Novel mechanism for the activation of rhodopsin kinase: Implications for other G protein-coupled receptor kinases (GRK's)
Novel mechanism for the activation of rhodopsin kinase: Implications for other G protein-coupled receptor kinases (GRK's)
复制标题
DOI：
10.1021/bi952480q
复制
发表时间：
1996-05-14
期刊：
影响因子：
2.9
通讯作者：
Akhtar, M
中科院分区：
文献类型：
--
作者：
Dean, KR;Akhtar, M
ATP，其不可水解的类似物，AMP-PNP;和白蛋白被发现，以促进视紫红质激酶从杆外节（ROS）含有光活化视紫红质（Rho*）的解离。这些功能体现在一个协议中的视紫红质激酶的回收含有ROS的孵育已进行了广泛的治疗。结果发现，从含有ATP，视紫红质激酶和光解ROS膜的混合物中回收的上清液催化Rho*-独立的肽磷酸化以及视紫红质的暗磷酸化。在上述两种测定中，该活化激酶的活性是在适当的标准测定中发现的最大固有活性的7-8%（即，视紫红质的光刺激磷酸化和Rho* 依赖性肽磷酸化）。激活的激酶恢复到其非活性的休息状态，在一个时间依赖性的方式，给一个tau（1/2）的衰减类似2分钟。激酶的内在活性，通过标准测定，但是，在此衰减期间保持不变。没有发现积极的证据表明，相互转换激活激酶失活激酶发生的磷酸化事件。累积的结果表明，视紫红质激酶ATP复合物与Rho* 的相互作用导致形成，可能是由于蛋白质结构的重组，可溶性活性激酶种类以时间依赖性的方式恢复到非活性的静息状态。
ATP, its nonhydrolyzable analogue, AMP-PNP;and albumin were found to promote the dissociation of rhodopsin kinase from rod outer segments (ROS) containing photoactivated-rhodopsin (Rho*). These features were embodied in a protocol for the recovery of rhodopsin kinase from incubations containing ROS which had been subjected to a wide range of treatments. It was found that the supernatants recovered from mixtures containing ATP, rhodopsin kinase, and photolyzed ROS membranes catalyzed a Rho*-independent peptide phosphorylation as well as dark-phosphorylation of rhodopsin. The activities of this activated kinase in the two aforementioned assays were 7-8% of the maximum intrinsic activity found in appropriate standard assays (i.e., light-stimulated phosphorylation of rhodopsin and Rho*-dependent peptide phosphorylation). The activated kinase reverted to its inactive resting-state in a time dependent fashion, giving a tau(1/2) of decay of similar to 2 min. The intrinsic activity of kinase as measured by the standard assay, however, remained constant during this decay period. No positive evidence was found to suggest that the interconversion activated kinase inactive kinase occurred by a phosphorylation event. Cumulatively, the results show that the interaction of rhodopsin kinase ATP complex with Rho* leads to the formation, presumably due to the reorganization of the protein structure, of a soluble active kinase species which reverts to the inactive resting state in a time-dependent fashion.