:324-333(1997) [PubMed] [EuropePMC]

• Expression and characterization of two beta-adrenergic receptor kinase isoforms using the baculovirus expression system.

  Kim C.M., Dion S.B., Onorato J.J., Benovic J.L.

  The beta-adrenergic receptor kinases, beta ARK1 and beta ARK2, are two recently cloned members of the G protein-coupled receptor kinase family. To further characterize these kinases, bovine beta ARK1 and beta ARK2 have been overexpressed in Sf9 insect cells using the baculovirus expression system. ... >> More

  The beta-adrenergic receptor kinases, beta ARK1 and beta ARK2, are two recently cloned members of the G protein-coupled receptor kinase family. To further characterize these kinases, bovine beta ARK1 and beta ARK2 have been overexpressed in Sf9 insect cells using the baculovirus expression system. High yields (5-7 mg/L cells) of purified kinase preparations were obtained by sequential chromatography of infected Sf9 cell supernatant fractions on S-Sepharose and Heparin-Sepharose. The expressed kinases were fully active as evidenced by their ability to specifically phosphorylate the agonist-occupied beta 2-adrenergic receptor (beta 2AR) and light-activated rhodopsin. Similar initial rates and maximal stoichiometries of beta 2AR phosphorylation were observed for both beta ARK1 and beta ARK2. Moreover, G protein beta gamma subunits enhanced the initial rates of both beta ARK1 and beta ARK2 mediated beta 2AR phosphorylation by approximately tenfold. In the presence of beta gamma subunits the maximal stoichiometry of beta 2AR phosphorylation was increased from approximately 4 mol phosphate/mol receptor to approximately 10 mol/mol. Detailed kinetic analysis of rhodopsin phosphorylation suggests that both kinases follow a sequential mechanistic pathway and have similar Kms for rhodopsin (approximately 14 microM) and MgATP (60-90 microM). Peptide phosphorylation studies demonstrate that both kinases prefer acidic amino acids amino terminal to a serine. Heparin was found to be the most potent inhibitor for both kinases with IC50s of 1.4 and 1.1 microM for beta ARK1 and beta ARK2, respectively. These studies demonstrate that beta ARK1 and beta ARK2 share very similar kinetic properties and suggest that they may have a similar substrate specificity in vivo. << Less

  Receptor 3:39-55(1993) [PubMed] [EuropePMC]

• Purification and characterization of the beta-adrenergic receptor kinase.

  Benovic J.L., Mayor F. Jr., Staniszewski C., Lefkowitz R.J., Caron M.G.

  The beta-adrenergic receptor kinase (beta-ARK) is a recently discovered enzyme which specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta-AR) as well as the light-bleached form of rhodopsin. beta-ARK is present in a wide variety of mammalian tissues. The kina ... >> More

  The beta-adrenergic receptor kinase (beta-ARK) is a recently discovered enzyme which specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta-AR) as well as the light-bleached form of rhodopsin. beta-ARK is present in a wide variety of mammalian tissues. The kinase can be purified from bovine cerebral cortex to greater than 90% homogeneity by sequential chromatography on Ultrogel AcA34, DEAE-Sephacel, CM-Fractogel, and hydroxylapatite. This results in an approximately 20,000-fold purification with an overall recovery of 12%. The purified kinase has an Mr approximately 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several findings indicate that this peptide contains the beta-ARK activity. First, on hydroxylapatite chromatography the enzyme activity coelutes with the Mr approximately 80,000 protein as revealed by Coomassie-Blue staining. Second, under phosphorylating conditions the Mr approximately 80,000 protein is phosphorylated. Finally, the Mr approximately 80,000 protein specifically interacts with reconstituted agonist-occupied beta-AR. Kinetic parameters of the enzyme for beta-AR are Km = 0.25 microM and Vmax = 78 nmol/min/mg whereas for rhodopsin the values are Km = 6 microM and Vmax = 72 nmol/min/mg. The Km value of the enzyme for ATP is approximately 35 microM using either beta-AR or rhodopsin as substrate. Receptor phosphorylation by beta-ARK is effectively inhibited by Zn2+, digitonin and a variety of salts. The availability of purified beta-ARK should greatly facilitate studies of its role in receptor desensitization. << Less

  J Biol Chem 262:9026-9032(1987) [PubMed] [EuropePMC]