Song Hongman, Belcastro Marycharmain, Young E J, Sokolov Maxim
Department of Ophthalmology, West Virginia University School of Medicine and West Virginia University Eye Institute, Morgantown, West Virginia 26506, USA.
J Biol Chem. 2007 Aug 10;282(32):23613-21. doi: 10.1074/jbc.M701974200. Epub 2007 Jun 14.
Phosducin is a major phosphoprotein of rod photoreceptors that interacts with the Gbetagamma subunits of heterotrimeric G proteins in its dephosphorylated state. Light promotes dephosphorylation of phosducin; thus, it was proposed that phosducin plays a role in the light adaptation of G protein-mediated visual signaling. Different functions, such as regulation of protein levels and subcellular localization of heterotrimeric G proteins, transcriptional regulation, and modulation of synaptic transmission have also been proposed. Although the molecular basis of phosducin interaction with G proteins is well understood, the physiological significance of light-dependent phosphorylation of phosducin remains largely hypothetical. In this study we quantitatively analyzed light dependence, time course, and subcellular localization of two principal light-regulated phosphorylation sites of phosducin, serine 54 and 71. To obtain physiologically relevant data, our experimental model exploited free-running mice and rats subjected to controlled illumination. We found that in the dark-adapted rods, phosducin phosphorylated at serine 54 is compartmentalized predominantly in the ellipsoid and outer segment compartments. In contrast, phosducin phosphorylated at serine 71 is present in all cellular compartments. The degree of phosducin phosphorylation in the dark appeared to be less than 40%. Dim light within rod operational range triggers massive reversible dephosphorylation of both sites, whereas saturating light dramatically increases phosphorylation of serine 71 in rod outer segment. These results support the role of phosducin in regulating signaling in the rod outer segment compartment and suggest distinct functions for phosphorylation sites 54 and 71.
视紫红质结合蛋白是视杆光感受器的一种主要磷蛋白，在其去磷酸化状态下与异源三聚体G蛋白的Gβγ亚基相互作用。光促进视紫红质结合蛋白的去磷酸化；因此，有人提出视紫红质结合蛋白在G蛋白介导的视觉信号转导的光适应中起作用。还提出了不同的功能，如调节异源三聚体G蛋白的蛋白质水平和亚细胞定位、转录调节以及突触传递的调节。尽管对视紫红质结合蛋白与G蛋白相互作用的分子基础有很好的理解，但视紫红质结合蛋白光依赖性磷酸化的生理意义在很大程度上仍然是假设性的。在本研究中，我们定量分析了视紫红质结合蛋白两个主要的光调节磷酸化位点丝氨酸54和71的光依赖性、时间进程和亚细胞定位。为了获得生理相关数据，我们的实验模型利用了自由活动的小鼠和大鼠，并对其进行了可控光照。我们发现，在暗适应的视杆中，丝氨酸54磷酸化的视紫红质结合蛋白主要分隔在椭球体和外段区室中。相比之下，丝氨酸71磷酸化的视紫红质结合蛋白存在于所有细胞区室中。在黑暗中视紫红质结合蛋白的磷酸化程度似乎小于40%。视杆工作范围内的暗光会触发这两个位点的大量可逆去磷酸化，而饱和光会显著增加视杆外段中丝氨酸71的磷酸化。这些结果支持了视紫红质结合蛋白在调节视杆外段区室信号转导中的作用，并表明磷酸化位点54和71具有不同的功能。
