(Received for publication, October 25, 1995; and in revised form, February 8, 1996)
From the
The GDP-dissociation-inhibitor (GDI) for Rho-like GTP-binding
proteins is capable of three different biochemical activities. These
are the inhibition of GDP dissociation, the inhibition of GTP
hydrolysis, and the stimulation of the release of GTP-binding proteins
from membranes. In order to better understand how GDI interactions with
Rho-like proteins mediate these different effects, we have set out to
develop a direct fluorescence spectroscopic assay for the binding of
the GDI to the Rho-like protein, Cdc42Hs. We show here that when the
GDI interacts with Cdc42Hs that contains bound N-methylanthraniloyl GDP (Mant-GDP), there is an 20%
quenching of the Mant fluorescence. The GDI-induced quenching is only
observed when Mant-GDP is bound to Spodoptera
frugiperda-expressed Cdc42Hs and is not detected when the Mant
nucleotide is bound to Escherichia coli-expressed Cdc42Hs and
thus shows the same requirement for isoprenylated GTP-binding protein
as that observed when assaying GDI activity. A truncated Cdc42Hs mutant
that lacks 8 amino acids from the carboxyl terminus and is insensitive
to GDI regulation also does not show changes in the fluorescence of its
bound Mant-GDP upon GDI addition. Thus, the GDI-induced quenching of
Mant-GDP provides a direct read-out for the binding of the GDI to
Cdc42Hs. Titration profiles of the GDI-induced quenching of the
Mant-GDP fluorescence are saturable and are well fit to a simple 1:1
binding model for Cdc42Hs-GDI interactions with an apparent K
value of 30 nM. A very similar K
value (28 nM) is measured when
titrating the GDI-induced quenching of the fluorescence of
Mant-guanylyl imidotriphosphate, bound to Cdc42Hs. These results
suggest that the GDI can bind to the GDP-bound and GTP-bound forms of
Cdc42Hs equally well. We also have used the fluorescence assay for GDI
interactions to demonstrate that the differences in functional potency
observed between the GDI molecule and a related human leukemic protein,
designated LD4, are due to differences in their binding affinities for
Cdc42Hs. This, together with the results from studies using GDI/LD4
chimeras, allow us to conclude that a limit region within the
carboxyl-terminal domain of the GDI molecule is responsible for its
ability to bind with higher affinity (compared with LD4) to Cdc42Hs.
The Ras-like low molecular weight GTP-binding proteins form a
superfamily whose members are involved in a plethora of biological
pathways that include the regulation of cell growth and
differentiation, vesicular transport, and cytoskeletal organization.
The GTP-binding proteins appear to act as molecular switches by cycling
between an inactive GDP-bound state and an active GTP-bound state. This
cycle is tightly regulated by distinct proteins. In particular, the
exchange of GDP to GTP is stimulated by guanine nucleotide exchange
factors (GEFs), ()and the hydrolysis of GTP back to GDP is
catalyzed by GTPase-activating proteins (GAPs). A third class of
regulatory proteins were discovered based on their ability to inhibit
GDP dissociation (and thus were originally designated as
GDP-dissociation inhibitors or GDIs). The GDIs are capable of two other
important biochemical activities, namely they inhibit GTP hydrolysis
and promote the dissociation of GTP-binding proteins from membranes.
Given these various activities, it has been speculated that the GDIs
might play a critical role in mediating the movement of GTP-binding
proteins between different cellular locations.
A major interest in our laboratory has been directed toward understanding the mechanism of regulation of the GTP-binding/GTPase cycle of the Cdc42Hs GTP-binding protein. Cdc42Hs is the human homolog of the Saccharomyces cerevisiae cell division cycle protein (Cdc42Sc) (Johnson and Pringle, 1990; Shinjo et al., 1990), which plays an essential role in the establishment of cell polarity by controlling the assembly of the bud site. The Schizosaccharomyces pombe Cdc42 protein is essential for both uni-directional and bi-directional cell growth (Miller and Johnson, 1994), and the mammalian Cdc42 protein has recently been implicated in filopodia formation (Kozma et al., 1995; Nobes and Hall, 1995) and in the stimulation of the nuclear kinases Jnk and p38 (Coso et al., 1995; Minden et al., 1995; Hill et al., 1995; Bagrodia et al., 1995a). Several regulators of the GTP-binding/GTPase cycle of Cdc42Hs have now been identified. These include the Dbl oncogene product (Hart et al., 1991a), which serves as a GEF for Cdc42 in mammalian cells, and the Cdc24 gene product, which functions as a GEF for Cdc42 in S. cerevisiae (Zheng et al., 1994). In addition, a specific GAP (designated the Cdc42Hs-GAP) has been purified (Hart et al., 1991b) and cloned (Barford et al., 1993), and a Cdc42Hs-GDI activity was purified from brain cytosol and shown to be identical to the RhoGDI (Leonard et al., 1992).
