Shi Guang W, Chen Jiayan, Concepcion Francis, Motamedchaboki Khatereh, Marjoram Paul, Langen Ralf, Chen Jeannie
Zilkha Neurogenetic Institute, Los Angeles, California 90033, USA.
J Biol Chem. 2005 Dec 16;280(50):41184-91. doi: 10.1074/jbc.M506935200. Epub 2005 Oct 11.
Phosphorylation of G-protein-coupled receptors (GPCRs) is a required step in signal deactivation. Rhodopsin, a prototypical GPCR, exhibits high gain phosphorylation in vitro whereby a hundred-fold molar excess of phosphates are incorporated into the rhodopsin pool per molecule of activated rhodopsin. The extent by which high gain phosphorylation occurs in the intact mammalian photoreceptor cell, and the molecular mechanism underlying this reaction in vivo, is not known. Trans-phosphorylation is a mechanism proposed for high gain phosphorylation, whereby rhodopsin kinase, upon phosphorylating the activated receptor, continues to phosphorylate nearby nonactivated rhodopsin. We used two different transgenic mouse models to test whether trans-phosphorylation occurs in the intact photoreceptor cell. The first transgenic model expressed a murine cone pigment, S-opsin, together with the endogenous rhodopsin in the rod cell. We showed that selective stimulation of rhodopsin also led to phosphorylation of S-opsin. The second mouse model expressed the constitutively active human opsin mutant K296E. K296E, in the arrestin-/- background, also led to phosphorylation of endogenous mouse rhodopsin in the dark-adapted retina. Both mouse models provide strong support of trans-phosphorylation as an underlying mechanism of high gain phosphorylation, and provide evidence that a substantial fraction of nonactivated visual pigments becomes phosphorylated through this mechanism. Because activated, phosphorylated receptors exhibit decreased catalytic activity, our results suggest that dephosphorylation would be an important step in the full recovery of visual sensitivity during dark adaptation. These results may also have implications for other GPCR signaling pathways.
G蛋白偶联受体（GPCRs）的磷酸化是信号失活的必要步骤。视紫红质作为一种典型的GPCR，在体外表现出高增益磷酸化，即每分子活化的视紫红质会有百倍摩尔过量的磷酸盐掺入视紫红质池中。在完整的哺乳动物光感受器细胞中高增益磷酸化发生的程度以及体内该反应的分子机制尚不清楚。转磷酸化是一种被提出用于高增益磷酸化的机制，即视紫红质激酶在磷酸化活化受体后，继续磷酸化附近未活化的视紫红质。我们使用了两种不同的转基因小鼠模型来测试转磷酸化是否发生在完整的光感受器细胞中。第一个转基因模型在杆状细胞中表达了一种鼠锥色素S - 视蛋白以及内源性视紫红质。我们发现对视紫红质的选择性刺激也会导致S - 视蛋白的磷酸化。第二个小鼠模型表达了组成型活性人视蛋白突变体K296E。在抑制蛋白缺失的背景下，K296E在暗适应的视网膜中也会导致内源性小鼠视紫红质的磷酸化。这两种小鼠模型都有力地支持了转磷酸化是高增益磷酸化的潜在机制，并提供证据表明相当一部分未活化的视觉色素通过这种机制发生磷酸化。由于活化的、磷酸化的受体表现出催化活性降低，我们的结果表明去磷酸化将是暗适应期间视觉敏感性完全恢复的重要步骤。这些结果可能也对其他GPCR信号通路有影响。
