Nair K Saidas, Mendez Ana, Blumer Joe B, Rosenzweig Derek H, Slepak Vladlen Z
Department of Molecular and Cellular Pharmacology and Neuroscience Program, University of Miami School of Medicine, Miami, Florida 33136, USA.
Invest Ophthalmol Vis Sci. 2005 Jan;46(1):383-9. doi: 10.1167/iovs.04-1006.
Heterotrimeric G proteins are regulated by receptors that act as guanine nucleotide exchange factors (GEFs) and by RGS proteins, which act as guanosine triphosphatase (GTPase) activating proteins (GAPs). Guanosine diphosphate (GDP) dissociation inhibitors (GDIs), such as activators of G protein signaling (AGS)-1 and -3 and Leu-Gly-Asn repeat-enriched (LGN) proteins regulate the Gi family of G proteins. AGS3 and LGN contain four characteristic G protein regulator (GPR) domains that are responsible for its GDI function. This study investigates the presence of a GDI for transducin in photoreceptor cells.
Western blot analysis of bovine and mouse retina was performed using specific antibodies to AGS and LGN proteins. The subcellular localization of LGN in retina was studied by immunofluorescence microscopy of mouse retinal sections and fractionation of retinal lysates, using sucrose density gradients. The interaction of LGN with transducin was studied using pull-down assays with GST-fused LGN constructs, co-immunoprecipitation and assays for GTPgammaS binding.
LGN, but not AGS3 and AGS1, was present in the retina, where it was localized mostly in the inner segments and outer plexiform layer of photoreceptor cells in both light and dark conditions. LGN was present in the cytosol, membrane, and the detergent-resistant cytoskeletal fraction. The amount of LGN relative to transducin was at least 1:15. The alpha subunit of transducin in its GDP-bound state interacted with endogenous and recombinant LGN, and the recombinant GPR domain of LGN reduced the rate of GTP exchange.
Photoreceptor inner segments contain LGN, which can bind to the alpha subunit of transducin and potentially regulate its function.
异三聚体G蛋白受作为鸟嘌呤核苷酸交换因子（GEF）的受体以及作为鸟苷三磷酸酶（GTPase）激活蛋白（GAP）的RGS蛋白调控。鸟苷二磷酸（GDP）解离抑制剂（GDI），如G蛋白信号激活剂（AGS）-1和-3以及富含亮氨酸-甘氨酸-天冬酰胺重复序列（LGN）的蛋白，可调节G蛋白的Gi家族。AGS3和LGN含有四个负责其GDI功能的特征性G蛋白调节（GPR）结构域。本研究调查了光感受器细胞中是否存在转导素的GDI。
使用针对AGS和LGN蛋白的特异性抗体对牛和小鼠视网膜进行蛋白质印迹分析。通过对小鼠视网膜切片进行免疫荧光显微镜检查以及使用蔗糖密度梯度对视网膜裂解物进行分级分离，研究了LGN在视网膜中的亚细胞定位。使用GST融合的LGN构建体进行下拉试验、共免疫沉淀和GTPγS结合试验，研究了LGN与转导素的相互作用。
LGN存在于视网膜中，而AGS3和AGS1不存在，在明、暗条件下，LGN主要定位于光感受器细胞的内段和外网状层。LGN存在于细胞质、细胞膜和抗去污剂细胞骨架组分中。LGN与转导素的相对量至少为1:15。处于GDP结合状态的转导素α亚基与内源性和重组LGN相互作用，LGN的重组GPR结构域降低了GTP交换速率。
光感受器内段含有LGN，其可与转导素α亚基结合并可能调节其功能。
