A NOVEL CYTOSOLIC REGULATOR, PIANISSIMO, IS REQUIRED FOR CHEMOATTRACTANT RECEPTOR AND G-PROTEIN-MEDIATED ACTIVATION OF THE 12 TRANSMEMBRANEDOMAIN ADENYLYL-CYCLASE IN DICTYOSTELIUM

Genetic analysis was applied to identify novel genes involved in G pro tein-linked pathways controlling development. Using restriction enzyme mediated integration (REMI), we have identified a new gene, Pianissim o (PiaA), involved in cAMP signaling in Dictyostelium discoideum. PiaA encodes a 130-kD cytosolic protein required for chemoattractant recep tor and G protein-mediated activation of the 12 transmembrane domain a denylyl cyclase. In piaA(-)null mutants, neither chemoattractant stimu lation of intact cells nor GTP gamma S treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects. Cy tosols of wild-type cells that contain Pia protein reconstitute the GT P gamma S stimulation of adenylyl cyclase activity in piaA(-)lysates, indicating that Pia is directly involved in the activation. Pia and CR AC, a previously identified cytosolic regulator, are both essential fo r activation of the enzyme as lysates of crac(-)piaA(-)double mutants require both proteins for reconstitution. Homologs of PiaA are found i n Saccharomyces cerevisiae and Schizosaccaromyces pombe; disruption of the S. cerevisiae homolog results in lethality, We propose that homol ogs of Pia and similar modes of regulation of these ubiquitous G prote in-linked pathways ape likely no exist in higher eukaryotes.