Liu Y, Arshavsky V Y, Ruoho A E
Department of Pharmacology, University Wisconsin Medical School, Madison, Wisconsin 53706, USA.
J Biol Chem. 1996 Oct 25;271(43):26900-7. doi: 10.1074/jbc.271.43.26900.
In photoreceptor cells, visual transduction occurs through photoexcitation of rhodopsin, GTP activation of the alpha subunit of transducin, and interaction between GTP-bound transducin alpha subunit and the inhibitory gamma subunit of phosphodiesterase. The gamma subunit of phosphodiesterase, in turn, accelerates the hydrolysis of GTP on the alpha subunit of transducin. Within the COOH-terminal residues (46-87) of the phosphodiesterase gamma subunit, Trp-70 has been implicated in phosphodiesterase activation, transducin alpha subunit-phosphodiesterase gamma subunit interaction, and the GTP hydrolysis accelerating activity. We have derivatized the phosphodiesterase gamma subunit with a reversible photoactivatable reagent, [125I]N-[(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) ]cysteine ([125I]ACTP), at cysteine (Cys-68). A light-dependent, cross-linked complex of guanosine 5'-(gamma-thio)triphosphate-bound transducin alpha subunit and ACTPderivatized phosphodiesterase gamma subunit formed after photolysis of a 1:1 stoichiometic complex of the two proteins. The specificity of complex formation between the transducin alpha subunit and the phosphodiesterase gamma subunit was demonstrated by specific protection by the C68A mutant of the phosphodiesterase gamma subunit. The cross-linked complex was treated with beta-mercaptoethanol to transfer the 125I photomoiety from the phosphodiesterase gamma subunit to the transducin alpha subunit. Combined techniques involving electrophoresis, chemical and enzymatic cleavage, and chemical and radiosequencing were used to identify photoinsertion sites on the alpha3 and alpha4/beta6 regions of the transducin alpha subunit. Three photo-labeled residues, His-244 (alpha3 helix), Met-308, and Arg-310 (alpha4/beta6 interface), were specifically identified as photoinsertion sites. Utilizing the crystal structure coordinates of the GTP-bound transducin alpha subunit and molecular modeling, we conclude that Cys-68 of the phosphodiesterase gamma subunit is located at a position between the exposed face of the alpha3 and alpha4 helices of the transducin alpha subunit. We propose that the phosphodiesterase gamma subunit interacts with GTP-bound transducin alpha subunit at multiple sites in which the cysteine 68 to tryptophan 70 sequence of the phosphodiesterase gamma subunit, which is critical for GTP hydrolysis accelerating activity, interacts in the alpha3/alpha4/beta6 region of GTP-bound transducin alpha subunit.
在光感受器细胞中，视觉转导通过视紫红质的光激发、转导蛋白α亚基的GTP激活以及GTP结合的转导蛋白α亚基与磷酸二酯酶抑制性γ亚基之间的相互作用来发生。反过来，磷酸二酯酶的γ亚基加速转导蛋白α亚基上GTP的水解。在磷酸二酯酶γ亚基的COOH末端残基（46 - 87）内，色氨酸70与磷酸二酯酶激活、转导蛋白α亚基 - 磷酸二酯酶γ亚基相互作用以及GTP水解加速活性有关。我们用一种可逆的光活化试剂[125I]N - [(3 - 碘 - 4 - 叠氮基苯丙酰胺基 - S - (2 - 硫代吡啶基)]半胱氨酸（[125I]ACTP）在半胱氨酸（Cys - 68）处对磷酸二酯酶γ亚基进行衍生化。在光解两种蛋白质的1:1化学计量复合物后，形成了鸟苷5' - （γ - 硫代）三磷酸结合的转导蛋白α亚基与ACTP衍生化的磷酸二酯酶γ亚基的光依赖性交联复合物。磷酸二酯酶γ亚基的C68A突变体的特异性保护证明了转导蛋白α亚基与磷酸二酯酶γ亚基之间复合物形成的特异性。用β - 巯基乙醇处理交联复合物，将125I光部分从磷酸二酯酶γ亚基转移到转导蛋白α亚基。涉及电泳、化学和酶促裂解以及化学和放射性测序的联合技术用于鉴定转导蛋白α亚基的α3和α4 / β6区域上的光插入位点。三个光标记残基，即组氨酸244（α3螺旋）、甲硫氨酸308和精氨酸310（α4 / β6界面）被明确鉴定为光插入位点。利用GTP结合的转导蛋白α亚基的晶体结构坐标和分子建模，我们得出结论，磷酸二酯酶γ亚基的Cys - 68位于转导蛋白α亚基的α3和α4螺旋暴露面之间的位置。我们提出，磷酸二酯酶γ亚基在多个位点与GTP结合的转导蛋白α亚基相互作用，其中磷酸二酯酶γ亚基的半胱氨酸68至色氨酸70序列对GTP水解加速活性至关重要，在GTP结合的转导蛋白α亚基的α3 / α4 / β6区域相互作用。
