Guanosine 5'-O-(3-thiotriphosphate) and guanosine 5'-O-(2-thiodiphosphate) activate G proteins and potentiate fibroblast growth factor-induced DNA synthesis in hamster fibroblasts.
Addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to intact Chinese hamster lung fibroblasts (CCL39) depolarized by high K+ concentrations results in activation of phosphoinositide-specific phospholipase C (PLC) (at GTP gamma S concentrations greater than 0.1 mM), inhibition of adenylate cyclase (between 10 microM and 0.5 mM), and activation of adenylate cyclase (above 0.5 mM). Since GTP gamma S-induced activation of PLC is dramatically enhanced upon receptor-mediated stimulation of PLC by alpha-thrombin, we conclude that in depolarized CCL39 cells GTP gamma S directly activates various guanine nucleotide-binding regulatory proteins (G proteins) coupled to PLC (Gp(s)) and to adenylate cyclase (Gi and Gs). Pretreatment of cells with pertussis toxin strongly inhibits GTP gamma S-induced activation of PLC and inhibition of adenylate cyclase. GTP gamma S cannot be replaced by other nucleotides, except by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which mimics after a lag period of 15-20 min all the effects of GTP gamma S, with the same concentration dependence and the same sensitivity to pertussis toxin. We suggest that GDP beta S is converted in cells into GTP beta S, which acts as GTP gamma S. Since cell viability is not affected by a transient depolarization, these observations provide a simple method to examine long-term effects of G protein activation on DNA synthesis. We show that a transient exposure of G0-arrested CCL39 cells to GTP gamma S or GDP beta S under depolarizing conditions is not sufficient by itself to induce a significant mitogenic response, but markedly potentiates the mitogenic action of fibroblast growth factor, a mitogen known to activate a receptor-tyrosine kinase. The potentiating effect is maximal after 60 min of pretreatment with 2 mM GTP gamma S. GDP beta S is equally efficient but only after a lag period of 15-20 min. Mitogenic effects of both guanine nucleotide analogs are suppressed by pertussis toxin. Since the activation of G proteins by GTP gamma S under these conditions vanishes after a few hours, we conclude that a transient activation of G proteins facilitates the transition G0----G1 in CCL39 cells, whereas tyrosine kinase-induced signals are sufficient to mediate the progression into S phase.
Paris
S
S
Centre de Biochimie, Université de Nice, France.
Pouysségur
J
J
D037361:Adenylate Cyclase Toxin; D000818:Animals; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects*; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D006150:Guanine Nucleotides; D007295:Inositol Phosphates; D007328:Insulin; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012965:Sodium Chloride; D005346:Fibroblast Growth Factors; D011189:Potassium Chloride; C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D004247:DNA; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D013917:Thrombin; D019204:GTP-Binding Proteins