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

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 ∼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 ∼5 × 10³ cAR3 sites per cell compared with ∼7 × 104 cAR1 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.