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
A. Shisheva et al., 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, BIOCHEM, 38(36), 1999, pp. 11711-11721
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.