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.

Okaichi, Kumio; Cubitt, A B; Pitt, G.; Firtel, Richard A.

doi:10.1091/mbc.3.7.735

Cited by 58 publications

(52 citation statements)

References 61 publications

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“…Furthermore, whereas cAMP-mediated increases in cAMP in vivo require Go,2, in vitro GTP~/S activation of adenylyl cyclase is uninhibited in all of the g~-cell lines (Pupillo et al 1992 and unpubl.). This latter result could be explained if B~/stimulated the enzyme (Okaichi et al 1992;PupiUo et al 1992). In vivo, excitation of cAR1 would cause the release of B~/from G~2, whereas in vitro GTP~/S would cause release of B~/ from any other G-protein.…”

Section: Discussionmentioning

confidence: 96%

A G-protein beta-subunit is essential for Dictyostelium development.

Lilly¹,

Wu²,

Welker³

et al. 1993

Genes Dev.

139

102

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Recent studies have demonstrated that G-protein-linked signal transduction pathways play a significant role in the developmental program of the simple eukaryotic organism Dictyostelium. We have reported previously the isolation of a G-protein 13-subunit and present here a more complete analysis of this gene. Low-stringency Southern blots and RFLP mapping studies suggest that the 13-subunit is a unique gene found on linkage group If. Its deduced amino acid sequence of 347 residues is -60% identical to those of the human, Drosophila, and Caenorhabditis elegans 13-subunits. The carboxy-terminal 300 residues are about 70% identical; the amino-terminal 50 residues are quite divergent, containing only 10 identities. At all stages of growth and development, a single 1.9-kb 13-subunit mRNA is present at a high level, and a specific antibody detects a single 37-kD protein. We propose that G-protein heterotrimers are formed when this 13-subunit couples with each of the eight distinct G-protein a-subunits that are transiently expressed during development. Targeted disruption of the 13-subunit gene had no effect on the viability of haploid cells, but resulted in the inability of cells to aggregate.

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Section: Discussionmentioning

confidence: 96%

A G-protein beta-subunit is essential for Dictyostelium development.

Lilly¹,

Wu²,

Welker³

et al. 1993

Genes Dev.

139

102

View full text Add to dashboard Cite

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“…Cyclic AMP mass assays were performed as published previously (29). Cells were starved for 0.5 h in phosphate buffer, washed, resuspended, and then stimulated with 50 M folic acid, and accumulated cAMP was measured using the Cayman cAMP enzyme immunoassay (Cayman Chemical Co., Ann Arbor, MI) according to the manufacturer's protocol.…”

Section: Purification Of His 6 -Tagged Erk2 Protein By Ni 2ϩmentioning

confidence: 99%

Activation of the Mitogen-activated Protein Kinase ERK2 by the Chemoattractant Folic Acid in Dictyostelium

