Expression and characterization of two beta-adrenergic receptor kinase isoforms using the baculovirus expression system.
Expression and characterization of two beta-adrenergic receptor kinase isoforms using the baculovirus expression system.
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发表时间：
1993
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通讯作者：
C. Kim;S. Dion;J. Onorato;J. Benovic
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作者：
C. Kim;S. Dion;J. Onorato;J. Benovic
β 肾上腺素能受体激酶 β ARK1 和 β ARK2 是最近克隆的 G 蛋白偶联受体激酶家族的两个成员。为了进一步表征这些激酶，使用杆状病毒表达系统在 Sf9 昆虫细胞中过表达牛 β ARK1 和 β ARK2。通过在 S-Sepharose 和肝素-Sepharose 上对受感染的 Sf9 细胞上清液级分进行连续层析，获得了高产率（5-7 mg/L 细胞）的纯化激酶制剂。表达的激酶具有完全活性，其特异性磷酸化激动剂占据的 β2-肾上腺素能受体 (β2AR) 和光激活视紫红质的能力证明了这一点。对于 β ARK1 和 β ARK2 观察到相似的 β 2AR 磷酸化初始速率和最大化学计量。此外，G蛋白βγ亚基将βARK1和βARK2介导的β2AR磷酸化的初始速率提高了大约十倍。在存在βγ亚基的情况下，β2AR磷酸化的最大化学计量从约4mol磷酸盐/mol受体增加到约10mol/mol。视紫红质磷酸化的详细动力学分析表明，两种激酶都遵循连续的机制途径，并且对视紫红质（约 14 microM）和 MgATP（60-90 microM）具有相似的 Kms。肽磷酸化研究表明，与丝氨酸相比，两种激酶更喜欢氨基末端的酸性氨基酸。肝素被发现是两种激酶最有效的抑制剂，β ARK1 和 β ARK2 的 IC50 值分别为 1.4 和 1.1 microM。这些研究表明 β ARK1 和 β ARK2 具有非常相似的动力学特性，并表明它们在体内可能具有相似的底物特异性。
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.