A novel cytosolic regulator, pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium
A novel cytosolic regulator, pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium
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DOI：
10.1101/gad.11.23.3218
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发表时间：
1997-12-01
影响因子：
10.5
通讯作者：
Devreotes, PN
中科院分区：
文献类型：
--
作者：
Chen, MY;Long, Y;Devreotes, PN
遗传分析被应用于确定新的基因参与G蛋白连接的途径控制发展。利用限制性内切酶介导的整合技术（REMI），我们在盘基网柄藻中发现了一个新的cAMP信号转导基因Pianissimo（PiaA）。PiaA编码一个130 kD的胞质蛋白，是趋化因子受体和G蛋白介导的12跨膜结构域腺苷酸环化酶激活所必需的。在piaA（-）无效突变体中，完整细胞的化学引诱物刺激和裂解物的GTP γ S处理都不能激活酶; PiaA的组成型表达逆转了这些缺陷。含有Pia蛋白的野生型细胞的胞质溶胶重建了piaA（-）裂解物中腺苷酸环化酶活性的GTP γ S刺激，表明Pia直接参与了激活。Pia和CRAC，一种先前鉴定的胞质调节剂，对于酶的活化都是必需的，因为crac（-）piaA（-）双突变体的裂解物需要这两种蛋白质进行重构。在酿酒酵母和粟酒裂殖酵母中发现了PiaA的同源物;我们认为Pia的同源物和这些普遍存在的G蛋白连接途径的类似调节模式在高等真核生物中可能不存在。
Genetic analysis was applied to identify novel genes involved in G protein-linked pathways controlling development. Using restriction enzyme mediated integration (REMI), we have identified a new gene, Pianissimo (PiaA), involved in cAMP signaling in Dictyostelium discoideum. PiaA encodes a 130-kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase. In piaA(-)null mutants, neither chemoattractant stimulation of intact cells nor GTP gamma S treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects. Cytosols of wild-type cells that contain Pia protein reconstitute the GTP gamma S stimulation of adenylyl cyclase activity in piaA(-)lysates, indicating that Pia is directly involved in the activation. Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme as lysates of crac(-)piaA(-)double mutants require both proteins for reconstitution. Homologs of PiaA are found in Saccharomyces cerevisiae and Schizosaccaromyces pombe; disruption of the S. cerevisiae homolog results in lethality, We propose that homologs of Pia and similar modes of regulation of these ubiquitous G protein-linked pathways ape likely no exist in higher eukaryotes.