TRANSDUCIN-ALPHA C-TERMINAL MUTATIONS PREVENT ACTIVATION BY RHODOPSIN - A NEW ASSAY USING RECOMBINANT PROTEINS EXPRESSED IN CULTURED-CELLS
TRANSDUCIN-ALPHA C-TERMINAL MUTATIONS PREVENT ACTIVATION BY RHODOPSIN - A NEW ASSAY USING RECOMBINANT PROTEINS EXPRESSED IN CULTURED-CELLS
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DOI：
10.1002/j.1460-2075.1995.tb00125.x
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发表时间：
1995-09-15
期刊：
影响因子：
11.4
通讯作者：
BOURNE, HR
中科院分区：
文献类型：
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作者：
GARCIA, PD;ONRUST, R;BOURNE, HR
我们使用一种新的测定方法测量了重组视紫红质对视网膜转导素（G（t），也是重组体）α亚基（α（t））的激活作用。用编码视蛋白和C-t的三个亚基（α（t）β（1）和γ（1））的DNA瞬时转染培养的细胞。在这些细胞的微粒体中，与11-顺式-视黄醛一起孵育，光引起G（t）的快速活化，如通过GTP γ S保护α（t）片段免于蛋白水解降解的能力所测量的。当在恒定照明下将全反式-视黄醛加入微粒体中时，也观察到G（t）的活化。激活取决于视蛋白和视网膜。具有已知的激活G（t）缺陷的视蛋白突变体在该测定中显示出类似的缺陷。模拟G（s）α-亚基中相应突变的α（t）突变在该测定中也产生定性相似的效果。作为旨在探索受体与G蛋白相互作用中结构和功能之间关系的策略的第一步，我们测试了C末端10个氨基酸中每一个都被丙氨酸取代的突变α（t）蛋白，该区域已知对与视紫红质的相互作用至关重要。在四个位置的丙氨酸取代中度（K341）或严重（L344，G348，L349）削弱了α（t）对视紫红质激活的敏感性。所有四种突变体都保留了被AIF 4激活的能力（4）（-）。另外两个取代（N343和F350）导致非常轻微的缺陷，而在其余四个位置（E342、K345、D346和C347）的取代没有影响。结合以前的观察，这些结果限制了alpha（t）的C-末端与视紫红质相互作用的模型。
We have measured the activation by recombinant rhodopsin of the alpha-subunit (alpha(t)) of retinal transducin (G(t), also recombinant) using a new assay. Cultured cells are transiently transfected with DNAs encoding opsin and the three subunits of C-t (alpha(t) beta(1) and gamma(1)). In the microsomes of these cells, incubated with 11-cis-retinal, light causes the rapid activation of G(t), as measured by the ability of GTP gamma S to protect alpha(t) fragments from proteolytic degradation. The activation of G(t) is also observed when all-trans-retinal is added to microsomes under constant illumination. Activation depends on both opsin and retinal. Opsin mutants with known defects in activating G(t) show similar defects in this assay. alpha(t) mutations that mimic the corresponding mutations in the alpha-subunit of G(s) also produce qualitatively similar effects in this assay. As a first step in a strategy aimed at exploring the relationships between structure and function in the interactions of receptors with G proteins, we tested mutant alpha(t) proteins with alanine substituted for each of the 10 amino acids at the C-terminus, a region known to be crucial for interactions with rhodopsin. Alanine substitution at four positions moderately (K341) or severely (L344, G348, L349) impairs the susceptibility of alpha(t) to activation by rhodopsin. All four mutants retain their ability to be activated by AIF4(4)(-). Two other substitutions (N343 and F350) resulted in very mild defects, while substitutions at the remaining four positions (E342, K345, D346 and C347) had no effect. In combination with previous observations, these results constrain models of the interaction of the C-terminus of alpha(t) with rhodopsin.