To better
understand how these different proteins regulate the GTP-binding/GTPase
cycle of Cdc42Hs, we have developed real time fluorescence
spectroscopic assays to monitor each step of the cycle. Initially, we
used the fluorescence of the single tryptophan residue of Cdc42Hs
(Trp) as an intrinsic reporter group for monitoring GTP
hydrolysis (Leonard et al., 1994). In addition, along the
lines of experiments performed with Ras (Antonny et al., 1991;
Rensland et al., 1991; Moore et al., 1993), we have
used the fluorescence of Mant-derivatized nucleotides to monitor the
kinetics of nucleotide binding and dissociation from Cdc42Hs (Leonard et al., 1994). More recently, we have covalently attached an
exogenous fluorescent probe to Cdc42Hs and used the fluorescence of the
probe to directly monitor the GTP-binding and GTPase activities of
Cdc42Hs (Nomanbhoy et al., 1996).
Although the methods discussed above have provided us with real time read-outs for the GEF-catalyzed GDP-GTP exchange and GAP-stimulated GTP hydrolytic reactions, they have not provided a means to directly monitor the binding of Cdc42Hs to its GDI (i.e. the RhoGDI). There is a great deal of interest in understanding the interactions of RhoGDI with Cdc42Hs and related proteins (i.e. Rac and RhoA) because of the multiple regulatory activities exhibited by the GDI, i.e. its ability to bind to both the GDP- and GTP-bound forms of Cdc42Hs and to elicit the release of both forms of Cdc42Hs from membranes. As a first step toward characterizing these interactions, we set out to develop a fluorescence assay that would allow us to directly monitor Cdc42Hs-RhoGDI complex formation in real time. In the present study, we describe such a read-out that takes advantage of a GDI-induced quenching of the fluorescence of Mant-nucleotides bound to Cdc42Hs. We have gone on to use this read-out to address two important questions regarding the actions of RhoGDI. The first concerns the relative ability of RhoGDI to bind to the GDP- and GTP-bound forms of Cdc42Hs. This question directly bears on the relevance of RhoGDI as a GTPase inhibitor of Cdc42Hs and on the ability of RhoGDI to cause activated (GTP-bound) forms of Cdc42Hs to be released from membranes. The second question that we have addressed relates to the mechanistic basis of the functional differences that have been observed between RhoGDI and a highly related molecule, designated LD4, in their actions toward Cdc42Hs. Specifically, it has been shown that although LD4 has GDI activity toward Cdc42Hs, it is significantly less potent than that of the RhoGDI. Recent chimera studies have demonstrated that the carboxyl-terminal domains of the RhoGDI and LD4 molecules are responsible for the difference in potency (Platko et al., 1995). In the present study, we have used the fluorescence read-out for GDI interactions to determine if the functional differences between RhoGDI and LD4 directly reflect differences in their binding affinity for Cdc42Hs. If so, this then would highlight the amino acid residues on the RhoGDI molecule that are critical for high affinity binding to GTP-binding proteins.
where F is the change in fluorescence over initial
fluorescence (-F/F
), F
is the initial value for
(-
F/F
), F
is the final value of
(-
F/F
), L
is the total concentration of RhoGDI, LD4, or the chimeras, and R
is the total concentration of Cdc42Hs-Mant-GDP
or Cdc42Hs-Mant-GppNp. Fits were generated using IgorPro wavemetrics
software.
