Rabbit anti-Human GIT1 polyclonal antibody

G protein-coupled receptor kinase-interactor 1 (Git1) is involved in cell motility control by serving as an adaptor that links signaling proteins such as Pix and PAK to focal adhesion proteins. We previously demonstrated that Git1 was a multiply tyrosine-phosphorylated protein, its primary phosphorylation site was Tyr-554 in the vicinity of the focal adhesion targeting-homology (FAH) domain, and this site was selectively dephosphorylated by protein tyrosine phosphatase receptor type Z (Ptprz). In the present study, we showed that Tyr-554 phosphorylation reduced the association of Git1 with the FAH-domain-binding proteins, paxillin and Hic-5, based on immunoprecipitation experiments using the Tyr-554 mutants of Git1. The Tyr-554 phosphorylation of Git1 was higher, and its binding to paxillin was consistently lower in the brains of Ptprz-deficient mice than in those of wild-type mice. We then investigated the role of Tyr-554 phosphorylation in cell motility control using three different methods: random cell motility, wound healing, and Boyden chamber assays. The shRNA-mediated knockdown of endogenous Git1 impaired cell motility in A7r5 smooth muscle cells. The motility defect was rescued by the exogenous expression of wild-type Git1 and a Git1 mutant, which only retained Tyr-554 among the multiple potential tyrosine phosphorylation sites, but not by the Tyr-554 phosphorylation-defective or phosphorylation-state mimic Git1 mutant. Our results suggested that cyclic phosphorylation-dephosphorylation at Tyr-554 of Git1 was crucial for dynamic interactions between Git1 and paxillin/Hic-5 in order to ensure coordinated cell motility.

Object. Promotion of the repair and regeneration of damaged adult neurons is a major goal of neurological science. In this study, the effects of G protein-coupled receptor kinase interacting protein 1 (GIT1) overexpression in human neuron cells were tested in human neuronal cells by using an adenoviral vector. Methods. A recombinant GIT1 and enhanced green fluorescent protein (EGFP) adenoviral vector (AdGIT1) was created by using a standard viral construction procedure. Human neuronal (NT2N) cells, which had been derived from an NT2 human teratocarcinoma cell line, were used in this experiment. Immunocytochemical methods were applied to identify NT2N cells with neural features and to probe the relationship among signaling proteins. Several biological activities were assessed, including neural spine formation, cell migration, and the levels of expression of growth-associated protein-43 (GAP-43) and active Cdc42. The number of cells with spine formation and the number of migrated cells were significantly higher in the AdGIT1-treated group of NT2N cells, than in untreated (control) NT2N cells or in AdEGFP-treated NT2N cells. The levels of GAP-43 and active Cdc42 expression were significantly higher in the AdGIT1-treated group than that in the other two cell groups. Conclusions. The results of this study demonstrate that GIT1 overexpression has the potential to promote neural spine formation and cell migration in human neuronal cells. At the same time, the increased level of GAP-43 in GIT1-overexpressed cells indicates that GIT1 may have the potential to improve growth and regeneration of damaged axons. The GIT1-beta-PIX-Cdc42-PAK pathway may play an important role in neuronal outgrowth.