Murga C, Ruiz-Gómez A, García-Higuera I, Kim C M, Benovic J L, Mayor F
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma, Madrid, Spain.
J Biol Chem. 1996 Jan 12;271(2):985-94. doi: 10.1074/jbc.271.2.985.
The beta-adrenergic receptor kinase (beta ARK) modulates beta-adrenergic and other G protein-coupled receptors by rapidly phosphorylating agonist-occupied receptors at the plasma membrane. We have recently shown that beta ARK also associates with intracellular microsomal membranes both "in vitro" and "in situ" (García-Higuera, I., Penela, P., Murga, C., Egea, G., Bonay, P., Benovic, J. L., and Mayor, F., Jr. (1994) J. Biol. Chem. 269, 1348-1355), thus suggesting a complex modulation of the subcellular distribution of beta ARK. In this report, we used recombinant [35S]methionine-labeled beta ARK to show that this kinase interacts rapidly with a high affinity binding site (Kd of 20 +/- 1 nM) present in salt-stripped rat liver microsomal membranes. Although beta ARK binding is not modulated by membrane preincubation with G protein activators, the activity of bound beta ARK toward rhodopsin or a synthetic peptide substrate was markedly enhanced upon stimulation of the endogenous heterotrimeric G proteins present in the microsomal membranes by AIF4- or mastoparan/guanosine 5'-(3-O-thio)triphosphate, thus strongly suggesting a functional link between these proteins and membrane-associated beta ARK. Interestingly, beta ARK association with microsomal membranes is not significantly affected by a fusion protein derived from the carboxyl terminus of beta ARK1 (the proposed location of the beta gamma subunit binding site), whereas it is markedly inhibited by fusion proteins corresponding to the amino-terminal region of the kinase. The main determinants of binding appear to be localized to an approximately 60-amino acid residue stretch (residues 88 to 145). Our results further indicate a functional relationship between beta ARK and heterotrimeric G proteins in different intracellular organelles, and suggest that additional proteins may be involved in modulating the cellular localization of the kinase through a new targeting domain of beta ARK.
β - 肾上腺素能受体激酶（βARK）通过在质膜上快速磷酸化激动剂占据的受体来调节β - 肾上腺素能受体及其他G蛋白偶联受体。我们最近发现，βARK在“体外”和“原位”均与细胞内微粒体膜相关联（加西亚 - 伊格拉，I.，佩内拉，P.，穆尔加，C.，埃加，G.，博奈，P.，贝诺维奇，J. L.，和梅奥尔，F.，Jr.（1994年）《生物化学杂志》269，1348 - 1355），因此提示βARK亚细胞分布存在复杂的调节机制。在本报告中，我们使用重组的[35S]甲硫氨酸标记的βARK来表明该激酶与存在于盐洗大鼠肝微粒体膜中的高亲和力结合位点（解离常数Kd为20±1 nM）快速相互作用。尽管βARK的结合不受膜与G蛋白激活剂预孵育的调节，但在用AlF4 - 或马斯托帕兰/鸟苷5' - （3 - O - 硫代）三磷酸刺激微粒体膜中存在的内源性异三聚体G蛋白后，结合的βARK对视紫红质或合成肽底物的活性显著增强，从而强烈提示这些蛋白与膜相关的βARK之间存在功能联系。有趣的是，βARK与微粒体膜的关联不受源自βARK1羧基末端的融合蛋白（推测为βγ亚基结合位点的位置）的显著影响，而对应于激酶氨基末端区域的融合蛋白则对其有明显抑制作用。结合的主要决定因素似乎定位于大约60个氨基酸残基的片段（第88至145位残基）。我们的结果进一步表明βARK与不同细胞内细胞器中的异三聚体G蛋白之间存在功能关系，并提示可能有其他蛋白通过βARK的新靶向结构域参与调节该激酶的细胞定位。
