DESENSITIZATION OF CONE VISUAL SIGNALING PATHWAYS

两种类型的细胞，杆状细胞和视锥细胞，在哺乳动物的视网膜上调节对光的反应。视杆细胞主要用于昏暗的光线，而视锥细胞则在明亮的光线下调节视觉。电生理学研究已经确定，视锥细胞对光线的反应比杆状细胞更快，恢复得也更快。造成这些差异的原因尚不清楚，但可能部分是由于直接参与视觉信号级联或进一步调节对光反应的调节机制的蛋白质之间相互作用动力学的改变。我们对哺乳动物光转导的大部分知识都来自于对视杆细胞的研究，主要是因为大多数哺乳动物的视网膜有90%到95%是视杆细胞。尽管已经克隆了许多锥体特异性蛋白，但很少有体外重构研究来确定这些蛋白是否与它们的杆状细胞对应物相似。最近，我们克隆了一种新的激酶GRK7，一种G蛋白偶联受体激酶（GRK），从13线地松鼠（一种视锥显性哺乳动物，其视网膜是94%的视锥），并在猪（一种视杆显性哺乳动物）的视网膜中证实了它的存在。原位杂交表明GRK7特异性定位于地鼠视网膜感光细胞层。使用针对GRK7的抗体的免疫荧光表明，该激酶特异性地定位于锥体中。这些结果表明GRK7可能是杆状特异视紫红质激酶GRK1的锥体同源物。通过比较GRK7和GRK1磷酸化视锥蛋白和视紫红质的能力，该基金开始解决在视杆细胞和视锥细胞中观察到的信号终止差异的原因。GRK7和GRK1在猪视网膜中的具体位置及其与视锥蛋白的共定位将采用间接免疫荧光法确定。利用GRK7和GRK1的重组制剂，建立体外视锥蛋白和视紫红质磷酸化的生化检测方法。在HEK-293或COS细胞中表达的GRK7中异戊二烯基修饰的类型以及异戊二烯化的作用将被研究。我们将研究自磷酸化位点及其对GRK7活性的影响。钙结合蛋白钙调蛋白和恢复蛋白对GRK7活性的影响也将被研究，该激酶在视锥蛋白上的底物位点将被分析。这些研究提供的新信息将有助于理解视锥细胞和视锥相关疾病过程中视觉信号的调节。

Two types of cells, rods and cones, mediate the response to light in the mammalian retina. Rods are used primarily in dim light, whereas the cones mediate vision in bright light. Electrophysiological studies have determined that cones respond to light more rapidly and recover more quickly than rods. The reasons for these differences are not well understood, but may be due in part to altered kinetics of interaction between the proteins that participate directly in the visual signaling cascade or in regulatory mechanisms that further modulate the response to light. Most of our knowledge of phototransduction in mammals is derived from studies of the rod cells, primarily because most mammalian retinas are 90 to 95 percent rods. Although a number of the cone-specific proteins have been cloned, there have been very few in vitro reconstitution studies to determine whether these proteins interact similarly to their rod cell counterparts. Recently, we cloned a novel kinase, GRK7, a G protein-coupled receptor kinase (GRK), from the 13-lined ground squirrel, a cone-dominant mammal whose retina is 94 percent cones, and also established its existence in the retina of the pig, a rod-dominant mammal. In situ hybridization showed that GRK7 is located specifically in the photoreceptor cell layer of the ground squirrel retina. Immunofluorescence, using an antibody directed against GRK7, demonstrates that this kinase is localized specifically in cones. These results suggest that GRK7 may be a cone homologue of GRK1, the rod-specific rhodopsin kinase. This grant begins to address the reasons for the differences in signal termination observed for rods and cones by comparing the ability of GRK7 and GRK1 to phosphorylate the cone opsins and rhodopsin. The specific locations of GRK7 and GRK1 and their colocalization with the cone opsins in pig retinas will be determined using indirect immunofluorescence. Biochemical assays will be established to study cone opsin and rhodopsin phosphorylation in vitro using recombinant preparations of GRK7 and GRK1. The type of isoprenyl modification in the GRK7 expressed in HEK-293 or COS cells and the role of isoprenylation will be investigated. The sites of autophosphorylation and their influence on GRK7 activity will be studied. The influence of the calcium binding proteins, calmodulin and recoverin, on the activity of GRK7 will also be investigated and the substrate sites for this kinase on the cone opsins will be analyzed. The new information provided by these studies will aid in understanding the regulation of visual signaling in cone cells and cone-related disease processes.