Intracellular translocation of the decapeptide carboxyl terminal of G i3α 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 i3α 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 i3α carboxyl terminal (G i3α 346–355; H-KNNLKECGLY-NH 2). Kinetic studies, using a CFDA-conjugated analogue of G i3α 346–355, confirmed the rapid and efficient intracellular translocation of TP10-G i3α 346–355 ( t 0.5 = 3 min). Translocated G i3α 346–355, but not other bioactive cargoes derived from PKC and the CB 1 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 i3α 346–355 did not influence the exocytosis of β-hexoseaminidase from RBL-2H3, a secretory event stimulated by other cell penetrant peptide cargoes and MP. We conclude that TP10-G i3α 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.