Maeda¹,

Firtel²

1997

Journal of Biological Chemistry

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In this study, we show that folic acid activates ERK2 in developmentally regulated manner and is required for ERK2 stimulation of adenylyl cyclase activity. Maximum levels of folate-stimulated ERK2 activity occur in cells from very early in development, prior to aggregation, and again at the tipped aggregate stages, corresponding to the stages in which folate receptors and the coupled G␣ subunit G␣4 are maximally expressed. During the activation by folic acid, ERK2 is phosphorylated on tyrosine residue(s) and contemporaneously shows a mobility shift on SDS-PAGE. Interestingly, this activation is not elicited in the absence of G␤ subunits, in contrast to the response to cAMP. This response also requires the G␣4 subunit known to be required for other folic acidmediated responses (Hadwiger, J., Lee, S., and Firtel, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10566 -10570). Furthermore, we show that the activation of ERK2 by cAMP is independent of the G␣4 subunit, while the activation of ERK2 by folate is independent of G␣2. Taken together, these data indicate that there are at least two pathways of ERK2 activation, heterotrimeric G proteindependent and -independent pathways. MAP 1 kinase cascades are used by eukaryotes to couple extracellular signals to diverse intracellular regulatory pathways. The diversity of these pathways is great and includes growth factor responses in mammalian cells, cell-type differentiation in Drosophila and Caenorhabditis elegans, mating and growth responses in yeast, and stress-mediated responses in yeast and mammalian cells. These cascades are mediated by various classes of G protein-and tyrosine kinase-coupled receptors (5-13). In Dictyostelium, three presumably independent MAP kinase cascades play essential regulatory roles during both growth and multicellular development. The MAP kinase kinase (MEK) DdMEK1 is required for chemotaxis toward cAMP during aggregation; ddmek1 null cells form very small aggregates but continue to differentiate into normally proportioned, but very small, fruiting bodies (14). DdMEK1 is specifically required at the time of cAMP stimulation for the activation of guanylyl cyclase and the production of cGMP, the second messenger for chemotaxis in these cells. Two MAP kinases, ERK1 and ERK2, have been identified and their functions characterized. ERK1 is required for vegetative growth and is thought to play roles during multicellular development (15), while ERK2 is required for the activation of adenylyl cyclase by the chemoattractant cAMP during aggregation, prespore-specific gene expression, and morphogenesis (2, 16). erk2 null cells are aggregation-deficient due to the inability to activate adenylyl cyclase and relay the cAMP signal but show normal activation of guanylyl cyclase, which couples to chemotaxis (17). erk2 null cells expressing a temperature-sensitive ERK2 show abnormal morphogenesis during the multicellular stages, as well as the inability to induce prespore gene expression when shifted to a nonpermissive temperature. ERK2 activity is induce ϳ...

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“…Guanylyl cyclase is stimulated by the receptor (Mato and Malchow, 1978). Previous studies have indicated that adaptation of the cGMP response occurs upstream of guanylyl cyclase (Van Haastert, 1983a), presumably at the receptor or at the Ga2-protein (Okaichi et al, 1992). Detailed kinetic studies of cAMP binding to D. discoideum cells suggest that a subpopulation of surface receptors is involved in the activation of guanylyl cyclase and that adaptation is associated at the interconversions between active and inactive receptor forms (Van Haastert et al, 1986).…”

Section: Introductionmentioning

confidence: 99%

A model for cAMP-mediated cGMP response in Dictyostelium discoideum.

Valkema

Haastert

1994

MBoC

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In Dictyostelium discoideum extracellular cyclic AMP (cAMP), as shown by previous studies, induces a transient accumulation of intracellular cyclic guanosine-5'-monophosphate (cGMP), which peaks at 10 s and recovers basal levels at 30 s after stimulation, even with persistent cAMP stimulation. Additional investigations have shown that the cAMP-mediated cGMP response is built up from surface cAMP receptor-mediated activation of guanylyl cyclase and hydrolysis of cGMP by phosphodiesterase. The regulation of these activities was measured in detail on a seconds time-scale, demonstrating complex adaptation of the receptor, allosteric activation of cGMP-phosphodiesterase by cGMP, and potent inhibition of guanylyl cyclase by Ca2 . In this paper we present a computer model that combines all experimental data on the cGMP response. The model is used to investigate the contribution of each structural and regulatory component in the final cGMP response. Four models for the activation and adaptation of the receptor are compared with experimental observations. Only one model describes the magnitude and kinetics of the response accurately. The effect of Ca21 on the cGMP response is simulated by changing the Ca21 concentrations outside the cell (Ca2+ influx) and in stores (IP3-mediated release) and changing phospholipase C activity. The simulations show that Ca21 mainly determines the magnitude of the cGMP accumulation; simulations are in good agreement with experiments on the effect of Ca2+ in electropermeabilized cells. Finally, when cGMP-phosphodiesterase activity is deleted from the model, the simulated cGMP response is elevated and prolonged, which is in close agreement with the experimental observations in mutant stmF that lacks this enzyme activity. We conclude that the computer model provides a good description of the observed response, suggesting that the main structural and regulatory components have been identified.

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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.

Cited by 58 publications

References 61 publications

A G-protein beta-subunit is essential for Dictyostelium development.

A G-protein beta-subunit is essential for Dictyostelium development.

Activation of the Mitogen-activated Protein Kinase ERK2 by the Chemoattractant Folic Acid in Dictyostelium

A model for cAMP-mediated cGMP response in Dictyostelium discoideum.

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