Nerve growth factor- and epidermal growth factor-stimulated translocation of the ADP-ribosylation factor-exchange factor GRP1 to the plasma membrane of PC12 cells requires activation of phosphatidylinositol 3-kinase and the GRP1 pleckstrin homology domain

Abstract

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)P3]. In order to study the effects of PtdIns(3,4,5)P3 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)P3, inositol 1, 3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], with high affinity (Kd 32. 2+/-5.2 nM) and inositol phosphate specificity [Kd values for Ins(1, 3,4,5,6)P5, InsP6, Ins(1,3,4)P3 and Ins(1,4,5)P3: 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)P4, data that are consistent with its proposed role as a putative PtdIns(3,4,5)P3 receptor. To address whether GRP1 binds PtdIns(3,4,5)P3 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)P3. This translocation is dependent on the activation of phosphatidylinositol 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) 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)P3 and hence constitutes a true PtdIns(3,4,5)P3 receptor.

ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling

“…The GST-GGA3 PBD fusion protein purified and coupled to glutathione beads as described (18). The transfected cells stimulated without or with HCG 10 IU/ml for 30 min at 37°C in the presence of or absence of inhibitor were lysed using ice-cold lysis buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 10 mM MgCl 2 ) with 1% protease inhibitors mix.…”

“…There are four cytohesin family members in humans (cytohesins 1-4), each of which contain a PH domain that binds to PIP3. Cytohesins 1-3 translocate from the cytoplasm to the plasma membrane in a PI3K-dependent manner, where they activate ARF6 (6,(17)(18)(19)(20)(21).…”

Luteinizing hormone/chorionic gonadotrophin receptor overexpressed in granulosa cells from polycystic ovary syndrome ovaries is functionally active

“…The GST-GGA3 PBD fusion protein purified and coupled to glutathione beads as previously described (Venkateswarlu et al, 1998). GCs isolated from PCOS and normal women were serum starved for 2h and stimulated without or with HCG 10IU/ml for 30min at 37 o C. The cells were then lysed using ice-cold modified RIPA lysis buffer with 1% mammalian protease inhibitors mix.…”

LL5β Is a Phosphatidylinositol (3,4,5)-Trisphosphate Sensor That Can Bind the Cytoskeletal Adaptor, γ-Filamin

“…There is now a substantial family of PtdIns(3,4,5)P 3 -binding proteins that have been shown to translocate to the plasma membrane in response to receptor stimulation of type I PI3K activity, including PKB (8,9), DAPP-1 (10 -13), PDK-1 (14), ARNO (15), , and GRP1 (17). All of the above PI3K effectors bind 3-phosphorylated lipids via a PH domain.…”