RhoGDI-binding-defective mutant of Cdc42Hs targets to membranes and activates filopodia formation but does not cycle with the cytosol of mammalian cells

Abstract

We have identified a mutant of the human G-protein Cdc42Hs, R66E, that fails to form a detectable complex with the GDP-dissociation inhibitor RhoGDI in cell-free systems or in intact cells. This point mutant is prenylated, binds guanine nucleotide and interacts with GTPase-activating protein in a manner indistinguishable from wild-type Cdc42Hs. Immunofluorescence localization studies revealed that this RhoGDI-binding-defective mutant is found predominantly in the Golgi apparatus, with a staining pattern similar to that of the wild-type protein. However, unlike wild-type Cdc42Hs, which is distributed in both the microsomal membrane and cytosolic fractions, studies using differential centrifugation show that prenylated R66E Cdc42Hs is found exclusively in association with lipid bilayers. Additionally, whereas the overexpression of RhoGDI results in an apparent translocation of wild-type Cdc42Hs from the Golgi apparatus into the cytosol, identical RhoGDI-overexpression conditions do not alter the Golgi localization of the R66E mutant. Furthermore, overexpression of this RhoGDI-binding-defective mutant of Cdc42Hs seems to activate redistribution of the actin cytoskeleton and filopodia formation in fibroblasts in a manner indistinguishable from the wild-type protein. Taken together, these results suggest that the interaction of Cdc42Hs with RhoGDI is not essential for proper membrane targeting of nascent prenylated Cdc42Hs in mammalian cells; neither is this interaction an essential part of the mechanism by which Cdc42Hs activates filopodia formation. However, it does seem that redistribution of Cdc42Hs to the cytosolic compartment is absolutely dependent on RhoGDI interaction.

The Parkinson’s disease-associated protein, leucine-rich repeat kinase 2 (LRRK2), is an authentic GTPase thatstimulates kinase activity

“…Subsequently, immunoprecipitates were incubated with 1µCi [α-32 P]GTP (3,000Ci/mmol) for 30min at 23°C followed by addition of 20mM MgCl 2 to stabilize and prevent dissociation of already bound nucleotide, as has been demonstrated for other Ras-related GTPases [27,28]. Binding was terminated by rapid filtration and washing with cold PBS containing 20mM MgCl 2 , three times, through 0.45µm microcentrifuge filter units (Millipore).…”

“…Infinite dilution and rapid filtration GTP hydrolysis experiments were performed similarly as described previously [27,28]. Briefly, immunoprecipitated LRRK2 was incubated with 1.5µCi [γ-32 P]GTP (4,500Ci/mmol) in assay buffer (100mM Tris-HCl, pH 7.5; 1mM DTT; 2mM EDTA; 10mM CTP; 100mM NaCl) for 20min at 23°C.…”

“…For GTP hydrolysis experiments approximately 2µg of GST-fusion proteins were incubated with 1.5µCi [γ-32 P]GTP (4,500Ci/mmol) in assay buffer as described above. In both cases, after 20min incubation, 10mM unlabeled GTP and 20mM MgCl 2 were added to infinitely dilute the radioactive nucleotide, therefore decreasing the possibility of further binding, and to stabilize and prevent dissociation of the already bound nucleotide, respectively [27,28]. For GTP binding experiments the entire reaction was diluted in 10ml icecold dilution buffer (20mM Tris-HCl, pH 7.5; 100mM NaCl; 10mM MgCl 2 ) and rapidly filtered through nitrocellulose filter discs on a Millipore model 1225 sampling manifold, while equivalent aliquots were removed at 0, 3, 10 and 30min time points for the GTP hydrolysis experiments.…”

“…Briefly, immunoprecipitated LRRK2 was incubated with 1.5µCi [γ-32 P]GTP (4,500Ci/mmol) in assay buffer (100mM Tris-HCl, pH 7.5; 1mM DTT; 2mM EDTA; 10mM CTP; 100mM NaCl) for 20min at 23°C. 10mM unlabeled GTP and 20mM MgCl 2 were then added to infinitely dilute the radioactive nucleotide, therefore decreasing the possibility of further binding, and to stabilize and prevent dissociation of the already bound nucleotide, respectively [27,28]. Equivalent aliquots were removed at 0, 3, 10, 30 and 60min time points.…”

Recruitment of Dbl by Ezrin and Dystroglycan Drives Membrane Proximal Cdc42 Activation and Filopodia Formation

“…49 Specific mutations in Cdc42 that block its interaction with RhoGDI alter the membrane distribution of Cdc42, but do not prevent its activation of filopodia. 50,51 The importance of the RhoGDI interaction in recycling Cdc42 is probably in the redistribution and/or delivery of Cdc42 to an appropriate site. Given that ERM proteins can form discrete complexes with RhoGDI and DG, it could be that the ERM-RhoGDI complex serves to deliver inactive Cdc42 to the GEF, in this case Dbl, and does so by docking with DG where it releases Cdc42 for activation and RhoGDI for recharging.…”