Morishita R, Fukada Y, Kokame K, Yoshizawa T, Masuda K, Niwa M, Kato K, Asano T
Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Japan.
Eur J Biochem. 1992 Dec 15;210(3):1061-9. doi: 10.1111/j.1432-1033.1992.tb17512.x.
Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their alpha subunits, though a notable multiplicity of gamma subunits has also been demonstrated. To investigate whether each subtype of alpha subunit is associated with a particular gamma subunit, various oligomeric G proteins, purified from bovine tissues, were subjected to gel electrophoresis in a Tricine buffer system. All G proteins examined were shown to have more than two kinds of gamma subunit. Of the brain G proteins, GoA, GoB, and Gi1 contain the same set of three gamma subunits, but Gi2 contains only two of these subunits. Lung Gi1 and Gi2 and spleen Gi2 and Gi3 had similar sets of two gamma subunits, one of which was distinct from the gamma subunits of brain G proteins. These observations indicate that each subtype of alpha subunit is associated with a variety of beta gamma subunits, and that the combinations differ among cells. For analyses of the structural diversity of the gamma subunits, beta gamma subunits were purified from the total G proteins of each tissue and subjected to reverse-phase HPLC under denaturing conditions, where none of the beta subunits were eluted from the column. Three distinct gamma subunits were isolated in this way from brain beta gamma subunits. In contrast, lung and spleen beta gamma subunits contained at least five gamma subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several gamma subunits, two subspecies appeared to be common to the three tissues. In fact, in each case, the partial amino acid sequence of the most abundant gamma subunit in each tissue was identical, and the sequences coincided exactly with that of 'gamma 6' [Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758-15761]. Fast-atom-bombardment mass spectrometry analysis indicated that this abundant gamma subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C-terminus, as was 'gamma 6' from brain. In addition to abundant gamma subunits, other tissue-specific gamma subunits were also shown to be geranylgeranylated by gas-chromatography-coupled mass spectrometry analysis of Raney nickel-treated gamma subunits. These results suggest that most gamma subunits associated with many different subtypes of alpha subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated gamma subunit.
异三聚体鸟嘌呤核苷酸结合调节蛋白（G蛋白）已根据其α亚基的特性被分为几个亚型，不过γ亚基也存在显著的多样性。为了研究每个α亚基亚型是否与特定的γ亚基相关联，从牛组织中纯化得到的各种寡聚G蛋白在Tricine缓冲液系统中进行凝胶电泳。所有检测的G蛋白都显示有两种以上的γ亚基。在脑G蛋白中，GoA、GoB和Gi1含有相同的一组三种γ亚基，但Gi2只含有其中两种亚基。肺组织中的Gi1和Gi2以及脾组织中的Gi2和Gi3具有相似的一组两种γ亚基，其中一种与脑G蛋白的γ亚基不同。这些观察结果表明，每个α亚基亚型都与多种βγ亚基相关联，并且这些组合在不同细胞之间存在差异。为了分析γ亚基的结构多样性，从每个组织的总G蛋白中纯化βγ亚基，并在变性条件下进行反相高效液相色谱分析，此时没有β亚基从柱上洗脱下来。通过这种方法从脑βγ亚基中分离出三种不同的γ亚基。相比之下，肺和脾的βγ亚基至少含有五种γ亚基，其洗脱位置和电泳迁移率在这两个组织之间无法区分。在几种γ亚基中，有两种亚型似乎在这三个组织中是共有的。事实上，在每种情况下，每个组织中最丰富的γ亚基的部分氨基酸序列是相同的，并且这些序列与“γ6”的序列完全一致[Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758 - 15761]。快原子轰击质谱分析表明，肺和脾中这种丰富的γ亚基在C末端进行了香叶基香叶基化和羧甲基化，就像脑中的“γ6”一样。除了丰富的γ亚基外，通过对经阮内镍处理的γ亚基进行气相色谱 - 质谱联用分析，还表明其他组织特异性γ亚基也进行了香叶基香叶基化。这些结果表明，与许多不同α亚基亚型相关联的大多数γ亚基在多种组织中进行了香叶基香叶基化，唯一的例外是视网膜，其中G蛋白转导素具有法尼基化的γ亚基。
