Phosphorylation of the G protein α-subunit, Gα2, ofDictyostelium discoideum requires a functional and activated Gα2

Gundersen, Robert E.

doi:10.1002/(sici)1097-4644(19970801)66:2<268::aid-jcb13>3.0.co;2-d

Cited by 6 publications

(2 citation statements)

References 45 publications

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“…Transformants were selected and grown with G418 at 20 μg/mL. Aggregation competent cells were generated as described . Cells were harvested by centrifugation at 700 xg and washed with development buffer (DB; 5 mM Na 2 HPO 4 , 5 mM KH 2 PO 4 , 2 mM MgSO 4 , and 0.2 mM CaCl 2 ), and resuspended at 2 × 10 7 cells/mL in DB.…”

Section: Methodsmentioning

confidence: 99%

“…Aggregation competent cells starved for 5 h were used to examine GTPγS inhibition of cAMP binding to Dictyostelium membranes. 24 The % [ 3 H]cAMP remaining bound was determined by subtracting cpms bound in the presence of GTPγS (50 µM) from the Total bound (+H 2 O) cpms divided by Total bound cpms and multiplied by 100. Nonspecific binding (+ 100 µM cAMP) is subtracted from both.…”

Section: Gtpγs Inhibition Of Camp Bindingmentioning

confidence: 99%

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Localization of palmitoylated and activated G protein α‐subunit in Dictyostelium discoideum

Alamer¹,

Kageyama

Gundersen³

2018

J of Cellular Biochemistry

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Guanine nucleotide-binding proteins (G proteins) act as molecular switches to regulate many fundamental cellular processes. The lipid modification, palmitoylation, can be considered as a key factor for proper G protein function and plasma membrane localization. In Dictyostelium discoidum, Gα2 is essential for the chemotactic response to cAMP in their developmental life cycle. However, the regulation of Gα2 with respect to palmitoylation, activation and Gβγ association is less clear. In this study, Gα2 is shown to be palmitoylated on Cys-4 by [ H]palmitate labeling. Loss of this palmitoylation site results in redistribution of Gα2 within the cell and poor D. discoideum development. Cellular re-localization is also observed for activated Gα2. In the membrane fraction, Gα2-wt (YFP) is highly enriched in a low-density membrane fraction, which is palmitoylation-dependent. Activated Gα2 monomer and heterotrimer are shifted to two different higher-density fractions. These results broaden our understanding of how G protein localization and function are regulated inside the cells.

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Section: Methodsmentioning

confidence: 99%

Section: Gtpγs Inhibition Of Camp Bindingmentioning

confidence: 99%

Localization of palmitoylated and activated G protein α‐subunit in Dictyostelium discoideum

Alamer¹,

Kageyama

Gundersen³

2018

J of Cellular Biochemistry

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Aggregation ofDictyostelium discoideum is dependent on myristoylation and membrane localization of the G protein ?-subunit, G?2

1999

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The heterotrimeric G protein, G2, from the eukaryotic organism Dictyostelium discoideum participates in signal transduction pathways which are essential to Dictyostelium's developmental life cycle. G2 is activated by cell surface cAMP receptors and in turn is required for the activation of a host of effectors, including adenylyl cyclase, guanylyl cyclase, and phospholipase C. Myristoylation of G protein alpha-subunits is known to affect alpha-subunit association with the beta gamma subunits and membrane localization. The putative site for N-terminal myristoylation of G alpha 2 was mutated from Gly to Ala (G2A) and expressed in the g alpha 2-null cell line, MYC2. Transformants expressing G alpha 2-G2A exhibit physiological and biochemical changes from wild-type cells. G alpha 2-G2A expressing cells fail to rescue the aggregation-minus phenotype of MYC2 cells on developmental agar plates. G alpha 2-G2A expressing cells are also not chemotactic to cAMP in a standard drop assay. G alpha 2-WT is found in both the pellet and supernatant fractions following lysis of the cells. G alpha 2-G2A however is found almost exclusively in the lysate supernatant. G alpha 2 is radiolabeled upon incubation of cells in [3H]myristate, while G alpha 2-G2A is not labeled. Examination of activation of the effectors adenylyl cyclase and guanylyl cyclase reveals that G alpha 2-G2A expressing cells partially activate adenylyl cyclase but show no cAMP-stimulation of guanylyl cyclase. The physiological deviations from wild-type can be explained by the variations in effector activation, possibly due to improper localization of the non-myristoylated G alpha 2-G2A to the cytosol.

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Activated Gα Subunits Can Inhibit Multiple Signal Transduction Pathways during Dictyostelium Development

Srinivasan

Gundersen

Hadwiger

1999

Developmental Biology

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Mutations impairing the GTPase activity of G protein Galpha subunits can result in activated Galpha subunits that affect signal transduction and cellular responses and, in some cases, promote tumor formation. An analogous mutation in the Dictyostelium Galpha4 subunit gene (Q200L substitution) was constructed and found to inhibit Galpha4-mediated responses to folic acid, including the accumulation of cyclic nucleotides and chemotactic cell movement. The Galpha4-Q200L subunit also severely inhibited responses to cAMP, including cyclic nucleotide accumulation, cAMP chemotaxis, and cellular aggregation. An analogous mutation in the Galpha2 subunit (Q208L substitution), previously reported to inhibit cAMP responses (K. Okaichi et al., 1992, Mol. Biol. Cell 3, 735-747), was also found to partially inhibit folic acid chemotaxis. Chemotactic responses to folic acid and cAMP and developmental aggregation were also inhibited by a mutant Galpha5 subunit with the analogous alteration (Q199L substitution). All aggregation-defective Galpha mutants were capable of multicellular development after a temporary incubation at 4 degrees C and this development was found to be dependent on wild-type Galpha4 function. This study indicates that mutant Galpha subunits can inhibit signal transduction pathways mediated by other Galpha subunits.

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Phosphorylation of the G protein α-subunit, Gα2, ofDictyostelium discoideum requires a functional and activated Gα2

Cited by 6 publications

References 45 publications

Localization of palmitoylated and activated G protein α‐subunit in Dictyostelium discoideum

Localization of palmitoylated and activated G protein α‐subunit in Dictyostelium discoideum

Aggregation ofDictyostelium discoideum is dependent on myristoylation and membrane localization of the G protein ?-subunit, G?2

Activated Gα Subunits Can Inhibit Multiple Signal Transduction Pathways during Dictyostelium Development

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