Figure 1: The Mant fluorescence of isoprenylated Cdc42Hs-Mant-GDP is sensitive to the interaction between RhoGDI and Cdc42Hs. A, normalized emission spectra for isoprenylated Cdc42Hs-Mant-GDP (80 nM) in the absence and the presence of RhoGDI (200 nM). The emission spectra were obtained by exciting the sample at 350 nm. B, 80 nM of either isoprenylated or nonisoprenylated Cdc42Hs-Mant-GDP was incubated in Buffer D. The time course for Mant fluorescence was observed (excitation = 350 nm, emission = 440 nm), and at the indicated time, RhoGDI was added to 200 nM.
As shown in Fig. 1B, the interaction between RhoGDI and Cdc42Hs that gives rise to the quenching of Mant-GDP fluorescence is rapid and occurs within the time period of mixing (<5 s). The RhoGDI-induced decrease in Mant-GDP fluorescence is also specific for isoprenylated Cdc42Hs, consistent with earlier suggestions that the geranyl-geranylation of Rho-subtype GTP-binding proteins is necessary for their interactions with GDI (Ueda et al., 1990). No quenching of the Mant-GDP fluorescence was observed when RhoGDI was added to Cdc42Hs that was expressed and purified from E. coli (Fig. 1B) or when added to Cdc42Hs that was expressed in Sf9 cells but purified from the aqueous fraction of the Triton-X 114 phase extraction (data not shown).
There were a number of other criteria that would be expected to be met if the RhoGDI-induced quenching of the Mant fluorescence reflected a direct interaction between GDI and Cdc42Hs. For example, it would be expected that the addition of isoprenylated (Sf9-expressed) Cdc42Hs that contained GDP (rather than the fluorescent Mant-GDP) would compete with the Cdc42Hs-Mant-GDP complex and thus block the RhoGDI-induced change in Mant fluorescence. As shown in Fig. 2A, the inclusion of excess isoprenylated GDP-bound Cdc42Hs (250 nMversus 15 nM Mant-GDP-bound Cdc42Hs) prior to the addition of RhoGDI completely inhibited the GDI-dependent quenching of the Mant fluorescence. This inhibition was specific for isoprenylated Cdc42Hs, because the addition of nonisoprenylated (E. coli-expressed) Cdc42Hs to 450 nM only slightly attenuated the quenching of Mant fluorescence by RhoGDI (Fig. 2B). In addition, when RhoGDI was first added to the Cdc42Hs-Mant-GDP complex, in order to effect a quenching of the Mant fluorescence, it was possible to rapidly reverse the GDI-induced quenching by subsequently adding an excess of isoprenylated GDP-bound Cdc42Hs (Fig. 2C).
Figure 2: The RhoGDI-induced changes in the Mant fluorescence of Cdc42Hs-Mant-GDP can be inhibited or reversed by isoprenylated Cdc42Hs-GDP. 15 nM isoprenylated Cdc42Hs-Mant-GDP was incubated in Buffer D in the presence of 250 nM isoprenylated Cdc42Hs (A) or 450 nM nonisoprenylated (E. coli-expressed) Cdc42Hs (B) or in the absence of Cdc42Hs (C). At the indicated time, RhoGDI was added to 60 nM. In the case of C, 250 nM isoprenylated Cdc42Hs was added after the addition of RhoGDI.
Finally, in a previous study, we
characterized a RhoGDI truncation mutant in which 8 amino acids had
been removed from the carboxyl terminus (RhoGDI8). Specifically,
we observed that RhoGDI
8 showed absolutely no GDI activity toward
Cdc42Hs (Platko et al., 1995). This indicated that the
carboxyl-terminal domain of RhoGDI contains residues that are critical
for the interaction between RhoGDI and Cdc42Hs, either through a direct
involvement in binding Cdc42Hs or through the maintenance of an
appropriate tertiary structure. We would therefore predict that due to
its inability to bind to Cdc42Hs, RhoGDI
8 would not induce a
quenching of the Mant fluorescence of Cdc42Hs-Mant-GDP. Fig. 3shows that this turned out to be the case, that is the
addition of RhoGDI
8 to isoprenylated Cdc42Hs-Mant-GDP did not
cause a significant decrease in Mant fluorescence. Thus, taken
together, our observations in Fig. 1Fig. 2Fig. 3indicate that the quenching of the Mant fluorescence by
RhoGDI is a direct reflection of the binding of GDI to Cdc42Hs.
