Novel features of amphiphilic peptide Mas7 in signalling via heterotrimeric G-proteins.
Amphiphilic peptide Mas7, a structural analogue of mastoparan is a known activator of heterotrimeric Gi-proteins and its downstream effectors. This study investigated the functional interaction of Mas7 with a plasma membrane protein from CHO cells, the endogenous mono-ADP-ribosyltransferase. The substrate of endogenous mono-ADP-ribosyltransferase was the ADP-ribosylated protein with a molecular mass of 36 kDa, which corresponded to the beta subunit of heterotrimeric G-proteins. The effect of Mas7 on endogenous mono-ADP-ribosyltransferase activity was in the micromolar range with a maximal activation of 205% over the basal. In pertussis treated plasma membranes, it was found that the effect of Mas7 on endogenous mono-ADP-ribosyltransferase was partially blocked, which suggests the involvement of G-proteins, such as Gi or G0. In addition, an immunoassay was developed for the visualization of interaction between the a subunit and the betagamma dimer of G-protein on a Ni-NTA support. The physical interaction was tested of Mas7 with the heterotrimeric G-protein alphai2 subunit, which was overexpressed together with beta1gamma2-His6 subunits in sf9 cells. An interaction between Gi2 heterotrimer and Mas7 was not observed, which was not in accordance with previously reported results of mastoparan obtained for Gi-proteins from bovine brain. In conclusion, the signal is mediated from Mas7 to endogenous mono-ADP-ribosyltransferase via pertussis sensitive G-proteins. Furthermore, it is hypothesized that Gi2 G-proteins are not involved in the process.
Bavec
Aljosa
A
Institute of Biochemistry, Medical Faculty, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia. aljosa.bavec@mf.uni-lj.si
D036002:ADP Ribose Transferases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D037342:Pertussis Toxin / Q000494:pharmacology; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins; D015398:Signal Transduction; D014162:Transfection
D036341:Intercellular Signaling Peptides and Proteins; C084070:Mas7 protein, synthetic; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; D036002:ADP Ribose Transferases; D037342:Pertussis Toxin; D020962:Heterotrimeric GTP-Binding Proteins