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Vol. 11, No. 23, pp. 3218-3231, December 1, 1997

RESEARCH PAPER
A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium

Mei-Yu Chen, Yu Long, and Peter N. Devreotes1

Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185 USA

Genetic analysis was applied to identify novel genes involved in G protein-linked pathways controlling development. Using restriction enzyme-mediated integration (REMI), we have identified a new gene, Pianissimo (PiaA), involved in cAMP signaling in Dictyostelium discoideum. PiaA encodes a 130-kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase. In piaA- null mutants, neither chemoattractant stimulation of intact cells nor GTPgamma S treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects. Cytosols of wild-type cells that contain Pia protein reconstitute the GTPgamma S stimulation of adenylyl cyclase activity in piaA- lysates, indicating that Pia is directly involved in the activation. Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme as lysates of crac- piaA- double mutants require both proteins for reconstitution. Homologs of PiaA are found in Saccharomyces cerevisiae and Schizosaccaromyces pombe; disruption of the S. cerevisiae homolog results in lethality. We propose that homologs of Pia and similar modes of regulation of these ubiquitous G protein-linked pathways are likely to exist in higher eukaryotes.

[Key Words: Chemoattractant receptor; G protein; adenylyl cyclase; signal transduction; Dictyostelium]

GENES & DEVELOPMENT 11:3218-3231 © 1997 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/97 $5.00
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