Figure 3:
RhoGDI8 does not quench the Mant
fluorescence of isoprenylated Cdc42Hs-Mant-GDP. 80 nM isoprenylated Cdc42Hs was incubated in Buffer D, and Mant
fluorescence was monitored (excitation = 350 nm, emission
= 440 nm). At the indicated time (arrow), either RhoGDI
was added to 200 nM or RhoGDI
8 was added to 400
nM.
Figure 4:
Determination of the dissociation constant (K) for the binding of RhoGDI to
Cdc42Hs-Mant-GDP. A, 80 nM isoprenylated Cdc42Hs was
incubated in Buffer D and Mant fluorescence was monitored (excitation
= 350 nm, emission = 440 nm). Approximately 40 nM aliquots of RhoGDI were added at 100-s intervals. B, the
fluorescence after the addition of each aliquot was averaged and a plot
of the fluorescence changes (-
F/F
) versus RhoGDI concentration was generated. The data were fit to calculate
the K
for the interaction between RhoGDI
and Cdc42Hs-Mant-GDP as described under ``Experimental
Procedures.''
Figure 5:
Determination of the dissociation constant (K) for the binding of RhoGDI to
Cdc42Hs-Mant-GppNp. A, 70 nM isoprenylated Cdc42Hs
was incubated in Buffer D and Mant fluorescence was monitored
(excitation = 350 nm, emission = 440 nm). 30 nM aliquots of RhoGDI were added at 100-s intervals. B, the
changes in Mant fluorescence were plotted as a function of RhoGDI, and
the data were fit to determine the K
for
the interaction between RhoGDI and Cdc42Hs-Mant-GppNp, as described in
the legend for Fig. 4B.
Figure 6:
Comparison of the binding of RhoGDI, LD4,
and different GDI/LD4 chimeras to Cdc42Hs-Mant-GDP. 80 nM isoprenylated Cdc42Hs was incubated in Buffer D, and Mant
fluorescence was monitored (excitation = 350 nm, emission
= 440 nm). The changes in the Mant fluorescence were quantitated
following the addition of aliquots of RhoGDI, LD4, or Chimeras B, C,
and D. Curve fitting and K determinations
were carried out as described in the legend for Fig. 4B.
In
a previous study (Platko et al., 1995), we had constructed a
number of chimeras between RhoGDI and LD4 in order to delineate a limit
region on the RhoGDI molecule that was responsible for its higher
potency (relative to LD4) toward Cdc42Hs. We found that a chimera that
contained the first 168 amino acids of LD4 and just the last 33 amino
acids from RhoGDI (designated Chimera B) was fully active as a GDI
toward Cdc42 (i.e. it behaved like wild type RhoGDI in
functional assays). A chimera that contained the first 171 amino acids
from RhoGDI, as well as residues 172-180 (from the
carboxyl-terminal 33 residues), and the last 24 residues from LD4
(designated Chimera C) also was fully active toward Cdc42Hs, whereas a
chimera that contained the first 171 amino acids from RhoGDI, followed
by 8 amino acids from LD4, and then the final 25 amino acids from
RhoGDI (designated as Chimera D) behaved like wild type LD4. Fig. 6shows an analysis of the binding of Chimeras B (triangles), C (diamonds), and D (inverted
triangles) to Cdc42Hs-Mant-GDP. The binding profiles obtained with
these chimeras exactly corresponded to the results obtained in
functional assays (Platko et al., 1995). From the best fits to
the binding data, we determined K values of 20
nM for Chimera B, 23 nM for Chimera C, and 380 nM for Chimera D. These results strongly argue that the differences
in functional potency between RhoGDI and LD4 toward Cdc42Hs are a
direct outcome of the different binding affinities of these regulatory
proteins.
