Agonist‐sensitive binding of a photoreactive GTP analog to a G‐protein α‐subunit in membranes of HL‐60 cells

Offermans, Stefan; Schäfer, Rainer; Hoffmann, Barbara; Bombien, E; Spicher, Karsten; Hinsch, Klaus‐Dieter; Schultz, Günter; Rosenthal, Walter

doi:10.1016/0014-5793(90)80054-m

Cited by 46 publications

(33 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the present study, in situ photoaffinity labeling with [␣ 32 P]GTPAA 1 (22,23) indicated a depolarization-induced accelerated exchange of GDP for [␣ 32 P]GTPAA in G␣ o1 -and G␣ o3 -proteins, implying a depolarization-induced activation of these G o -proteins. [␣ 32 P]-GTPAA was introduced into transiently permeabilized synaptoneurosomes as described before (10).…”

supporting

confidence: 51%

Activation of Go-proteins by Membrane Depolarization Traced by in Situ Photoaffinity Labeling of Gαo-proteins with [α32P]GTP-azidoanilide

Anis¹,

Nürnberg²,

Visochek³

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

GTP-binding trimeric proteins have been implicated in signal transduction from receptors in the cell membrane to intracellular effectors and ion channels in a variety of cells (1-5). The mechanism involves signal-induced G-protein activation initiated by an exchange of GDP for GTP on the ␣ subunit of the protein (5-7). Subsequent GTPase activity of the G␣ subunit converts the activated G-proteins into their inactive, GDPbound state (7). Activation of G-proteins has been induced experimentally by stimulation of G-protein-coupled receptors in the cell membrane (2,3,8,9). Evidence indicating activation of G-proteins in response to membrane depolarization were previously observed in brain stem synaptoneurosomes (10 -12).G␣ o -proteins are widely distributed in the central nervous system (15-17). Three subtypes showing marked homology but exhibiting functional differences have been identified (13,14,18). The G␣ o1 subtype appears to be involved in the coupling of muscarinic receptors to Ca ϩ2 channels, and the G␣ o2 subtype mediates inhibition of Ca ϩ2 current activated by somatostatin receptors (19). The function of the G␣ o3 subtype is not clear (14). Phospholipase C activation mediated by activation of G oproteins has been demonstrated in the cell-free system (20), and G o -protein-mediated activation of protein kinase C has been observed in Chinese hamster ovary (CHO) cells (21).In the present study, in situ photoaffinity labeling with [␣ 32 P]GTPAA 1 (22, 23) indicated a depolarization-induced accelerated exchange of GDP for [␣ 32 P]GTPAA in G␣ o1 -and G␣ o3 -proteins, implying a depolarization-induced activation of these G o -proteins. [␣ 32 P]-GTPAA was introduced into transiently permeabilized synaptoneurosomes as described before (10). Unlike the endogenously bound guanine nucleotides, [␣ 32 P]GT-PAA, covalently bound to G␣-proteins by photoaffinity labeling, was not displaced during SDS-polyacrylamide gel electrophoresis, providing a possible tool for identification of in situ activated G-proteins.In view of findings indicating a reciprocal influence of depolarization-induced activation of VGSC and uncoupling of Gproteins from muscarinic receptors (12, 24, 25), we examined the possibility that VGSC can be involved in depolarizationinduced activation of G␣ o -proteins. Our results indicated that depolarization-induced activation of G o -proteins can be prevented by preventing VGSC activation. In addition, in depolarized brain-stem and cortical synaptoneurosomes, the ␣ subunit of VGSC cross-linked most efficiently with G␣ o -proteins. In isolated synaptoneurosomal membranes, VGSC-␣ subunit cross-linked most efficiently with GDP␤S-bound rather than GTP␥S-bound G␣ o -proteins. These findings suggest repeated

show abstract

supporting

confidence: 51%

Activation of Go-proteins by Membrane Depolarization Traced by in Situ Photoaffinity Labeling of Gαo-proteins with [α32P]GTP-azidoanilide

Anis¹,

Nürnberg²,

Visochek³

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Basal photolabeling of both proteins remained low during the whole time course (4-45 min), whereas stimulation of [a-32P]GTP azidoanilide incorporation into a12 and a13 by U46619 and thrombin was detectable after 4-8 min and reached a maximum not before 30-45 min. Since effective agonist-dependent binding ofhydrolysisresistant GTP analogues to a subunits of G, and Go proteins can be more readily measured in the presence of GDP (8,31,32), we tested the effect of increasing GDP concentrations on the U46619-stimulated photolabeling of a12 and a13 (Fig. 6B).…”

Section: Methodsmentioning

confidence: 99%

G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets.

