CAMP RECEPTOR SUBTYPES AND DICTYOSTELIUM DEVELOPMENT

CAMP 受体亚型和盘基网柄菌的发育

基本信息

批准号：
3286874
负责人：
Peter N Devreotes
金额：
$ 22.66万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-02-01 至 1995-01-31
项目状态：
已结题

项目摘要

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.

细胞外 cAMP 在盘基网柄菌发育中发挥多种作用，作为化学引诱剂、细胞间信号分子、形态发生素，和基因表达的诱导剂。我们最近发现盘基网柄菌依次表达多个表面 cAMP 受体 (cAR) 在其发展计划期间。多种cAR亚型的发现可以解释 cAMP 的多功能性。 cAR 类似于哺乳动物 G 蛋白连接受体，如视紫红质、β-肾上腺素能受体和毒蕈碱乙酰胆碱受体。 cAR 亚型具有很强的同源性贯穿假定的七个跨膜域并连接环，但在 C 端推定的细胞质结构域中显着不同。我们将完成三个cAR亚型（cAR1、 cAR2 和 cAR3)，设计 cAR 通用探针和亚型特异性探针，分析基因组 DNA 印迹，克隆第四个 cAR 亚型 (cAR4)，并生成特异性针对每种亚型的抗体。我们将验证每个 cAR 都是一个 cAMP 结合蛋白，确定其表达的时间过程，是否经历配体诱导的修饰及其亚细胞分布。为了评估每个 cAR 亚型在发育中的作用盘基网柄菌程序，我们将专门删除或更改它。首先，我们将确定 cAR1 中表达哪些亚型反义细胞系，然后构建特定的反义细胞系每个亚型。我们将使用同源重组并创建亚型我们将用功能基因补充“无效”细胞。我们将克隆粘菌相关物种的 cAR 亚型并比较盘基网柄菌的结构，也将筛选各种动物物种以确定它们是否含有表面 cAMP 受体。我们将描述 cAR1 在表达时所执行的功能超出生长细胞的正常发育环境。它是药理学特异性、结合动力学、附着位点光亲和标记、配体诱导的磷酸化和丝氨酸位点磷酸化、配体诱导的下调和重新分布将被探索。为了评估 cAR1 与 G-alpha-2 的耦合，我们将在生长的细胞中共表达这两种蛋白质。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点，嵌合受体更精确地定位位点，并进行位点定向和随机点突变来完善模型。与这些相结合研究中，我们将进行生物物理表征每个受体的“细胞质”结构域。