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EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain
- Source :
- Europe PubMed Central
-
Abstract
- ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4, 5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4, 5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane. This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIP3 production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by coexpression with a dominant negative p85. This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain.
- Subjects :
- Green Fluorescent Proteins
Plasma protein binding
Biology
PC12 Cells
Wortmannin
Phosphatidylinositol 3-Kinases
chemistry.chemical_compound
Animals
Guanine Nucleotide Exchange Factors
Inositol
Nerve Growth Factors
Phosphatidylinositol
Microscopy, Confocal
Epidermal Growth Factor
Cell Membrane
Biological Transport
Cell Biology
Transfection
Recombinant Proteins
Protein Structure, Tertiary
Rats
Cell biology
Pleckstrin homology domain
Luminescent Proteins
chemistry
Second messenger system
Indicators and Reagents
Guanine nucleotide exchange factor
Cell Adhesion Molecules
Phosphorus Radioisotopes
Protein Binding
Subjects
Details
- Database :
- OpenAIRE
- Journal :
- Europe PubMed Central
- Accession number :
- edsair.doi.dedup.....4330ce25d094f8fffc0c7748f12c618d