SPATIAL AND TEMPORAL EXPRESSION OF THE DICTYOSTELIUM-DISCOIDEUM G-ALPHA PROTEIN SUBUNIT G-ALPHA-2 - EXPRESSION OF A DOMINANT-NEGATIVE PROTEIN INHIBITS PROPER PRESTALK TO STALK DIFFERENTIATION
SPATIAL AND TEMPORAL EXPRESSION OF THE DICTYOSTELIUM-DISCOIDEUM G-ALPHA PROTEIN SUBUNIT G-ALPHA-2 - EXPRESSION OF A DOMINANT-NEGATIVE PROTEIN INHIBITS PROPER PRESTALK TO STALK DIFFERENTIATION
复制标题
DOI：
10.1091/mbc.5.1.7
复制
发表时间：
1994-01-01
影响因子：
3.3
通讯作者：
FIRTEL, RA
中科院分区：
文献类型：
--
作者：
CARREL, F;DHARMAWARDHANE, S;FIRTEL, RA
先前的结果表明，Got 蛋白亚基 G α 2 是盘基网柄菌中聚集所必需的，并且对于在此发育阶段将细胞表面 cAMP 受体与体内下游效应器偶联至关重要。 G α 2 表达至少四种不同的转录物，这些转录物在发育过程中受到差异性调节；其中两个转录物仅在多细胞阶段表达，并且它们的表达仅限于前茎细胞。我们部分解剖了 G α 2 启动子，并使用荧光素酶基因融合鉴定了在多细胞阶段专门表达的组件。当该启动子区域与 lacZ 偶联时，β-gal 表达仅限于多细胞阶段并定位于前茎细胞，其模式与 ecmA 前茎特异性启动子相似。我们发现，在发育早期阶段，G α 2 突变蛋白 [G α 2(G206T)] 在野生型细胞中的表达会阻断腺苷酸环化酶和鸟苷酸环化酶的聚集和 cAMP 介导的激活，表明其作为显性负活性 G α 亚基发挥作用。当这种突变的 G α 蛋白从 ecmA 茎前特异性启动子表达时，会观察到茎在顶盛期间的异常分化。来自 SP60 前孢子启动子的突变型 G α 2 或来自 ecmA 或 SP60 启动子的野生型 G α 2 的表达导致没有可检测的表型。结果表明，G α 2 在前茎细胞的顶峰阶段发挥着重要作用，并且可能在多细胞发育过程中介导这些过程的 cAMP 受体激活。
Previous results have shown that the Got protein subunit G alpha 2 is required for aggregation in Dictyostelium discoideum and is essential for coupling cell-surface cAMP receptors to downstream effecters in vivo during this stage of development. G alpha 2 expresses at least four distinct transcripts that are differentially regulated during development; two of the transcripts are expressed exclusively in the multicellular stages and their expression is restricted to prestalk cells. We partially dissected the G alpha 2 promoter and identified a component that is expressed exclusively during the multicellular stages using luciferase gene fusions. When this promoter region is coupled to lacZ, beta-gal expression is restricted to the multicellular stages and localized in prestalk cells with a pattern similar to that of the ecmA prestalk-specific promoter. We show that expression in wild-type cells of the G alpha 2 mutant protein [G alpha 2(G206T)] during the early stages of development blocks aggregation and cAMP-mediated activation of adenylyl cyclase and guanylyl cyclase, suggesting it functions as a dominant negatively active G alpha subunit. When this mutant G alpha protein is expressed from the ecmA prestalk-specific promoter, abnormal stalk differentiation during culmination is observed. Expression of the mutant G alpha 2 from the SP60 prespore promoter or wild-type G alpha 2 from either the ecmA or the SP60 promoter results in no detectable phenotype. The results suggest that G alpha 2 plays an essential role during the culmination stage in prestalk cells and may mediate cAMP receptor activation of these processes during multicellular development.