Mint2 Promotes The Retention Of TrkA In Golgi Apparatus And Affects NGF-induced Neurite Outgrowth

The interaction between Mint2 and TrkA was confirmed in rat DRG and basal forebrain by co-immunoprecipitation experiment. Mint2 and TrkA were co-localized in DRG neurons by double fluorescent immunohistochemistry. To explore the function of this interaction EGFP-Mint2, myc-Mint2 and EGFP-TrkA plasmids were constructed. The cellular distribution and location were consistent with described previously, and EGFP-TrkA internalized after NGF treated. PC12 differentiation assay showed Mint2 may negatively affect NGF-induced neurite outgrowth of PC12 cells. Neurite outgrowth of DRG neurons was significantly decreased when Mint2 overexpressed and increased if Mint2 was knocked down. Therefore, these results further demonstrated that Mint2 could negatively affect NGF-induced neurite outgrowth.Immunocytochemistry showed myc-Mint2 mainly locate in Golgi apparatus and GFP-TrkA mainly accumulate in cytoplasma and co-localize with WGA and myc-Mint2 before and after NGF treated. Mint2 inhibited the surface expression of GFP-TrkA before NGF sitmulation, which was confirmed by surface protein biotin assay. To make clear when the retention happened, the media of the transfected PC12 cells was changed without serum and then Sulfo-NHS-Biotin was added to lable the surface proteins. The morphological results show GFP-TrkA is co-localized with WGA before NGF treated when co-transfected with myc-Mint2, comparing with co-transfected with vector. After NGF treated GFP-TrkA was internalized, and the internalized TrkA was distributed dispersedly in cell plasma and not congregated with WGA. These results indicated that the Golgi body retention was happeded before NGF treated.