Agonist‐stimulated high‐affinity GTPase in <i>Dictyostelium</i> membranes

Snaar-Jagalska, B. Ewa; Jakobs, Karl H.; Haastert, Peter J.M. van

doi:10.1016/0014-5793(88)80302-8

Cited by 21 publications

(21 citation statements)

References 38 publications

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“…GTP hydrolysis in Dictyostelium membranes is performed by at least two enzymes, one with a high affinity for GTP and one with low affinity. Only the high-affinity GTPase is stimulated by cAMP [12]. To examine if CMF regulates the low-or high-affinity GTPases, wild-type cells were starved, washed free of CMF, lysed, and the resulting membranes assayed for the hydrolysis of [y-32 P]GTP.…”

Section: Resultsmentioning

confidence: 99%

“…In the absence of CMF, cAMP induced a 15% increase in high-affinity GTP binding (Fig. 2 and [20,23]). Addition of CMF caused a slight but insignificant increase in cAMP-stimulated GTP binding (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The binding of GTP to membranes was measured following SnaarJagalska and Van Haastert [23] with the following modifications. Vegetative cells were harvested by centrifugation at 500 Xg in PBM, washed once in PBM, and starved in PBM by shaking at a density of 10 7 cells/ml.…”

Section: Gtp Bindingmentioning

confidence: 99%

“…GTPase activity was determined as described by Snaar-Jagalska et al [12], with the exception that cells were starved and crude membranes were prepared as described above for GTP binding. The membranes were washed once with 10 mM triethanolamine HC1, 0.5 mM EDTA pH 7.4 and the final pellet resuspended in 10 mM triethanolamine HC1, pH 7.4, to the equivalent of 10 8 cells/ml.…”

Section: Gtpase Assaymentioning

confidence: 99%

“…The GTP which bound to Gcc2 in response to cAMP is then hydrolysed to GDP. Dictyostelium membranes contain several membraneassociated GTPases [12][13][14]. These include a basal level of a low-affinity GTPase, and a high-affinity GTPase activity which is stimulated by a pulse of cAMP [12].…”

Section: Introductionmentioning

confidence: 99%

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A cell‐density sensing factor regulates the lifetime of a chemoattractant‐induced Gα‐GTP conformation

1997

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Starving Dictyostelium discoideum cells monitor the local density of other starving cells by simultaneously secreting and sensing CMF. CMF regulates signal transduction through the chemoattractant cAMP receptor, cARl. cARl activates a heterotrimeric G protein by stimulating Goc2 to release GDP and bind GTP. We show here that the rate of cAMP-stimulated GTP hydrolysis in membranes from cells exposed to CMF is roughly 4 times slower than in membranes from untreated cells, even though the rate of GTP binding is the same. This hydrolysis is abolished in cells lacking Goc2. Our data thus suggest that CMF regulates cAMP signal transduction in part by prolonging the lifetime of the Ga2-GTP complex.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Gtp Bindingmentioning

confidence: 99%

Section: Gtpase Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A cell‐density sensing factor regulates the lifetime of a chemoattractant‐induced Gα‐GTP conformation

1997

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Guanylate cyclase activity in permeabilizedDictyostelium discoideum cells

et al. 1996

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Dictyostelium discoideum cells respond to chemoattractants by transient activation of guanylate cyclase. Cyclic GMP is a second messenger that transduces the chemotactic signal. We used an electropermeabilized cell system to investigate the regulation of guanylate cyclase. Enzyme activity in permeabilized cells was dependent on the presence of a nonhydrolysable GTP analogue (e.g., GTP gamma S), which could not be replaced by GTP, GDP, or GMP. After the initiation of the guanylate cyclase reaction in permeabilized cells only a short burst of activity is observed, because the enzyme is inactivated with a t1/2 of about 15 s. We show that inactivation is not due to lack of substrate, resealing of the pores in the cell membrane, product inhibition by cGMP, or intrinsic instability of the enzyme. Physiological concentrations of Ca2+ ions inhibited the enzyme (half-maximal effect at 0.3 microM), whereas InsP3 had no effect. Once inactivated, the enzyme could only be reactivated after homogenization of the permeabilized cells and removal of the soluble cell fraction. This suggests that a soluble factor is involved in an autonomous process that inactivates guanylate cyclase and is triggered only after the enzyme is activated. The initial rate of guanylate cyclase activity in permeabilized cells is similar to that in intact, chemotactically activated cells. Moreover, the rate of inactivation of the enzyme in permeabilized cells and that due to adaptation in vivo are about equal. This suggests that the activation and inactivation of guanylate cyclase observed in this permeabilized cell system is related to that of chemotactic activation and adaptation in intact cells.

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G‐proteins in the signal‐transduction pathways of Dictyostelium discoideum

1988

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The functional interaction of surface cAMP receptors with effector enzymes via G-proteins was investigated in Dictyostelium discoideum. Several experimental conditions were used to investigate signal transduction, such as reduced temperatures, use of down-regulated cells and of mutants. The results are presented as a model describing the complex interaction between multiple forms of the surface cAMP receptor and different G-proteins that are responsible for the generation of the second messengers, cAMP, cGMP, InsP3 and Ca2+.

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Agonist‐stimulated high‐affinity GTPase in Dictyostelium membranes

Abstract: GTP hydrolysis in Dictyostelium discoideum membranes is caused by a low (K,,,> 1 mM) and a high affinity (K,,, 6.5 PM) GTPase. CAMP enhances GTP hydrolysis apparently by increasing the affinity of the high affinity GTPase (

Cited by 21 publications

References 38 publications

A cell‐density sensing factor regulates the lifetime of a chemoattractant‐induced Gα‐GTP conformation

A cell‐density sensing factor regulates the lifetime of a chemoattractant‐induced Gα‐GTP conformation

Guanylate cyclase activity in permeabilizedDictyostelium discoideum cells

G‐proteins in the signal‐transduction pathways of Dictyostelium discoideum

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