title: Intracellular translocation of the decapeptide carboxyl terminal of Gi3 alpha induces the dual phosphorylation of p42/p44 MAP kinases.
abstract: The carboxyl terminal of heterotrimeric G protein alpha subunits binds both G protein-coupled receptors and mastoparan (MP), a tetradecapeptide allostere. Moreover, peptides corresponding to the carboxyl domains of G(i)3alpha and G(t) display intrinsic biological activities in cell-free systems. Thus, the purpose of this study was to develop a cell penetrant delivery system to further investigate the biological properties of a peptide mimetic of the G(i)3alpha carboxyl terminal (G(i)3alpha(346-355); H-KNNLKECGLY-NH2). Kinetic studies, using a CFDA-conjugated analogue of G(i)3alpha(346-355), confirmed the rapid and efficient intracellular translocation of TP10-G(i)3alpha(346-355) (t(0.5) = 3 min). Translocated G(i)3alpha(346-355), but not other bioactive cargoes derived from PKC and the CB1 cannabinoid receptor, promoted the dual phosphorylation of p42/p44 MAPK without adverse changes in cellular viability. The relative specificity of this novel biological activity was further confirmed by the observation that translocated G(i)3alpha(346-355) did not influence the exocytosis of beta-hexoseaminidase from RBL-2H3, a secretory event stimulated by other cell penetrant peptide cargoes and MP. We conclude that TP10-G(i)3alpha(346-355) is a valuable, non-toxic research tool with which to study and modulate signal transduction pathways mediated by heterotrimeric G proteins and MAPK.
authors.affiliation: Research Institute in Healthcare Science, University of Wolverhampton, Wulfruna Street, Wolverhampton, WV1 1SB, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Jones
authors.forename: Michelle
authors.initials: M
authors.lastname: Farquhar
authors.forename: Ashley
authors.initials: A
authors.lastname: Martin
authors.forename: John
authors.initials: J
authors.lastname: Howl
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D021381:Protein Transport / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D001619:beta-N-Acetylhexosaminidases; C498819:GNAI3 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go