Activation of G proteins by (Rp) and (Sp) diastereomers of guanosine 5'-[beta-thio]triphosphate in hamster fibroblasts. Differential stereospecificity of Gi, Gs and Gp.
The effects of guanosine 5'-[beta-thio]triphosphate (GTP beta[S]) on G proteins have been examined in Chinese hamster lung fibroblasts (CCL39 line) permeabilized with alpha-toxin from Staphylococcus aureus. Although much less effective than guanosine 5'-[gamma-thio]triphosphate (GTP gamma[S]), both (Rp) and (Sp) diastereomers of GTP beta[S] were found to activate three G protein-mediated pathways: inhibition of forskolin-stimulated adenylate cyclase (mediated by Gi), potentiation of receptor-mediated activation of adenylate cyclase (mediated by Gs), and activation of phosphoinositide breakdown (mediated by Gp). Activation of Gi and Gs occurred above 3 microM-GTP beta[S], but activation of Gp only occurred above 100 microM-GTP beta[S]. Moreover, the order of effectiveness of the two diastereomers was not the same for the three G protein-mediated processes. Whereas both Gi and Gs were more effectively activated (about 5-fold) by (Sp)-GTP beta[S] than by (Rp)-GTP beta[S], Gp showed a marked preference for the (Rp) isomer. Indeed, (Rp)-GTP beta[S] induced the formation of inositol phosphates with a shorter latency and was a better competitor of GDP for binding to Gp than the (Sp) isomer. These results point to different guanine nucleotide-binding properties for Gi and Gs on the one hand and Gp on the other. At least two distinct Gp proteins, differing by their sensitivity to pertussis toxin, are present in CCL39 cells. Since pretreatment of cells with pertussis toxin completely suppressed the effects of (Rp)-GTP beta[S] on Gi, while only slightly attenuating its effects on Gp, we believe that it is the pertussis toxin-insensitive Gp which prefers the (Rp) isomer. Therefore (Rp)-GTP beta[S] may be a valuable tool for the selective activation and the biochemical characterization of this pertussis toxin-insensitive Gp.
Paris
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CNRS-Institut de Pharmacologie, Valbonne, France.
Eckstein
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D037361:Adenylate Cyclase Toxin; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002463:Cell Membrane Permeability; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000096:biosynthesis; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives* / Q000494:pharmacology; D007295:Inositol Phosphates / Q000096:biosynthesis; D037342:Pertussis Toxin; D015398:Signal Transduction / Q000187:drug effects; D013237:Stereoisomerism; D013379:Substrate Specificity; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D007295:Inositol Phosphates; D010566:Virulence Factors, Bordetella; C034883:guanosine 5'-O-(2-thiotriphosphate); D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins