DESENSITIZATION OF CONE VISUAL SIGNALING PATHWAYS

两种类型的细胞，视杆细胞和视锥细胞，介导了哺乳动物视网膜对光的反应。 视杆细胞主要在昏暗的光线下使用，而视锥细胞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比视杆细胞对光的反应更快，恢复更快。 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反应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的动力学改变。 我们对哺乳动物中光传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90%到95%都是视杆细胞。虽然一些锥特异性蛋白质已被克隆，有很少的体外重建研究，以确定这些蛋白质的相互作用是否类似于他们的视杆细胞对应物。 最近，我们克隆了一种新的激酶，GRK 7，一种G蛋白偶联受体激酶（GRK），从13-lined地松鼠，一种视锥细胞占主导地位的哺乳动物，其视网膜是94%的视锥细胞，也建立了它的存在于视网膜的猪，一种杆占主导地位的哺乳动物。 原位杂交结果表明GRK 7特异性地定位于黄鼠视网膜的感光细胞层。 免疫荧光，使用针对GRK 7的抗体，证明这种激酶是本地化的具体锥。 这些结果表明，GRK 7可能是视杆细胞特异性视紫红质激酶GRK 1的锥体同源物。 这项资助通过比较GRK 7和GRK 1磷酸化视锥视蛋白和视紫红质的能力，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。 GRK 7和GRK 1的具体位置及其与视锥视蛋白在猪视网膜中的共定位将使用间接免疫荧光来确定。 将建立生物化学测定以使用GRK 7和GRK 1的重组制剂在体外研究视锥视蛋白和视紫红质磷酸化。 将研究HEK-293或COS细胞中表达的GRK 7中异戊二烯基修饰的类型以及异戊二烯化的作用。 将研究自磷酸化的位点及其对GRK 7活性的影响。 钙结合蛋白，钙调素和recoverin，对GRK 7的活性的影响也将进行研究，并分析视锥细胞视蛋白上的这种激酶的底物位点。 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.