Biochemistry Group, Department of Neurosciences, Carl von Ossietzky University Oldenburg Oldenburg, Germany.
Biochemistry Group, Department of Neurosciences, Carl von Ossietzky University Oldenburg Oldenburg, Germany ; Research Center Neurosensory Science, Carl von Ossietzky University Oldenburg Oldenburg, Germany.
Front Mol Neurosci. 2014 Feb 26;7:4. doi: 10.3389/fnmol.2014.00004. eCollection 2014.
Membrane bound guanylate cyclases are expressed in rod and cone cells of the vertebrate retina and mutations in several domains of rod outer segment guanylate cyclase 1 (ROS-GC1 encoded by the gene GUCY2D) correlate with different forms of retinal degenerations. In the present work we investigated the biochemical consequences of three point mutations, one is located in position P575L in the juxtamembrane domain close to the kinase homology domain and two are located in the cyclase catalytic domain at H1019P and P1069R. These mutations correlate with various retinal diseases like autosomal dominant progressive cone degeneration, e.g., Leber Congenital Amaurosis and a juvenile form of retinitis pigmentosa. Wildtype and mutant forms of ROS-GC1 were heterologously expressed in HEK cells, their cellular distribution was investigated and activity profiles in the presence and absence of guanylate cyclase-activating proteins were measured. The mutant P575L was active under all tested conditions, but it displayed a twofold shift in the Ca(2) (+)-sensitivity, whereas the mutant P1069R remained inactive despite normal expression levels. The mutation H1019P caused the cyclase to become more labile. The different biochemical consequences of these mutations seem to reflect the different clinical symptoms. The mutation P575L induces a dysregulation of the Ca(2) (+)-sensitive cyclase activation profile causing a slow progression of the disease by the distortion of the Ca(2) (+)-cGMP homeostasis. In contrast, a strong reduction in cGMP synthesis due to an inactive or structurally unstable ROS-GC1 would trigger more severe forms of retinal diseases.
膜结合型鸟苷酸环化酶在脊椎动物视网膜的杆状和锥状细胞中表达，几种杆状细胞外段鸟苷酸环化酶 1（由基因 GUCY2D 编码的 ROS-GC1）的结构域突变与不同形式的视网膜变性相关。在本研究中，我们研究了三个点突变的生化后果，一个位于靠近激酶同源结构域的跨膜结构域中的 P575L 位置，另外两个位于环化酶催化结构域中的 H1019P 和 P1069R。这些突变与各种视网膜疾病相关，如常染色体显性进行性锥体细胞变性，例如莱伯先天性黑蒙和青少年型视网膜色素变性。野生型和突变型 ROS-GC1 均在 HEK 细胞中异源表达，研究了它们的细胞分布，并在存在和不存在鸟苷酸环化酶激活蛋白的情况下测量了它们的活性谱。突变体 P575L 在所有测试条件下均具有活性，但在 Ca(2)+敏感性方面表现出两倍的偏移，而突变体 P1069R 尽管表达水平正常，但仍保持无活性。突变体 H1019P 导致环化酶变得更加不稳定。这些突变的不同生化后果似乎反映了不同的临床症状。突变体 P575L 导致 Ca(2)+敏感的环化酶激活谱失调，通过破坏 Ca(2)+cGMP 平衡而导致疾病缓慢进展。相比之下，由于无活性或结构不稳定的 ROS-GC1 导致 cGMP 合成严重减少，会引发更严重形式的视网膜疾病。
