INTRACELLULAR ADENOSINE 3',5'-PHOSPHATE FORMATION IS ESSENTIAL FOR DOWN-REGULATION OF SURFACE ADENOSINE 3',5'-PHOSPHATE RECEPTORS IN DICTYOSTELIUM

Dictyostelium discoideum cells contain cell surface cyclic AMP (cAMP) receptors that bind cAMP as a first messenger and intracellular cAMP r eceptors that bind cAMP as a second messenger. Prolonged incubation of Dictyostelium cells with cAMP induces a sequential process of phospho rylation, sequestration and down-regulation of the surface receptors. The role of intracellular cAMP in down-regulation of surface receptors was investigated. Down-regulation of receptors does not occur under c onditions that specifically inhibit the formation of intracellular cAM P (the drug caffeine or mutant cells lacking adenylate cyclase) or con ditions that inhibit the function of intracellular cAMP (mutants lacki ng protein kinase A activity). Cell-permeable non-hydrolysable cAMP de rivatives were used to investigate further the requirement of intracel lular cAMP for down-regulation. The Sp isomer of 6-thioethylpurineribo side 3',5'-phosphorothioate (6SEth-cPuMPS) does not bind to the surfac e receptor, enters the cell and has relative high affinity for protein kinase A. 6SEth-cPuMPS alone has no effect on downregulation. However , together with an agonist of the surface receptor, the analogue induc es down-regulation in caffeine-treated wild-type cells and in mutant c ells lacking adenylate cyclase, but not in mutant cells lacking protei n kinase A. These results indicate that intracellular cAMP formation a nd activation of protein kinase A are essential for down-regulation of the surface cAMP receptor.