CAMP RECEPTOR SUBTYPES AND DICTYOSTELIUM DEVELOPMENT

胞外cAMP在网柄苔藓的发育中起着多种作用， 作为一种化学诱导剂，一种细胞间的信号分子，一种形态因子， 以及基因表达的诱导剂。我们最近发现， 网柄苔藓顺序表达多种表面cAMP受体(CARS) 在它的发展计划中。多种汽车亚型的发现 或许可以解释夏令营的多功能性。这些汽车看起来像哺乳动物的G蛋白 相关受体，如视紫红质、β-肾上腺素能受体和 粘液型乙酰胆碱受体。汽车亚型之间有很强的同源性 在假设的七个膜上跨越结构域和连接 环状结构，但在C末端假定的细胞质结构域上有显著不同。 我们将完成三个CAR亚型(cAR1， CAR2和CAR3)，设计汽车通用和亚型专用探头，分析 基因组DNA杂交，克隆第四个CAR亚型(CAR4)，并产生特异性 针对每个亚型的抗体。我们会核实每辆车都是营地 结合蛋白，决定其表达的时间进程，它是否 经历配体诱导的修饰，及其亚细胞分布。 为了评估每种汽车亚型在发展中的作用 程序，我们将专门删除或更改它。 首先，我们将确定在cAR1中表示哪些子类型 建立反义细胞系，构建特异性反义细胞系 每个子类型。我们将使用同源重组并创建亚型 “空”细胞，我们将用功能基因来补充。我们将进行克隆 粘菌相关物种的汽车亚型，并比较 的结构，还将筛选出各种 以确定它们是否含有表面cAMP受体。 我们将描述cAR1在表达时执行的那些功能 在生长的细胞中脱离正常的发育环境。它的 药理特异性、结合动力学、结合部位 光亲和标记、配体诱导的磷酸化和丝氨酸的位点 磷酸化和配体诱导的下调和重新分配将 被探索。为了评估cAR1与G-α-2的偶联，我们将 这两种蛋白质在生长中的细胞中共同表达。我们正在设计结构- 功能研究，以确定受体的哪些区域参与 每个功能：3‘-删除以粗略定位重要位置，嵌合体 受体，以更准确地定位部位，并定位和 随机点突变来完善模型。与这些结合在一起 研究后，我们将开展生物物理表征 每个受体的“细胞质”结构域。

Extracellular cAMP plays multiple roles in Dictyostelium development, acting as a chemoattractant, a cell-cell signaling molecule, a morphogen, and an inducer of gene expression. We have recently discovered that Dictyostelium sequentially expresses multiple surface cAMP receptors (cARs) during its developmental program. The discovery of multiple cAR subtypes may explain the versatility of cAMP. The cARs resemble mammalian G-protein linked receptors such as rhodopsin, the beta-adrenergic receptor, and the mucarinic acetylcholine receptor. The cAR subtypes are strongly homologous throughout the putative seven membrane spanning domains and connecting loops but differ strikingly in the C-terminal putative cytoplasmic domains. We will complete the cloning and sequencing of three cAR subtypes (cAR1, cAR2, and cAR3), design cAR general and subtype specific probes, analyze genomic DNA blots, clone a fourth cAR subtype (cAR4), and generate specific antibodies against each subtype. We will verify that each cAR is a cAMP binding protein, determine its time course of expression, whether it undergoes ligand-induced modification, and its subcellular distribution. In order to assess the role of each cAR subtype in the developmental program of Dictyostelium, we will specifically delete or alter it. Initially, we will determine which subtypes are expressed in a cAR1 antisense cell line and then construct a specific antisense cell line for each subtype. We will use homologous recombination and create subtype "null" cells which we will complement with functional genes. We will clone the cAR subtypes from related species of slime molds and compare the structures of those of Dictyostelium and will also screen a variety of animal species to determine whether they contain surface cAMP receptors. We will characterize those functions cAR1 carries out when expressed outside of its normal developmental context in growing cells. Its pharmacological specificity, kinetics of binding, site of attachment of photoaffinity labels, ligand-induced phosphorylation and sites of serine phosphorylation, and ligand-induced-down-regulation and redistribution will be explored. In order to assess the coupling of cAR1 to G-alpha-2, we will co-express the two proteins in growing cells. We are designing structure- function studies to determine which regions of the receptor participate in each function: 3'-deletions to roughly localize important sites, chimeric receptors to more precisely localize the sites, and site-directed and random point mutations to refine the models. In conjunction with these studies, we will carry out biophysical characterization of the "cytoplasmic" domains of each receptor.