Mastoparan may activate GTP hydrolysis by Gi-proteins in HL-60 membranes indirectly through interaction with nucleoside diphosphate kinase.
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.
Klinker
J F
JF
Institut für Pharmakologie, Freie Universität Berlin, Germany.
Hagelüken
A
A
Grünbaum
L
L
Heilmann
I
I
Nürnberg
B
B
Harhammer
R
R
Offermanns
S
S
Schwaner
I
I
Ervens
J
J
Wenzel-Seifert
K
K
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D036341:Intercellular Signaling Peptides and Proteins; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D010455:Peptides; D037342:Pertussis Toxin; D013481:Superoxides / Q000378:metabolism; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014862:Wasp Venoms / Q000494:pharmacology*
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D010566:Virulence Factors, Bordetella; D014862:Wasp Venoms; D013481:Superoxides; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; C037500:mastoparan; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D009701:Nucleoside-Diphosphate Kinase; D000251:Adenosine Triphosphatases; D019204:GTP-Binding Proteins; D002118:Calcium