Amino acid substitutions in the Dictyostelium G alpha subunit G alpha 2 produce dominant negative phenotypes and inhibit the activation of adenylyl cyclase, guanylyl cyclase, and phospholipase C.
Previous studies have demonstrated that the Dictyostelium G alpha subunit G alpha 2 is essential for the cAMP-activation of adenylyl cyclase and guanylyl cyclase and that g alpha 2 null mutants do not aggregate. In this manuscript, we extend the analysis of the function of G alpha 2 in regulating downstream effectors by examining the in vivo developmental and physiological phenotypes of both wild-type and g alpha 2 null cells carrying a series of mutant G alpha 2 subunits expressed from the cloned G alpha 2 promoter. Our results show that wild-type cells expressing G alpha 2 subunits carrying mutations G40V and Q208L in the highly conserved GAGESG (residues 38-43) and GGQRS (residues 206-210) domains, which are expected to reduce the intrinsic GTPase activity, are blocked in multicellular development. Analysis of down-stream effector pathways essential for mediating aggregation indicates that cAMP-mediated activation of guanylyl cyclase and phosphatidylinositol-phospholipase C (PI-PLC) is almost completely inhibited and that there is a substantial reduction of cAMP-mediated activation of adenylyl cyclase. Moreover, neither mutant G alpha 2 subunit can complement g alpha 2 null mutants. Expression of G alpha 2(G43V) and G alpha 2(G207V) have little or no effect on the effector pathways and can partially complement g alpha 2 null cells. Our results suggest a model in which the dominant negative phenotypes resulting from the expression of G alpha 2(G40V) and G alpha 2(Q208L) are due to a constitutive adaptation of the effectors through a G alpha 2-mediated pathway. Analysis of PI-PLC in g alpha 2 null mutants and in cell lines expressing mutant G alpha 2 proteins also strongly suggests that G alpha 2 is the G alpha subunit that directly activates PI-PLC during aggregation. Moreover, overexpression of wild-type G alpha 2 results in the ability to precociously activate guanylyl cyclase by cAMP in vegetative cells, suggesting that G alpha 2 may be rate limiting in the developmental regulation of guanylyl cyclase activation. In agreement with previous results, the activation of adenylyl cyclase, while requiring G alpha 2 function in vivo, does not appear to be directly carried out by the G alpha 2 subunit. Our data are consistent with adenylyl cyclase being directly activated by either another G alpha subunit or by beta gamma subunits released on activation of the G protein containing G alpha 2.
Department of Biology, University of California, San Diego, La Jolla 92093-0634.
K
K
Okaichi
A B
AB
Cubitt
G S
GS
Pitt
R A
RA
Firtel
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000201:enzymology* / Q000254:growth & development; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D006162:Guanylate Cyclase / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011953:Receptors, Cyclic AMP / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000378:metabolism*
D011953:Receptors, Cyclic AMP; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase