Kieselbach T, Irrgang K D, Rüppel H
Max-Volmer-Institut für Biophysikalische und Physikalische Chemie, Technische Universität Berlin, Germany.
Eur J Biochem. 1994 Nov 15;226(1):87-97. doi: 10.1111/j.1432-1033.1994.tb20029.x.
In retinal rods, photoexcited rhodopsin (R*) is inactivated upon phosphorylation by rhodopsin kinase and the subsequent binding of arrestin. We have studied the structural role of a cationic region of bovine arrestin (Val170-Arg182) using anti-peptide IgGs specifically recognizing this segment and the corresponding oligopeptide. Our results clearly indicate that amino acids Val170-Arg182 are shielded within the arrestin-rhodopsin-complex and very likely belong to a binding domain of arrestin for phosphorylated R*. The purified anti-peptide IgGs strongly reacted with isolated arrestin but did not recognize arrestin when bound to phosphorylated R*. In agreement with these experiments, the oligopeptide Val170-Arg182 was found to compete with arrestin for binding to phosphorylated R*. Increasing concentrations of this peptide caused an oligomerization of phosphorylated rhodopsin when illuminated by white light as well as in the dark. Unphosphorylated rhodopsin did not oligomerize up to a 400-fold molar ratio of peptide/rhodopsin. Limited proteolysis of the phosphorylated carboxy-terminus of rhodopsin with endoproteinase Asp-N caused a significant decrease in the peptide-induced formation of oligomers. Therefore, Val170-Arg182 of bovine arrestin probably interacts with the phosphorylated carboxy-terminus of rhodopsin. The data presented support the proposal of Palczewski et al. (1991c) considering the region Lys163-Arg182 in bovine arrestin to be a possible binding domain for phosphorylated R*.
在视网膜视杆细胞中，光激发的视紫红质（R*）在被视紫红质激酶磷酸化并随后与抑制蛋白结合后失活。我们使用特异性识别该片段和相应寡肽的抗肽IgG研究了牛抑制蛋白阳离子区域（Val170 - Arg182）的结构作用。我们的结果清楚地表明，氨基酸Val170 - Arg182在抑制蛋白 - 视紫红质复合物中被屏蔽，并且很可能属于抑制蛋白与磷酸化R的结合域。纯化的抗肽IgG与分离的抑制蛋白强烈反应，但在与磷酸化R结合时不识别抑制蛋白。与这些实验一致，发现寡肽Val170 - Arg182与抑制蛋白竞争结合磷酸化R*。当用白光照射以及在黑暗中时，该肽浓度的增加导致磷酸化视紫红质的寡聚化。未磷酸化的视紫红质在肽/视紫红质摩尔比高达400倍时不会寡聚化。用天冬氨酸蛋白酶 - N对视紫红质的磷酸化羧基末端进行有限的蛋白水解导致肽诱导的寡聚体形成显著减少。因此，牛抑制蛋白的Val170 - Arg182可能与视紫红质的磷酸化羧基末端相互作用。所呈现的数据支持Palczewski等人（1991c）的提议，即认为牛抑制蛋白中的Lys163 - Arg182区域是磷酸化R*的可能结合域。
