Abstract 238: Angiogenesis Requires G-Protein-Coupled Receptor Kinase 2-Interacting Protein 1 (GIT1): A Novel GIT1-ERK1/2-Cortactin Pathway Controls Endothelial Cell Directional Migration

Abstract

Introduction— GIT1 is a multidomain scaffold protein involved in diverse cellular processes including cell adhesion and migration. GIT1 is required for vascular endothelial cell growth factor (VEGF) signaling in endothelial cells (EC) and translocates to lamellipodia during directional migration. Lamellipodia are regions of very rapid actin remodeling which is mediated in part by the cortical actin remodeling protein, cortactin. We hypothesize that GIT1 mediates directional migration of EC by regulating cortactin localization and activation. Methods and Results— To determine the role of GIT1 in directional migration of EC, we used a Dunn’s chamber and in vitro scratch wound healing assay. In Dunn’s chamber, the migration of GIT1 siRNA transfected Human Umbilical Vein EC (HUVEC) was decreased by 66±9.1% compared to control siRNA transfected EC in response to 50ng/ml VEGF gradient. Furthermore, the extent of wound healing was decreased by 63±7.8% in GIT1 knock out (GIT1-KO) mouse lung microvascular EC (MLMEC). We next studied the localization pattern of cortactin in MLMEC from GIT1 wild type (GIT1-WT) and GIT1-KO mice. In GIT1-KO MLMEC the number of cells with cortactin localized by immunofluorescence (IF) at the leading edge during wound healing was reduced by 73±5.7% compared to GIT1-WT. Dual IF for cortactin and GIT1 at the leading edge of HUVEC showed extensive co-localization. In response to 50ng/ml VEGF for 15 min, serum deprived HUVEC exhibited a 75.6±11.3% increase in GIT1-cortactin association as demonstrated by co-immunoprecipitation (IP). Among GIT1 domains, the Spa homology domain (SHD) is unique to GIT1 and is required for signal transduction. We next transfected GIT1-KO MLMEC with GIT1(1-770), GIT1(1-420) that contains SHD, and GIT1(1-250+420-770) that lacks SHD. IF and IP analysis of MLMEC showed that SHD was required for cortactin membrane localization and cortactin-GIT1 association. Finally, we showed that GIT1 regulates VEGF induced ERK1/2 mediated phosphorylation of cortactin Ser405, a requirement for cortactin activation. Conclusion— The unique SHD of GIT1 is essential for VEGF induced EC migration by assembling a GIT1-ERK1/2-cortactin complex suggesting that SHD of GIT1 may be a therapeutic target to limit pathological angiogenesis.