García-Higuera I, Penela P, Murga C, Egea G, Bonay P, Benovic J L, Mayor F
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1994 Jan 14;269(2):1348-55.
beta-Adrenergic receptor kinase (beta ARK) is a regulatory enzyme involved in the modulation of beta-adrenergic and other G protein-coupled receptors. It has been described that beta ARK is a cytosolic protein that transiently translocates to the plasma membrane in order to specifically phosphorylate agonist-occupied receptors. In this report, we used beta ARK-specific antibodies to demonstrate that a significant amount of this kinase is present in rat liver microsomal membranes. beta ARK seems to be peripherally associated with the cytosolic side of microsomal membranes since it can be stripped from the membranes by mild salt treatment. Cell-free association experiments indicate that the interaction of beta ARK is reversible, saturable, and strongly inhibited by protease or heat treatment of the microsomes, thus suggesting that beta ARK interacts with a protein component of the microsomal membrane. Gradient fractionation studies indicate that the highest beta ARK-specific activity co-migrates with endoplasmic reticulum enzymatic markers. Furthermore, indirect immunofluorescence and immunogold electron microscopy experiments performed in cultured cells using affinity-purified anti-beta ARK antibodies are consistent with this subcellular localization pattern. Taken together, our data suggest that several beta ARK pools (i.e. microsome-bound, plasma membrane-bound, and cytosolic) may exist inside the cell. Such results are in line with recent reports showing that proteins involved in plasma membrane signal transduction, such as heterotrimeric G proteins, are also associated with membranes of different intracellular organelles.
β-肾上腺素能受体激酶（βARK）是一种参与调节β-肾上腺素能受体和其他G蛋白偶联受体的调节酶。据描述，βARK是一种胞质蛋白，它会短暂地转运至质膜，以便特异性磷酸化被激动剂占据的受体。在本报告中，我们使用βARK特异性抗体来证明大鼠肝微粒体膜中存在大量这种激酶。βARK似乎在外周与微粒体膜的胞质侧相关联，因为它可以通过温和的盐处理从膜上剥离。无细胞结合实验表明，βARK的相互作用是可逆的、可饱和的，并且会被蛋白酶或对微粒体进行热处理强烈抑制，因此表明βARK与微粒体膜的一种蛋白质成分相互作用。梯度分级分离研究表明，最高的βARK特异性活性与内质网酶标记物共迁移。此外，使用亲和纯化的抗βARK抗体在培养细胞中进行的间接免疫荧光和免疫金电子显微镜实验与这种亚细胞定位模式一致。综上所述，我们的数据表明细胞内可能存在多个βARK库（即与微粒体结合的、与质膜结合的和胞质的）。这些结果与最近的报告一致，这些报告表明参与质膜信号转导的蛋白质，如异源三聚体G蛋白，也与不同细胞内细胞器的膜相关联。
