Department of Pharmacology, Center for Molecular Neuroscience and Vision Research Center, Vanderbilt University, Nashville, Tennessee 37232, USA.
J Neurosci. 2012 Aug 1;32(31):10758-66. doi: 10.1523/JNEUROSCI.0565-12.2012.
Arrestins belong to a family of multifunctional adaptor proteins that regulate internalization of diverse receptors including G-protein-coupled receptors (GPCRs). Defects associated with endocytosis of GPCRs have been linked to human diseases. We used enhanced green fluorescent protein-tagged arrestin 2 (Arr2) to monitor the turnover of the major rhodopsin (Rh1) in live Drosophila. We demonstrate that during degeneration of norpA(P24) photoreceptors the loss of Rh1 is parallel to the disappearance of rhabdomeres, the specialized visual organelle that houses Rh1. The cause of degeneration in norpA(P24) is the failure to activate CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) and retinal degeneration C (RDGC) because of a loss of light-dependent Ca(2+) entry. A lack of activation in CaMKII, which phosphorylates Arr2, leads to hypophosphorylated Arr2, while a lack of activation of RDGC, which dephosphorylates Rh1, results in hyperphosphorylated Rh1. We investigated how reversible phosphorylation of Rh1 and Arr2 contributes to photoreceptor degeneration. To uncover the consequence underlying a lack of CaMKII activation, we characterized ala(1) flies in which CaMKII was suppressed by an inhibitory peptide, and showed that morphology of rhabdomeres was not affected. In contrast, we found that expression of phosphorylation-deficient Rh1s, which either lack the C terminus or contain Ala substitution in the phosphorylation sites, was able to prevent degeneration of norpA(P24) photoreceptors. This suppression is not due to a loss of Arr2 interaction. Importantly, co-expression of these modified Rh1s offered protective effects, which greatly delayed photoreceptor degeneration. Together, we conclude that phosphorylation of Rh1 is the major determinant that orchestrates its internalization leading to retinal degeneration.
抑制蛋白属于多功能衔接蛋白家族，能调控包括 G 蛋白偶联受体（GPCR）在内的多种受体的内化。GPCR 内化缺陷与人类疾病相关。我们使用增强型绿色荧光蛋白标记的抑制蛋白 2（Arr2）来监测活果蝇中主要视紫红质（Rh1）的周转。我们证明，在 norpA(P24)光感受器变性过程中，Rh1 的丢失与视小杆的消失平行，视小杆是容纳 Rh1 的特化视觉细胞器。norpA(P24)变性的原因是由于光依赖性 Ca2+内流的丧失，CaMKII（Ca2+/钙调蛋白依赖性蛋白激酶 II）和视网膜变性 C（RDGC）无法激活。由于抑制蛋白 2（Arr2）的磷酸化缺乏，CaMKII 的缺乏导致低磷酸化的 Arr2，而 RDGC 的缺乏，其能使 Rh1 去磷酸化，导致高磷酸化的 Rh1。我们研究了 Rh1 和 Arr2 的可逆磷酸化如何导致光感受器变性。为了揭示 CaMKII 激活缺乏的后果，我们通过抑制肽抑制 CaMKII 对 ala(1) 蝇进行了特征描述，结果表明视小杆的形态没有受到影响。相比之下，我们发现表达缺乏磷酸化的 Rh1s，即缺乏 C 端或磷酸化位点含有 Ala 取代的 Rh1s，能够防止 norpA(P24)光感受器的变性。这种抑制不是由于 Arr2 相互作用的丧失。重要的是，这些修饰的 Rh1s 的共表达提供了保护作用，大大延迟了光感受器的变性。总之，我们得出结论，Rh1 的磷酸化是协调其内化导致视网膜变性的主要决定因素。
