DESENSITIZATION OF CONE VISUAL SIGNALING PATHWAYS

两种类型的细胞，视杆细胞和视锥细胞，在哺乳动物的视网膜中调节对光的反应。视杆主要用在昏暗的光线下，而视锥则在强光下调节视觉。电生理学研究已经确定，锥体对光的反应更快，恢复得也更快。这些差异的原因尚不清楚，但部分原因可能是直接参与视觉信号级联的蛋白质之间相互作用的动力学改变，或者是进一步调节对光的反应的调节机制。我们对哺乳动物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有90%到95%的视杆细胞。虽然已经克隆了许多视锥细胞特异的蛋白质，但很少有体外重组研究来确定这些蛋白质是否与它们的杆状细胞相似地相互作用。最近，我们克隆了一种新的激酶GRK7，它是一种G蛋白偶联受体激酶(GRK)，来自13行地松鼠，它是一种视锥占优势的哺乳动物，其视网膜占94%，并证实了它在杆状占优势的哺乳动物猪的视网膜中的存在。原位杂交显示GRK7特异定位于黄鼠视网膜的光感受器细胞层。使用针对GRK7的抗体的免疫荧光表明，该激酶特异性地位于锥体中。这些结果表明，GRK7可能是视紫红质特异的视紫红质激酶GRK1的锥形同系物。这项资助通过比较GRK7和GRK1磷酸化视锥视蛋白和视紫红质的能力，开始解决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的具体位置以及它们与视锥视蛋白在猪视网膜中的共存将用间接免疫荧光法确定。GRK7和GRK1的重组制剂将用于研究视锥视蛋白和视紫红质的体外磷酸化。在HEK-293或COS细胞中表达的GRK7中的异戊二烯修饰类型以及异戊二烯修饰的作用将被研究。将研究自身磷酸化的位置及其对GRK7活性的影响。还将研究钙结合蛋白钙调蛋白和恢复素对GRK7活性的影响，并分析该激酶在锥体视蛋白上的底物位置。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.