title: General role of GDP dissociation inhibitor 2 in membrane release of Rab proteins: modulations of its functional interactions by in vitro and in vivo structural modifications.
abstract: The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins in the functional cycle and recycling of Rab GTPases. Previous studies have demonstrated that GDI-1 can operate with multiple Rab proteins. In this study we have addressed a plausible general activity of GDI-2 in supporting Rab membrane release and have analyzed the requirements of sequence-conserved vs variable regions of GDI-2 in these functional interactions. The in vitro function of expressed recombinant GDI-2 wild-type-, point-, or deletion-mutant proteins was investigated toward several Rab family members, divergent in structure, localized and operating on different membranes, including Rab2, Rab4, Rab5, Rab8, Rab9, and Rab11. We demonstrate here a general and nearly invariant ability of GDI-2(WT) to release from membranes this subset of diverse Rabs. Deletion of an 18-residue segment from the C-terminal variable region yielded a fully functional or only slightly defective GDI-2. Conversely, substitution of Met at position 250 of the conserved region markedly abrogated the activity toward all Rabs. Surprisingly, a replacement of an adjacent conserved residue (Y249V) resulted in a selective Rab-dependent response and a profound gain of function toward specific Rabs. To further test whether the endogenous GDI-2 can adopt a gain-of-function conformation, we pharmacologically stimulated intact 3T3-L1 adipocytes to induce GDI-2 tyrosine phosphorylation. We found a pronounced increase of the Rab4 soluble form and its soluble complexes with the tyrosine-phosphorylated GDI-2. Together, these results indicate that (a) GDI-2 displays a general activity to release Rabs from membranes, (b) GDI-2-conserved residues, but not the C-terminal variable region, are essential for this activity, and (c) structural modifications in GDI-2 can enhance its functional activity, directing selective interactions with individual Rabs.
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, 540 East Canfield Street, Detroit, Michigan 48201, USA. ashishev@moose.med.wayne.edu
authors.forename: A
authors.initials: A
authors.lastname: Shisheva
authors.forename: S R
authors.initials: SR
authors.lastname: Chinni
authors.forename: C
authors.initials: C
authors.lastname: DeMarco
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
chemical_list: D017931:DNA Primers; C098085:Gdi2 protein, mouse; D020730:Guanine Nucleotide Dissociation Inhibitors; D019204:GTP-Binding Proteins