NGF-and EGF-stimulated stimulated translocation of the ARF-exchange factor GRP1 to the plasma membrane of PC12 cells requires activation of PI3-kinase and a functional GRP1 PH domain

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells. GRP1 is a member of a family of ARF guanine-nucleotide-exchange factors that binds in vitro the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P-3]. In order to study the effects of PtdIns(3,4,5)P-3 on the function of GRP1, we have cloned the human homologue of GRP1, encoding for a protein which is 98.8 % identical to mouse brain GRP1. Human GRP1 binds, via its pleckstrin homology (PH) domain, the inositol head group of PtdIns(3,4,5)P-3, inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P-4], with high affinity 32.2+/-5.2 nM) and inositol phosphate specificity [K-d values for Ins(1,3,4,5,6)P-5, InsP(6), Ins(1,3,4)P-3 and Ins(1,4,5)P-3: 283+/-32, >10000, > 10000 and > 10000 nM, respectively). Furthermore, GRP1 can accommodate addition of glycerol or diacetylglycerol to the 1-phosphate of Ins(1,3,4,5)P-4, data that are consistent with its proposed role as a putative PtdIns(3,4,5)P-3 receptor. To address whether GRP1 binds PtdIns(3,4,5)P-3 in vivo, we have expressed a chimaera of green fluorescent protein (GFP) fused to the N-terminus of GRP1 in PC12 cells and, using confocal microscopy, examined its resultant localization in live cells. Stimulation with either nerve growth factor or epidermal growth factor (both at 100 ng/ml) results in a rapid, PH-domain dependent, translocation of GFP-GRP1 from the cytosol to the plasma membrane, which occurs with a time course that parallels the production of PtdIns(3,4,5)P-3. This translocation is dependent on the activation of phosphatidylinositol 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 mu M) and by the co-expression with dominant negative p85. Taken together these data strongly suggest that GRP1 interacts in vivo with plasma membrane-located PtdIns(3,4,5)P-3 and hence constitutes a true PtdIns(3,4,5)P-3 receptor.