Changes in cyclic AMP receptor properties during adaptation in Dictyostelium discoideum

1990 ◽  
Vol 95 (4) ◽  
pp. 623-629
Author(s):  
M.E. Luderus ◽  
M.J. Spijkers ◽  
R. Van Driel
Keyword(s):  
Cyclic Amp ◽  
G Protein ◽  
Dictyostelium Discoideum ◽  
Receptor Binding ◽  
Molecular Basis ◽  
Guanine Nucleotides ◽  
Cyclic Process ◽  
Binding Properties ◽  
Receptor System ◽  
Receptor Affinity

In developing Dictyostelium discoideum cells, binding of cyclic AMP to the chemotactic receptor has been shown to oscillate. These oscillations represent cycles of activation, adaptation and deadaptation of the cyclic AMP receptor system. We studied the molecular basis of these oscillatory changes in cyclic AMP receptor binding. We developed a rapid method of lysing cells during the course of the oscillations. This method guaranteed good preservation of ligand binding properties of the cyclic AMP receptor. We found that oscillations in cyclic AMP binding resulted from changes in receptor affinity. The total number of receptors did not significantly change during oscillations. Our experiments also showed that both GTP and GDP abolished oscillations in receptor binding completely, presumably by acting via a G protein. The guanine nucleotides reduced the affinity of the receptor at all time-points of the oscillation cycle to the minimal, i.e. adapted, level. We conclude that the cyclic process of activation, adaptation and de-adaptation in D. discoideum, at cyclic AMP receptor level, involves changes in receptor-G protein interaction. During adaptation, the affinity of the cyclic AMP receptor decreases and the receptor becomes insensitive to guanine nucleotides.

scholarly journals Selection of Gβ Subunits with Point Mutations That Fail to Activate Specific Signaling Pathways In Vivo: Dissecting Cellular Responses Mediated by a Heterotrimeric G Protein in Dictyostelium discoideum

1998 ◽  
Vol 9 (10) ◽  
pp. 2949-2961 ◽  
Author(s):  
Tian Jin ◽  
Mario Amzel ◽  
Peter N. Devreotes ◽  
Lijun Wu
Keyword(s):  
Adenylyl Cyclase ◽  
G Protein ◽  
Dictyostelium Discoideum ◽  
Actin Polymerization ◽  
Point Mutations ◽  
Cellular Responses ◽  
Receptor System ◽  
Heterotrimeric G Protein ◽  
Downstream Effectors

In Dictyostelium discoideum, a unique Gβ subunit is required for a G protein–coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Gα2, and Gβ genes completely impair all these responses. To dissect specificity in Gβγ signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gβ genes and isolated Gβ alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gβ subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gβγ by making point mutations on Gβ. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gβs and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtβγ interacts with its regulators, Gα and phosducin.

scholarly journals Dictyostelium discoideum mutant synag 7 with altered G-protein-adenylate cyclase interaction

1988 ◽  
Vol 91 (2) ◽  
pp. 287-294
Author(s):  
B.E. Snaar-Jagalska ◽  
P.J. Van Haastert
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
G Protein ◽  
Dictyostelium Discoideum ◽  
High Speed ◽  
Wild Type ◽  
High Affinity ◽  
Gtp Binding

Previous results have shown that Dictyostelium discoideum mutant synag 7 is defective in the regulation of adenylate cyclase by receptor agonists in vivo and by GTP gamma S in vitro; the guanine nucleotide activation of adenylate cyclase is restored by the high-speed supernatant from wild-type cells. Here we report that in synag 7 membranes: (1) cyclic AMP receptors had normal levels and were regulated by guanine nucleotides as in wild-type; (2) GTP binding and high-affinity GTPase were reduced but still stimulated by cyclic AMP; (3) the supernatant from wild-type cells restored GTP binding to membranes of this mutant, and partly restored high-affinity GTPase activity; (4) the supernatant of synag 7 was ineffective in these reconstitutions and did not influence GTP binding and GTPase activities in mutant or wild-type membranes. These results suggest that the defect in mutant synag 7 is located between G-protein and adenylate cyclase, and not between receptor and G-protein. A factor in the supernatant is absent in synag 7 and appears to be essential for normal GTP binding, GTPase and activation of adenylate cyclase. This soluble heat-labile factor may represent a new molecule required for receptor- and G-protein-mediated activation of adenylate cyclase.

scholarly journals A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum. I. Properties.

1984 ◽  
Vol 259 (1) ◽  
pp. 654-661 ◽  
Author(s):  
I H Majerfeld ◽  
B H Leichtling ◽  
J A Meligeni ◽  
E Spitz ◽  
H V Rickenberg
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Dependent Protein Kinase ◽  
Dependent Protein

scholarly journals Involvement of A pertussis Toxin Sensitive G-Protein in the Inhibition of Inwardly Rectifying K+Currents by Platelet-Activating Factor in Guinea-Pig Atrial Cardiomyocytes

1994 ◽  
Vol 3 (1) ◽  
pp. 45-51
Author(s):  
M. Gollasch ◽  
T. Kleppisch ◽  
D. Krautwurst ◽  
D. Lewinsohn ◽  
J. Hescheler
Keyword(s):  
Cyclic Amp ◽  
Guinea Pig ◽  
G Protein ◽  
Pertussis Toxin ◽  
Platelet Activating Factor ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Guinea Pig Heart ◽  
Ventricular Cells ◽  
Inwardly Rectifying

Platelet-activating factor (PAF) inhibits single inwardly rectifying K+channels in guinea-pig ventricular cells. There is currently little information as to the mechanism by which these channels are modulated. The effect of PAF on quasi steady-state inwardly rectifying K+currents (presumably of the IK1type) of auricular, atrial and ventricular cardiomyocytes from guinea-pig were studied. Applying the patch-clamp technique in the whole-cell configuration, PAF (10 nM) reduced the K+currents in all three cell types. The inhibitory effect of PAF occurred within seconds and was reversible upon wash-out. It was almost completely abolished by the PAF receptor antagonist BN 50730. Intracellular infusion of atrial cells with guanine 5′-(β-thio)diphosphate (GDPS) or pretreatment of cells with pertussis toxin abolished the PAF dependent reduction of the currents. Neither extracellularly applied isoproterenol nor intracellularly applied adenosine 3′,5′-cyclic monophosphate (cyclic AMP) attenuated the PAF effect. In multicellular preparations of auricles, PAF (10 nM) induced arrhythmias. The arrhythmogenic activity was also reduced by BN 50730. The data indicate that activated PAF receptors inhibit inwardly rectifying K+currents via a pertussis toxin sensitive G-protein without involvement of a cyclic AMP-dependent step. Since IK1is a major component in stabilizing the resting membrane potential, the observed inhibition of this type of channel could play an important role in PAF dependent arrhythmogenesis in guinea-pig heart.

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