Identification of amino acid residues of Gsα critical to repression of adipogenesis
Identification of amino acid residues of Gsα critical to repression of adipogenesis
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DOI：
10.1074/jbc.273.19.11685
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发表时间：
1998-05-08
影响因子：
4.8
通讯作者：
Wang, HY
中科院分区：
文献类型：
--
作者：
Liu, XX;Malbon, CC;Wang, HY
GSα调节3T3-L1小鼠胚胎成纤维细胞向脂肪细胞的分化，这一过程被称为脂肪生成，通过用G蛋白GIα2的相应区域替换Gsα区域而产生的嵌合体的表达，参与抑制脂肪生成的Gsα结构域被定位于该分子的146-235序列(Wang，H-y，Johnson，G.L.，Liu，X.，Malbon，C.(1996)J.Biol)。化学。271、22022-22029)。作为丙氨酸扫描诱变的前奏，由Giα2的125-213序列三分构建的Gsα中的嵌合体仅稳定表达，并评估了该嵌合体对地塞米松和甲基异丁基黄嘌呤联合诱导剂抑制脂肪生成的能力。含有GIα2序列150-177的嵌合体抑制脂肪生成，而含有GIα2序列125-149或178-213的嵌合体未能抑制脂肪生成的诱导，首先对这两个关键结构域进行丙氨酸扫描突变，然后通过单突变分析证实。鉴定出Gsα特有的6个残基分别为Asn(167)、Cys(200)、Leu(203)、Ser(205)、Val(214)和Lys(216)。Leu(208)和Ser(205)是串联需要的，因为单独对丙氨酸的诱变对阻遏活性没有影响。剩下的四个残基是抑制活性所必需的；其中任何一个残基的突变都会取消GSA抑制脂肪生成的能力，尽管不影响Gsα突变形式调控腺苷环化酶的能力，利用在Giα和Gsα晶体结构中发现的保守标志，Leu(203)和Ser(205)簇似乎暴露、紧密排列并位于开关I区域。Asn(16 7)，Val(2 14)和Lys(2 16)投射到Gsα上暴露在铜亚单位的Gtp-S配位状态下的区域。我们推测，这些残基构成了Gsα和控制脂肪生成的效应器之间的一个重要的接触域，该结构域尚未确定。
Gs alpha regulates the differentiation of 3T3-L1 mouse embryonic fibroblasts to adipocytes, a process termed adipogenesis, Through the expression of chimera created by substituting regions of Gs alpha with corresponding regions of the G protein Gi alpha 2, the domain of Gs alpha involved in repression of adipogenesis was localized to sequence 146-235 of the molecule (Wang, H-y., Johnson, G. L., Liu, X., Malbon, C. C. (1996) J. Biol. Chem. 271, 22022-22029). As a prelude to alanine-scanning mutagenesis, chimeras in Gs alpha constructed from trisection of the sequence 125-213 of Gi alpha 2 mere expressed stably, and clones were evaluated for the ability of the chimera to repress adipogenesis in response to the inducers, dexamethasone and methylisobutylxanthine, in combination. The chimera containing sequence 150-177 of Gi alpha 2 repressed adipogenesis, whereas the chimeras with either sequence 125-149 or 178-213 of Gi alpha 2 failed to repress induction of adipogenesis, Alanine-scanning mutagenesis of these two critical domains was performed first in clusters and then confirmed by analysis of single mutations. Six residues unique to Gs alpha were identified as critical to repression of adipogenesis, Asn(167), Cys(200), Leu(203), Ser(205), Val(214), and Lys(216). Leu(208) and Ser(205) are required in tandem, as mutagenesis to alanine of either one alone was without effect on repressor activity. The remaining four residues are required for repressor activity; mutation of any one of these abolishes the ability of Gsa to repress adipogenesis, although not affecting the ability of the mutant form of Gs alpha to regulate adenylylcyclase, Using conserved landmarks found in the crystal structures of Gi alpha and Gs alpha, the Leu(203) and Ser(205) cluster appears to be exposed, closely aligned and located in switch I region. Asn(167), Val(214), and Lys(216) project to regions on Gs alpha that are exposed in the GTP gamma S-liganded state of the cu subunit. We speculate that these residues constitute an important contact domain between Gs alpha and the effector controlling adipogenesis, which is yet to be identified.