Spatial and temporal expression of the Dictyostelium discoideum G alpha protein subunit G alpha 2: expression of a dominant negative protein inhibits proper prestalk to stalk differentiation.
Previous results have shown that the G alpha protein subunit G alpha 2 is required for aggregation in Dictyostelium discoideum and is essential for coupling cell-surface cAMP receptors to downstream effectors in vivo during this stage of development. G alpha 2 expresses at least four distinct transcripts that are differentially regulated during development; two of the transcripts are expressed exclusively in the multicellular stages and their expression is restricted to prestalk cells. We partially dissected the G alpha 2 promoter and identified a component that is expressed exclusively during the multicellular stages using luciferase gene fusions. When this promoter region is coupled to lacZ, beta-gal expression is restricted to the multicellular stages and localized in prestalk cells with a pattern similar to that of the ecmA prestalk-specific promoter. We show that expression in wild-type cells of the G alpha 2 mutant protein [G alpha 2(G206T)] during the early stages of development blocks aggregation and cAMP-mediated activation of adenylyl cyclase and guanylyl cyclase, suggesting it functions as a dominant negatively active G alpha subunit. When this mutant G alpha protein is expressed from the ecmA prestalk-specific promoter, abnormal stalk differentiation during culmination is observed. Expression of the mutant G alpha 2 from the SP60 prespore promoter or wild-type G alpha 2 from either the ecmA or the SP60 promoter results in no detectable phenotype. The results suggest that G alpha 2 plays an essential role during the culmination stage in prestalk cells and may mediate cAMP receptor activation of these processes during multicellular development.
Carrel
F
F
Department of Biology, University of California, San Diego, La Jolla 92093-0634.
Dharmawardhane
S
S
Clark
A M
AM
Powell-Coffman
J A
JA
Firtel
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D002449:Cell Aggregation; D000242:Cyclic AMP / Q000378:metabolism; D004023:Dictyostelium / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis*; D019204:GTP-Binding Proteins / Q000096:biosynthesis*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D019204:GTP-Binding Proteins