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

The GDP dissociation inhibitors (GDIs) represent an important class of regu latory proteins in the functional cycle and recycling of Rab GTPases. Previ ous studies have demonstrated that GDI-1 can operate with multiple Rab prot eins. In this study we have addressed a plausible general activity of GDI-2 in supporting Rab membrane release and have analyzed the requirements of s equence-conserved vs variable regions of GDI-2 in these functional interact ions. The in vitro function of expressed recombinant GDI-2 wild-type-, poin t-, or deletion-mutant proteins was investigated toward several Rab family members, divergent in structure, localized and operating on different membr anes, including Rab2, Rab4, Rab5, Rab8, Rab9, and Rab11. We demonstrate her e a general and nearly invariant ability of GDI-2(WT) to release from membr anes this subset of diverse Rabs. Deletion of an 18-residue segment from th e C-terminal variable region yielded a fully functional or only slightly de fective GDI-2. Conversely, substitution of Met at position 250 of the conse rved region markedly abrogated the activity toward all Rabs. Surprisingly, a replacement of an adjacent conserved residue (Y249V) resulted in a select ive 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-func tion conformation, we pharmacologically stimulated intact 3T3-L1 adipocytes to induce GDI-2 tyrosine phosphorylation. We found a pronounced increase o f the Rab4 soluble form and its soluble complexes with the tyrosine-phospho rylated GDI-2. Together, these results indicate that (a) GDI-2 displays a g eneral activity to release Rabs from membranes, (b) GDI-2-conserved residue s, but not the C-terminal variable region, are essential for this activity, and (c) structural modifications in GDI-2 can enhance its functional activ ity, directing selective interactions with individual Rabs.