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Mastoparan may activate GTP hydrolysis by Gi-proteins in HL-60 membranes indirectly through interaction with
J F Klinker1, A Hagelüken, L Grünbaum
1Institut für Pharmakologie, Freie Universität Berlin, Germany.
Abstract:
The wasp venom, mastoparan (MP), activates reconstituted pertussis toxin (PTX)-sensitive G-proteins in a receptor-independent manner. We studied the effects of MP and its analogue, mastoparan 7 (MP 7), on G-protein activation in HL-60 cells and a reconstituted system and on nucleoside diphosphate kinase (NDPK)-catalysed GTP formation. MP activated high-affinity GTP hydrolysis in HL-60 membranes with an EC50 of 1-2 microM and a maximum at 10 microM. Unlike the effects of the formyl peptide receptor agonist, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), on GTPase, those of MP were only partially PTX-sensitive. MP-induced rises in cytosolic Ca2+ concentration and superoxide-anion formation in intact HL-60 cells were also only incompletely PTX-sensitive. N-Ethylmaleimide inhibited MP-stimulated GTP hydrolysis to a greater extent than that stimulated by fMet-Leu-Phe. Unlike the latter, MP did not enhance incorporation of GTP azidoanilide into, and cholera toxin-catalysed ADP-ribosylation of, Gi-protein alpha-subunits in HL-60 membranes. By contrast to fMet-Leu-Phe, MP did not or only weakly stimulated binding of guanosine 5'-[gamma-thio]triphosphate to Gi-protein alpha-subunits. MP 7 was considerably more effective than MP at activating the GTPase of reconstituted Gi/G(o)-proteins, whereas in HL-60 membranes, MP and MP 7 were similarly effective. MP and MP 7 were similarly effective at activating [3H]GTP formation from [3H]GDP and GTP in HL-60 membranes and by NDPK purified from bovine liver mitochondria. Our data suggest the following: (1) MP activates Gi-proteins in HL-60 cells, but (2) the venom does not simply mimic receptor activation. (3) MP and MP 7 may activate GTP hydrolysis in HL-60 membranes indirectly through interaction with NDPK. (4) MP 7 is a more effective direct activator of PTX-sensitive G-proteins than MP, whereas with regard to NDPK, MP and MP 7 are similarly effective.
Insights
Mastoparan (MP) activates G-proteins in HL-60 cells, but not like typical receptor agonists. MP and MP 7 may indirectly activate GTP hydrolysis via nucleoside diphosphate kinase (NDPK).
Area of Science:
- Biochemistry
- Cell Signaling
- Pharmacology
Background:
- Mastoparan (MP), a wasp venom peptide, is known to activate G-proteins independently of receptors.
- Pertussis toxin (PTX)-sensitive G-proteins play crucial roles in various cellular signaling pathways.
- HL-60 cells are a human promyelocytic leukemia cell line commonly used to study cellular responses.
Purpose of the Study:
- To investigate the effects of mastoparan (MP) and its analogue mastoparan 7 (MP 7) on G-protein activation.
- To compare the mechanisms of MP-induced G-protein activation with receptor-mediated activation.
- To explore the potential involvement of nucleoside diphosphate kinase (NDPK) in MP-induced signaling.
Main Methods:
- Assessing GTP hydrolysis and GTP formation in HL-60 cell membranes and reconstituted systems.
- Utilizing PTX-sensitive G-proteins and N-Ethylmaleimide (NEM) to probe signaling pathways.
- Measuring cytosolic Ca2+ concentration and superoxide-anion formation in intact HL-60 cells.
- Investigating cholera toxin-catalyzed ADP-ribosylation and guanosine 5'-[gamma-thio]triphosphate binding to Gi-protein alpha-subunits.
Main Results:
- MP activated GTP hydrolysis in HL-60 membranes, with effects only partially sensitive to PTX.
- MP-induced cellular responses (Ca2+ and superoxide) were also incompletely PTX-sensitive.
- MP and MP 7 showed similar effectiveness in activating GTP formation via NDPK, but MP 7 was more potent in directly activating PTX-sensitive G-proteins in reconstituted systems.
Conclusions:
- MP activates Gi-proteins in HL-60 cells, but its mechanism differs from typical receptor-mediated activation.
- MP and MP 7 may activate GTP hydrolysis indirectly through interaction with NDPK.
- MP 7 is a more effective direct activator of PTX-sensitive G-proteins compared to MP.