Artemyev N O, Mills J S, Thornburg K R, Knapp D R, Schey K L, Hamm H E
Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago 60680.
J Biol Chem. 1993 Nov 5;268(31):23611-5.
Activation of cGMP phosphodiesterase (PDE) by the rod G-protein transducin is a key event in visual signal transduction in vertebrate photoreceptor cells. Interaction between the GTP-bound form of the alpha-subunit of transducin (alpha t*) and the PDE inhibitory gamma-subunit (P gamma) is a major component of PDE activation. The central polycationic region of P gamma, P gamma-24-45, has been implicated as one of the sites involved in alpha t*.P gamma interaction. Here we determine the site on alpha t* that interacts with P gamma-24-45 using a photo-cross-linking approach. The synthetic peptides Cys(ACM)Tyr-P gamma-24-45-Cys (where ACM indicates acetamidomethyl group) and Cys-P gamma-24-45 were labeled with 4-(N-maleimido)benzophenone at the COOH and NH2 termini, respectively, and then cross-linked to alpha t. When the photoprobe was attached to the COOH terminus of the peptide, a specific high yield cross-linked product (80%) was formed between the peptide and alpha t GTP gamma S (guanosine 5'-O-(thiotriphosphate)). A lower yield of cross-linking (35%) was seen between the peptide and alpha t GDP. The site of cross-linking between Cys(ACM)Tyr-P gamma-24-45-Cys and alpha t GTP gamma S was localized to within alpha t-306-310 using a variety of chemical and proteolytic cleavages of the cross-linked product, analysis of the fragments with SDS-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry.
视杆细胞G蛋白转导素激活环磷酸鸟苷磷酸二酯酶（PDE）是脊椎动物光感受器细胞视觉信号转导中的关键事件。转导素α亚基的GTP结合形式（αt*）与PDE抑制性γ亚基（Pγ）之间的相互作用是PDE激活的主要组成部分。Pγ的中央聚阳离子区域Pγ-24-45被认为是参与αt*.Pγ相互作用的位点之一。在这里，我们使用光交联方法确定αt*上与Pγ-24-45相互作用的位点。合成肽Cys(ACM)Tyr-Pγ-24-45-Cys（其中ACM表示乙酰氨基甲基）和Cys-Pγ-24-45分别在COOH和NH2末端用4-(N-马来酰亚胺)二苯甲酮标记，然后与αt交联。当光探针连接到肽的COOH末端时，在肽与αt GTPγS（鸟苷5'-O-(硫代三磷酸)）之间形成了一种特定的高产率交联产物（80%）。在肽与αt GDP之间观察到较低的交联产率（35%）。使用交联产物的各种化学和蛋白酶切割、SDS-聚丙烯酰胺凝胶电泳分析片段以及基质辅助激光解吸电离质谱，将Cys(ACM)Tyr-Pγ-24-45-Cys与αt GTPγS之间的交联位点定位到αt-306-310范围内。
