Identification and targeted gene disruption of cAR3, a cAMP receptor subtype expressed during multicellular stages of Dictyostelium development

Abstract

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.