Offermanns

Laugwitz

Spicher

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

410

289

View full text Add to dashboard Cite

Using subtype-specific antisera, we were able to identify the recently described alpha subunits of G12 and G13 in platelet membranes as 43-kDa proteins. Activation of the thromboxane A2 and the thrombin receptors in platelet membranes led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha 12 and alpha 13, indicating that both receptors couple to G12 and G13. In addition, both activated receptors were demonstrated to couple to one or more members of the Gq family. In the absence of receptor agonists, incorporation of [alpha-32P]GTP azidoanilide into alpha 12 and alpha 13 was low over a long time period (up to 45 min) due to an obviously low basal nucleotide exchange rate, whereas an agonist-stimulated photolabeling of alpha 12 and alpha 13 could be observed after 4-8 min and reached a maximum after 30-45 min. Effective activation of G12 and G13 via the thromboxane A2 and the thrombin receptors was not dependent on the presence of GDP. Our results provide evidence that G12 and G13 play a functional role in transmembrane signal transduction and suggest that both proteins are involved in pathways leading to platelet activation.

show abstract

“…Although fMLP, C5a and LTB4 all effectively activate Gi-proteins as assessed by measurement of high- affinity GTP hydrolysis and GTP [yS] binding and photolabeling of cr-subunits with GTP azidoanilide [4][5][6], substantial differences in the effects of fMLP and LTB 4 have been observed. Specifically, LTB4, unlike fMLP, does not enhance CTX-catalysed ADPribosylation of Gi-protein ol-subunits in membranes of DMSO-differentiated HL-60 cells [7,8].…”

mentioning

confidence: 99%

Differential activation of dibutyryl cAMP-differentiated HL-60 human leukemia cells by chemoattractants

Klinker

Schwaner

Offermanns

et al. 1994

Biochemical Pharmacology

View full text Add to dashboard Cite

Ahstraet--Dibutyryl cAMP-differentiated HL-60 human leukemia cells possess receptors for the chemoattractants N-formyl-c-methionyl-L-leucyl-g-phenylalanine (fMLP), C5a and leukotriene B 4 (LTB4). We compared the effects of these chemoattractants in HL-60 membranes and in intact HL-60 cells, fMLP, C5a and LTB4 stimulated GTP hydrolysis and guanosine 5'-O-[3-thio]triphosphate (GTP [gS]) binding in HL-60 membranes with similar effectiveness and in a pertussis toxin (PTX)-sensitive manner. They also stimulated photolabeling of the oc-subunits of the guanine nucleotidebinding proteins (G-proteins), G~2 and G~3 with similar effectiveness. Chloride salts of monovalent cations differentially enhanced and inhibited chemoattractant-induced GTP hydrolyses. C5a was less effective than fMLP in enhancing cholera toxin-catalysed ADP-ribosylation of Gia 2 and Gi,3, and LTB4 was ineffective, fMLP was more effective than C5a and LTB4 in stimulating Ca 2+ influx in HL-60 cells. C5a-and LTB4-induced rises in cytosolic Ca 2+ concentration ([Ca2+]~) were PTX-sensitive, whereas the effect of fMLP was partially PTX-insensitive. LTB4-induced rises in [Ca2+]i were more sensitive towards homologous desensitization than those induced by C5a, and the effect of fMLP was resistant in this regard. C5a was considerably less effective than fMLP in activating superoxide anion formation and azurophilic granule release, and LTB 4 was ineffective. Our data suggest that fMLP, C5a and LTB4 effectively activate the G-proteins, G~2 and Gi3, in HL-60 cells and that fMLP may additionally activate PTX-insensitive G-proteins. fMLP, C5a and LTB4 are full, partial and incomplete secretagogues, respectively, and these differences may be due to differences in homologous receptor desensitization and qualitative G~-protein activation.

show abstract

Agonist‐sensitive binding of a photoreactive GTP analog to a G‐protein α‐subunit in membranes of HL‐60 cells

Cited by 46 publications

References 28 publications

Activation of Go-proteins by Membrane Depolarization Traced by in Situ Photoaffinity Labeling of Gαo-proteins with [α32P]GTP-azidoanilide

Activation of Go-proteins by Membrane Depolarization Traced by in Situ Photoaffinity Labeling of Gαo-proteins with [α32P]GTP-azidoanilide

G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets.

Differential activation of dibutyryl cAMP-differentiated HL-60 human leukemia cells by chemoattractants

Contact Info

Product

Resources

About