Abstract In classic view, inhibitory heterotrimeric G protein alpha subunits 1, 2, and 3 (Gi1α, Gi2α and Gi3α) couple to Giα protein-coupled receptors (GiPCR) and then transduce signals from GiPCRs to downstream pathways in hormone and chemo-attractant signaling. Recent genetic evidence demonstrates that Giα proteins are crucial to embryo development and growth as well as tumorigenesis. However, the molecular mechanism underlying the implication of Giα proteins in this physiological and pathological process is largely unknown. Here we report that Giα proteins differentially regulate receptor tyrosine kinases (RTKs)-mediated activation of growth factor signaling. Gi1α and Gi3α double deficient (Gi1/3 DKO) mouse embryonic fibroblasts (MEFs), in which Gi2α expression is also lower than that in wild type cells, exhibited a clear defect in ERK1/2 activation by EGF, HB-EGF, insulin and IGF-I, but not aFGF, bFGF or high dose of serum (FBS). Further, Gi1/3 DKO MEFs were largely defective in Akt-mTORC1 activation by aFGF, bFGF, EGF, HB-EGF, serum, but not IGF-1 and insulin. The defect in Akt or ERK1/2 activation in Giα deficient MEFs was rescued upon reintroduction of Gi1α and Gi3α proteins. Interestingly, Giα deficiency impaired activation of Akt-mTROC1 and ERK1/2 by PDGF at a low, but not a high dose. Using a RNA interference strategy, we showed that knockdown of Giα proteins in either wt MEFs or triple negative breast cancer cells (MDA-MB231-D3H2-LN) led to a defect in Akt-mTROC1 and ERK1/2 activation, which is similar to that in Giα deficient cells in response to EGF, bFGF or IGF-1. Additional experimental analysis revealed that Giα proteins formed a complex with RTKs, Gab1, FRS2 and Shp2 in breast cancer cells in response to EGF, bFGF, or IGF-1. Gi1/3 DKO MEFs showed a defect in EGF- and IGF-1-induced interaction of Gab1 with SHP2, which is required for ERK1/2 activation by EGF and IGF-1, and a defect in bFGF-induced interaction of Grb2 with Gab1, which is required for Akt-mTORC1 activation by aFGF and bFGF. Finally, knockdown of Giα proteins impaired in vitro growth and invasion of breast cancer cells in response to bFGF, EGF, and IGF-1. Thus, Giα proteins are a differential mediator of RTK signaling in normal and breast cancer cells. Citation Format: Zhanwei Wang, Rica Dela-Cruz, meisheng Jiang, Wen-Ming Chu. Giα proteins are a differential mediator of activation of RTK signaling in normal and breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 529. doi:10.1158/1538-7445.AM2013-529