A long term goal of our laboratory has been to develop
fluorescence spectroscopic read-outs to aid in the biochemical
characterization of different proteins that regulate the
GTP-binding/GTPase cycle of Cdc42Hs. An important aim was to develop
such a read-out for the RhoGDI molecule, because it is capable of a
number of interesting regulatory activities and because we already had
obtained a good deal of structure-function information from chimera
studies of the GDI and a related molecule, LD4 (Platko et al.,
1995). In the present work, we describe a real time spectroscopic
read-out for RhoGDI interactions with Cdc42Hs bound to the fluorescent
nucleotide, Mant-GDP. We show that as an outcome of RhoGDI binding, a
significant quenching (20%) of the Mant-GDP fluorescence occurs.
The RhoGDI-induced quenching shows all the characteristics expected for
a proper interaction between this regulator and the GTP-binding
protein, namely that it is specific for the isoprenylated form of
Cdc42Hs and is not detected when using a carboxyl-terminal truncated
mutant of the GDI, which was previously shown to be incapable of
binding.
Using this fluorescence assay, one of the important
questions that we set out to address was how the binding of the RhoGDI
to the GTP-bound (activated) form of Cdc42Hs compared with its binding
to the GDP-bound (inactive) form. We found that these interactions
occur with virtually identical affinities, thus suggesting that the
ability of the RhoGDI to bind to the Cdc42Hs-GTP species and inhibit
the GTPase activity may be as important (in terms of biological
regulation) as the previously recognized binding of this regulator to
the Cdc42Hs-GDP complex. One possibility is that the RhoGDI interaction
is essential for ``shuttling'' Cdc42Hs between different
membrane locations within the cell. We recently have demonstrated that
a predominant location of the GDP-bound form of Cdc42Hs is the Golgi ()and that this location is influenced by the Arf
GTP-binding protein, which has been implicated in intracellular
trafficking. These findings, together with the biological effects that
Cdc42Hs mediates at the plasma membrane (filopodia formation) (Nobes
and Hall, 1995; Kozma et al., 1995), suggest that the
GTP-binding protein may need to cycle between these different membrane
locations. Thus, the ability of the RhoGDI to bind equally well to both
the GDP-bound and GTP-bound forms of Cdc42Hs, coupled with its ability
to release Cdc42Hs from membranes (Leonard et al., 1992), may
enable this regulatory protein to facilitate the movement of the
appropriate nucleotide-bound form of Cdc42Hs to the proper membrane
target site. However, it also is possible that as yet unidentified
cellular proteins influence the interactions between Cdc42Hs and the
RhoGDI and either enhance or inhibit its ability to bind to specific
nucleotide states of Cdc42Hs.
We have found that we can also use this fluorescence assay to directly monitor the binding of the human leukemia protein, LD4, to Cdc42Hs. The LD4 protein shows a relatively high degree of sequence identity to RhoGDI, with the sequence homology extending throughout the entire length of these molecules. Interestingly, however, we have shown that LD4 is much less effective as a GDI toward Cdc42Hs, being 10-20-fold less potent. These differences were first narrowed down to the carboxyl-terminal 33 amino acid residues of the two molecules, based on studies using GDI/LD4 chimeras to assay GDI activity toward Cdc42Hs (Platko et al., 1995). It was then shown that these differences could be attributed to as few as six amino acid differences that existed between residues 172-180 of RhoGDI and the corresponding residues 169-178 of LD4. Moreover, a single amino acid change at residue 174 of LD4 to the corresponding residue of RhoGDI could impart nearly full GDI activity to the LD4 molecule. Our present fluorescence studies now show that the differences in functional potency between the RhoGDI and LD4 can be entirely attributed to differences in their abilities to bind to Cdc42Hs. This then argues that residues 172-180 of the RhoGDI molecule are responsible for the ability of this regulatory protein to bind with high affinity to the GTP-binding protein. Because two of these residues are conserved in LD4, these results argue that as few as six amino acids are responsible for the significantly higher affinity of RhoGDI (compared with LD4) for Cdc42Hs. At present, we still do not know the identity of all of the amino acid residues on the GDI molecule and Cdc42Hs that are critical for the binding interaction between these proteins. However, we intend to use this read-out to screen a variety of mutants of GDI and the GTP-binding protein to gain a more complete picture of the contact sites that are involved. We also hope to use this read-out in conjunction with fluorescence resonance energy transfer measurements in membrane preparations to address the molecular mechanism that underlies the GDI-stimulated release of Cdc42Hs from phospholipid bilayers.