MODIFICATION OF CAMP RECEPTORS IN DICTYOSTELIUM

在SDS/PAGE分析中，主要细胞表面cAMP受体 双中柱细胞表现为一对带，命名为R(MW=43000)。在……里面 体内，在没有胞外cAMP的情况下，R型占主导地位。 CAMP与受体的结合触发了它的修饰，并在15 分钟，85%的人迁移为D形。修改似乎是 磷酸化；D形式的磷酸化至少是6倍 比R型更强烈。黄曲霉毒素的动力学和浓度依赖性 这种配体诱导的受体修饰表明，这是一种机制 这导致了对cAMP的两种主要反应的适应：趋化性和 激活腺苷酸环化酶。 一种针对纯化的受体产生的特异性抗血清将用于 筛选Lambda GTll cDNA文库。杂交选育、亲和纯化 相关抗原决定簇，以及蛋白质和核苷酸的比较 序列将被用来证明阳性斑块正在表达 受体的片段。受体基因将被克隆，并 基因组和cdna的核苷酸序列比较。基础和配基诱导 受体磷酸化的位置将通过二维表征 免疫沉淀法体内磷酸化受体的多肽图谱 在试管中。这些位置在主序列中的位置 受体将通过对磷酸肽的部分测序来确定。 将合成制备数量的关键磷酸肽，并 用作受体激酶体外分析的底物或抑制剂(S) 并制备针对该激酶的抗独特型抗血清(S)。这个 受体修饰在生理反应适应中的作用 将通过对体内受体磷酸化的特异性抑制进行测试 并对该反应中存在缺陷的突变体进行了研究。网柄网柄菌属 细胞将被改变或反义受体序列和 检测受体的特定突变或缺失的影响。 这些研究旨在阐明受体和 配体诱导的受体修饰在调节趋化、活化中的作用 腺苷环化酶和基因表达。

When analysed on SDS/PAGE, the major cell surface cAMP receptor in Dicytostelium appears as a doublet of bands, designated R (MW=43000). In vivo, in the absence of extracellular cAMP, the R form predominates. Binding of cAMP to the receptor triggers its modification and, within 15 minutes, 85% migrates as the D form. The modification appears to be phosphorylation; the D form is phosphorylated at least 6-fold more intensely than the R form. The kinetics and concentration dependence of this ligand-induced receptor modification suggest that it is the mechanism that causes adaptation of the two major responses to cAMP: chemotaxis and activation of adenylate cyclase. A specific antiserum, raised against the purified receptor, will be used to screen a Lambda GTll cDNA library. Hybrid-selection, affinity purification of linked antigenic determinants, and comparison of protein and nucleotide sequence will be used to prove that positive plaques are expressing fragments of the receptor. The receptor gene will be cloned and the nucleotide sequence of genomic and cDNA compared. Basal and ligand-induced sites of receptor phosphorylation will be characterized by 2-dimensional peptide mapping of immunoprecipitated receptor phosphorylated in vivo and in vitro. The location of these sites within the primary sequence of the receptor will be determined by partial sequencing of the phosphopeptides. Preparative amounts of the critical phosphopeptides will be synthesized and used as substrates or inhibitors in in vitro assays for receptor kinase(s) and to generate anti-idiotype antisera directed against the kinase(s). The role of receptor modification in adaptation of the physiological responses will be tested by specific inhibition of in vivo receptor phosphorylation and investigation of mutants defective in this reaction. Dictyostelium cells will be transformed with altered or anti-sense receptor sequences and the effects of specific mutation or deletion of the receptor examined. These studies are designed to elucidate the role of receptors and ligand-induced receptor modification in regulating chemotaxis, activation of adenylate cyclase, and gene expression.