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

Ronald L. Johnson, Charles L. Saxe, Rachel Gollop, Alan R. Kimmel, Peter N. Devreotes

Research output: Contribution to journalArticlepeer-review

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 ∼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.

Original languageEnglish (US)
Pages (from-to)273-282
Number of pages10
JournalGenes and Development
Volume7
Issue number2
DOIs
StatePublished - 1993

Keywords

  • Dictyostelium
  • Morphogenesis
  • Receptors
  • cAMP

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology

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