RESUMO
BACKGROUND: Assembly and disassembly of microtubules (MTs) is critical for neurite outgrowth and differentiation. Evidence suggests that nerve growth factor (NGF) induces neurite outgrowth from PC12 cells by activating the receptor tyrosine kinase, TrkA. G protein-coupled receptors (GPCRs) as well as heterotrimeric G proteins are also involved in regulating neurite outgrowth. However, the possible connection between these pathways and how they might ultimately converge to regulate the assembly and organization of MTs during neurite outgrowth is not well understood. RESULTS: Here, we report that Gßγ, an important component of the GPCR pathway, is critical for NGF-induced neuronal differentiation of PC12 cells. We have found that NGF promoted the interaction of Gßγ with MTs and stimulated MT assembly. While Gßγ-sequestering peptide GRK2i inhibited neurite formation, disrupted MTs, and induced neurite damage, the Gßγ activator mSIRK stimulated neurite outgrowth, which indicates the involvement of Gßγ in this process. Because we have shown earlier that prenylation and subsequent methylation/demethylation of γ subunits are required for the Gßγ-MTs interaction in vitro, small-molecule inhibitors (L-28 and L-23) targeting prenylated methylated protein methyl esterase (PMPMEase) were tested in the current study. We found that these inhibitors disrupted Gßγ and ΜΤ organization and affected cellular morphology and neurite outgrowth. In further support of a role of Gßγ-MT interaction in neuronal differentiation, it was observed that overexpression of Gßγ in PC12 cells induced neurite outgrowth in the absence of added NGF. Moreover, overexpressed Gßγ exhibited a pattern of association with MTs similar to that observed in NGF-differentiated cells. CONCLUSIONS: Altogether, our results demonstrate that ßγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and modulating MT rearrangement.
