Subject(s)
3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Eye Proteins/metabolism , rab GTP-Binding Proteins/metabolism , 3',5'-Cyclic-GMP Phosphodiesterases/genetics , 3',5'-Cyclic-GMP Phosphodiesterases/isolation & purification , Cell Membrane/metabolism , Chromatography, Affinity/methods , Cyclic Nucleotide Phosphodiesterases, Type 6 , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Eye Proteins/genetics , Eye Proteins/isolation & purification , Fluorescent Antibody Technique , HeLa Cells , Humans , Protein Binding , Protein Subunits , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Rod Cell Outer Segment/enzymology , Saccharomyces cerevisiae , Solubility , Two-Hybrid System Techniques , rab GTP-Binding Proteins/biosynthesis , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/isolation & purificationABSTRACT
Small Rab GTPases are involved in the regulation of membrane trafficking. They cycle between cytosolic and membrane-bound forms. These membrane association/dissociation are tightly controlled by regulatory proteins. To search for proteins interacting with Rab13, a small GTPase associated with vesicles in fibroblasts and predominantly with tight junctions in epithelial cells, we screened a HeLa two-hybrid cDNA library and isolated a clone encoding a protein of 17.4 kDa. This protein, almost identical to the bovine rod cGMP phosphodiesterase delta subunit, was named human delta-PDE. The delta-PDE binds specifically to Rab13. It exhibits two putative C-terminal sequences necessary for the interaction with PDZ (PSD95, Dlg, ZO-1) domains contained in many proteins localized to specific plasma membrane microdomains. Immunofluorescence microscopic studies revealed that the vesicular stomatitis virus (VSV)-tagged delta-PDE is localized in vesicular structures accumulated near the plasma membrane in epithelial cells. Deletion of the PDZ binding motifs impair VSV-delta-PDE subcellular distribution. Purified recombinant delta-PDE had the capacity to dissociate Rab13 from cellular membranes. Our data support the proposal that delta-PDE, but not GDP dissociation inhibitor, may serve to control the dynamic of the association of Rab13 with cellular membranes.
