Briscoe C, Moniakis J, Kim J Y, Brown J M, Hereld D, Devreotes P N, Firtel R A
Section of Cell and Developmental Biology, Center for Molecular Genetics, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093-0634, USA.
Dev Biol. 2001 May 1;233(1):225-36. doi: 10.1006/dbio.2001.0217.
cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on multicellular development when expressed in wild-type cells. These findings suggest that the phosphorylated C-terminus of cAR1 may be involved in regulating aspects of receptor-mediated processes, is not essential for GBF function, and may play a role in mediating subsequent development.
环磷酸腺苷（cAMP）受体通过偶联的异源三聚体G蛋白介导一些信号通路，而其他一些则不依赖G蛋白。后一类包括转录因子GBF和STATa的激活。在盘基网柄菌聚集形成的细胞丘内，微摩尔水平的cAMP激活GBF功能，从而诱导聚集后基因的转录并启动多细胞分化。STATa是一种调控终分化和ecmB表达的因子，其激活也发生在丘期细胞中，是由cAMP受体依赖性酪氨酸磷酸化和核定位所导致的。在丘发育过程中，cAMP受体cAR1处于低亲和力状态，并且在其C末端的多个丝氨酸残基上发生磷酸化。本文探讨了cAMP受体磷酸化在cAMP介导的GBF活性刺激、STATa酪氨酸磷酸化以及细胞类型特异性基因表达中可能发挥的作用。为了实现这一目标，我们在一个体内介导聚集后基因表达的内源性cAMP受体被缺失的菌株中表达了cAR1突变体。然后我们检测了这些细胞进行形态发生以及诱导聚集后和细胞类型特异性基因表达和STATa酪氨酸磷酸化的能力。对C末端尾巴缺失或配体介导的磷酸化位点发生突变的cAR1突变体的分析表明，cAR1的C末端对于GBF介导的聚集后基因表达或STATa酪氨酸磷酸化并非必需，但可能在调节细胞类型特异性基因表达和形态发生中发挥作用。一种C末端尾巴持续磷酸化的突变受体，在悬浮的脉冲细胞中表现出STATa酪氨酸磷酸化的持续激活，并且诱导细胞类型特异性基因表达的能力显著受损。当在野生型细胞中表达时，持续磷酸化的受体对多细胞发育也发挥部分显性负效应。这些发现表明，cAR1的磷酸化C末端可能参与调节受体介导过程的各个方面，对GBF功能并非必需，并且可能在介导后续发育中发挥作用。
