Expression of a mutated ras gene in Dictyostelium discoideum alters the binding of cyclic AMP to its chemotactic receptor.
Dictyostelium discoideum cells contain a ras gene that codes for a polypeptide that is highly homologous to the human ras proteins. Extra copies of the wild-type gene or a gene carrying a missense mutation in codon 12 (ras-Gly12 and ras-Thr12, respectively) have been introduced into Dictyostelium cells by transformation. We have investigated the properties of the chemotactic cell surface cyclic AMP receptor in crude membrane preparations of wild-type Dictyostelium cells and ras-Gly12 and ras-Thr12 transformants. In vitro, an ATP- and Ca2+-dependent reduction of the number of cyclic AMP receptors was observed in membranes from all three cell types. The number of available receptors was decreased maximally by about 50%. In the presence of ATP the half-maximal Ca2+ concentration required for this process was about 10(-5) M in wild-type and ras-Gly12 membranes, and less than 10(-7) M in ras-Thr12 membranes. Addition of GTP (but not GDP) or the phorbol ester PMA (phorbol-12-myristate-13-acetate) reduced the Ca2+ requirement of the process in wild-type and ras-Gly12 membranes to the physiological level of less than 10(-7) M. In membranes derived from ras-Thr12 cells addition of GTP or PMA had no effect. The results indicate that D. discoideum cells contain a cyclic AMP receptor-controlling pathway that can be activated in vitro and involves a GTP-binding protein and a Ca2+ plus ATP-dependent activity, possibly protein kinase C. It is concluded that the ras protein specifically interacts with this pathway; the pathway appears to be constitutively activated by the mutated ras gene product.
Ludérus
M E
ME
Laboratory of Biochemistry, University of Amsterdam, The Netherlands.
Reymond
C D
CD
Van Haastert
P J
PJ
Van Driel
R
R
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002633:Chemotaxis; D000242:Cyclic AMP / Q000378:metabolism*; D004023:Dictyostelium / Q000235:genetics*; D011905:Genes, ras; D009154:Mutation; D011953:Receptors, Cyclic AMP / Q000378:metabolism*
D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP