Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding
Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding
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DOI：
10.1074/jbc.m110.151043
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发表时间：
2011-01-14
影响因子：
4.8
通讯作者：
Gurevich, Vsevolod V.
中科院分区：
文献类型：
--
作者：
Bayburt, Timothy H.;Vishnivetskiy, Sergey A.;Gurevich, Vsevolod V.
g蛋白偶联受体（GPCR）寡聚化已经在各种各样的实验环境中被观察到，但这种现象在a类GPCR生命周期不同阶段的功能意义仍有待阐明。视紫红质（Rh）是一种典型的视觉转导a类GPCR，也能够形成二聚体和高阶低聚物。最近的研究表明，Rh单体足以激活其同源的G蛋白，转导蛋白，这促使我们测试相同的单体状态是否足以使视紫红质磷酸化和抑制蛋白1结合。在本研究中，我们发现单体活性视紫红质被视紫红质激酶（GRK1）磷酸化，其效率与天然盘膜中的视紫红质一样高。纳米圆盘中的单体磷酸化光活化Rh （P-Rh*）本质上与阻滞蛋白1以及天然圆盘膜中的P-Rh*结合。我们还使用基于荧光的测定方法测量了纳米圆盘中arrestin-1对P-Rh*的亲和力，发现arrestin-1与单体P-Rh*的相互作用具有低纳摩尔亲和力和1:1的化学计量，正如之前在天然圆盘膜中确定的那样。因此，与转导蛋白激活类似，GRK1对视紫红质的磷酸化和高亲和力的arrestin-1结合只需要一个视紫红质单体。
G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.