Johnson R L, Saxe C L, Gollop R, Kimmel A R, Devreotes P N
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Genes Dev. 1993 Feb;7(2):273-82. doi: 10.1101/gad.7.2.273.
Extracellular cAMP acts through cell-surface receptors to coordinate the developmental program of Dictyostelium. A cAMP receptor (cAR1), which is expressed during early aggregation, has been cloned and sequenced previously. We have identified a new receptor subtype, cAR3, that has approximately 56% and 69% amino acid identity with cAR1 and cAR2, respectively. cAR1, cAR2, or cAR3 expressed from plasmid in growing Dictyostelium cells can be photoaffinity labeled with 8-N3[32P]cAMP and phosphorylated when stimulated with cAMP. cAR3 RNA was not present during growth but appeared during late aggregation. Its expression peaked at 9 hr and then fell to a reduced level that was maintained until culmination. The expression of cAR3 protein followed a similar pattern, but with a 3-hr lag, and reached a maximum at the mound stage. In contrast, cAR1 protein was expressed predominantly during early aggregation and at low levels during later stages. At their respective peaks of expression, there were approximately 5 x 10(3) cAR3 sites per cell compared with approximately 7 x 10(4) cAR2 sites per cell. The cAR3 gene was disrupted by homologous recombination in several different parental cell lines. Surprisingly, the car3- cell lines display no obvious phenotype.
细胞外的环磷酸腺苷（cAMP）通过细胞表面受体发挥作用，以协调盘基网柄菌的发育程序。一种在早期聚集阶段表达的cAMP受体（cAR1），此前已被克隆和测序。我们鉴定出了一种新的受体亚型cAR3，它与cAR1和cAR2的氨基酸序列一致性分别约为56%和69%。在生长的盘基网柄菌细胞中，从质粒表达的cAR1、cAR2或cAR3能够被8-N3[32P]cAMP进行光亲和标记，并在受到cAMP刺激时发生磷酸化。cAR3 RNA在生长期间不存在，但在聚集后期出现。其表达在9小时达到峰值，然后降至较低水平并维持到发育成熟。cAR3蛋白的表达遵循类似模式，但有3小时的延迟，并在丘状体阶段达到最大值。相比之下，cAR1蛋白主要在早期聚集阶段表达，在后期阶段表达水平较低。在它们各自的表达峰值时，每个细胞约有5×10(3)个cAR3位点，而每个细胞约有7×10(4)个cAR2位点。cAR3基因在几种不同的亲本细胞系中通过同源重组被破坏。令人惊讶的是，car3-细胞系没有表现出明显的表型。
