Similarities of the Guanine Nucleotide Binding Sites Responsible for Adenylate Cyclase Activation and Carbamylcholine Binding Inhibition in Rat Heart Membranes

Abstract In the fission yeast Schizosaccharomyces pombe, genetic studies have identified genes that are required for glucose repression of fbp1 transcription. The git2 gene, also known as cyr1, encodes adenylate cyclase. Adenylate cyclase converts ATP into the second messenger cAMP as part of many eukaryotic signal transduction pathways. The git1, git3, git5, git7, git8 and git10 genes act upstream of adenylate cyclase, presumably encoding an adenylate cyclase activation pathway. In mammalian cells, adenylate cyclase enzymatic activity is regulated by heterotrimeric guanine nucleotide-binding proteins (G proteins). In the budding yeast Saccharomyces cerevisiae, adenylate cyclase enzymatic activity is regulated by monomeric, guanine nucleotide-binding Ras proteins. We show here that git8 is identical to the gpa2 gene that encodes a protein homologous to the alpha subunit of a G protein. Mutations in two additional genes, git3 and git5 are suppressed by gpa2+ in high copy number. Furthermore, a mutation in either git3 or git5 has an additive effect in strains deleted for gpa2 (git8), as it significantly increases expression of an fbp1-lacZ reporter gene. Therefore, git3 and git5 appear to act either in concert with or independently from gpa2 (git8) to regulate adenylate cyclase activity.

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Effects of tiazofurin on guanine nucleotide binding regulatory proteins in HL-60 cells

Blood ◽

10.1182/blood.v75.3.583.583 ◽

1990 ◽

Vol 75 (3) ◽

pp. 583-588 ◽

Cited By ~ 23

Author(s):

SM Kharbanda ◽

ML Sherman ◽

DW Kufe

Keyword(s):

Adenylate Cyclase ◽

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Binding Sites ◽

Nucleotide Binding ◽

Membrane Receptors ◽

Guanine Nucleotide ◽

Guanine Nucleotide Binding ◽

Stimulation Of

Abstract Guanine nucleotide binding proteins (G proteins) are regulatory molecules that couple membrane receptors to effector systems such as adenylate cyclase and phospholipase C. The alpha subunits of G proteins bind to guanosine 5′-diphosphate (GDP) in the unstimulated state and guanosine 5′ triphosphate (GTP) in the active state. Tiazofurin (2-beta- D-ribofuranosylthiazole-4-carboxamide), a specific inhibitor of inosine monophosphate (IMP) dehydrogenase, decreases guanylate synthesis from IMP in HL-60 promyelocytic leukemia cells and depletes intracellular guanine nucleotide pools. This study demonstrates that treatment of HL- 60 cells with tiazofurin is associated with a fourfold increase in membrane binding sites for the nonhydrolyzable analogue GDP beta S. This increase in binding sites was associated with a 3.2-fold decrease in GDP beta S binding affinity. Similar findings were obtained with GTP gamma S. These effects of tiazofurin treatment on guanine nucleotide binding were also associated with decreased adenosine diphosphate- ribosylation of specific G protein substrates by cholera and pertussis toxin. The results further demonstrate that tiazofurin treatment results in inhibition of G protein-mediated transmembrane signaling mechanisms. In this regard, stimulation of adenylate cyclase by prostaglandin E2 was inhibited by over 50% in tiazofurin-treated cells. Furthermore, tiazofurin treatment resulted in inhibition of N- formylmethionylleucylphenylalanine-induced stimulation of phospholipase C. Taken together, these results indicate that tiazofurin acts at least in part by inhibiting the ability of G proteins to function as transducers of intracellular signals.

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Cloning and sequence analyses of cDNAs for interferon- and virus-induced human Mx proteins reveal that they contain putative guanine nucleotide-binding sites: functional study of the corresponding gene promoter.

Journal of Virology ◽

10.1128/jvi.64.3.1171-1181.1990 ◽

1990 ◽

Vol 64 (3) ◽

pp. 1171-1181 ◽

Cited By ~ 76

Author(s):

M A Horisberger ◽

G K McMaster ◽

H Zeller ◽

M G Wathelet ◽

J Dellis ◽

...

Keyword(s):

Binding Sites ◽

Gene Promoter ◽

Nucleotide Binding ◽

Functional Study ◽

Guanine Nucleotide ◽

Sequence Analyses ◽

Nucleotide Binding Sites ◽

Guanine Nucleotide Binding

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Effects of tiazofurin on guanine nucleotide binding regulatory proteins in HL-60 cells

Blood ◽

10.1182/blood.v75.3.583.bloodjournal753583 ◽

1990 ◽

Vol 75 (3) ◽

pp. 583-588

Author(s):

SM Kharbanda ◽

ML Sherman ◽

DW Kufe

Keyword(s):

Adenylate Cyclase ◽

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Binding Sites ◽

Nucleotide Binding ◽

Membrane Receptors ◽

Guanine Nucleotide ◽

Guanine Nucleotide Binding ◽

Stimulation Of

Guanine nucleotide binding proteins (G proteins) are regulatory molecules that couple membrane receptors to effector systems such as adenylate cyclase and phospholipase C. The alpha subunits of G proteins bind to guanosine 5′-diphosphate (GDP) in the unstimulated state and guanosine 5′ triphosphate (GTP) in the active state. Tiazofurin (2-beta- D-ribofuranosylthiazole-4-carboxamide), a specific inhibitor of inosine monophosphate (IMP) dehydrogenase, decreases guanylate synthesis from IMP in HL-60 promyelocytic leukemia cells and depletes intracellular guanine nucleotide pools. This study demonstrates that treatment of HL- 60 cells with tiazofurin is associated with a fourfold increase in membrane binding sites for the nonhydrolyzable analogue GDP beta S. This increase in binding sites was associated with a 3.2-fold decrease in GDP beta S binding affinity. Similar findings were obtained with GTP gamma S. These effects of tiazofurin treatment on guanine nucleotide binding were also associated with decreased adenosine diphosphate- ribosylation of specific G protein substrates by cholera and pertussis toxin. The results further demonstrate that tiazofurin treatment results in inhibition of G protein-mediated transmembrane signaling mechanisms. In this regard, stimulation of adenylate cyclase by prostaglandin E2 was inhibited by over 50% in tiazofurin-treated cells. Furthermore, tiazofurin treatment resulted in inhibition of N- formylmethionylleucylphenylalanine-induced stimulation of phospholipase C. Taken together, these results indicate that tiazofurin acts at least in part by inhibiting the ability of G proteins to function as transducers of intracellular signals.

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