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J. Biol. Chem., Vol. 275, Issue 42, 32816-32821, October 20, 2000
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From the Program in Molecular Medicine and Department of
Biochemistry and Molecular Biology, University of Massachusetts Medical
School, Worcester, Massachusetts 01655
Received for publication, March 22, 2000, and in revised form, July 13, 2000
GRP1 and the related proteins ARNO and
cytohesin-1 are ARF exchange factors that contain a pleckstrin homology
(PH) domain thought to target these proteins to cell membranes through
binding polyphosphoinositides. Here we show the PH domains of all three proteins exhibit relatively high affinity for dioctanoyl
phosphatidylinositol 3,4,5-triphosphate
(PtdIns(3,4,5)P3), with KD
values of 0.05, 1.6 and 1.0 µM for GRP1, ARNO, and
cytohesin-1, respectively. However, the GRP1 PH domain was unique among
these proteins in its striking selectivity for
PtdIns(3,4,5)P3 versus phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P2), for which it exhibits
about 650-fold lower apparent affinity. Addition of a glycine to the
Gly274-Gly275 motif in GRP1 greatly increased
its binding affinity for PtdIns(4,5)P2 with little effect
on its binding to PtdIns(3,4,5)P3, while deletion of a
single glycine in the corresponding triglycine motif of the ARNO PH
domain markedly reduced its binding affinity for
PtdIns(4,5)P2 but not for PtdIns(3,4,5)P3. In
intact cells, the hemagglutinin epitope-tagged PH domain of GRP1 was
recruited to ruffles in the cell surface in response to insulin, as
were full-length GRP1 and cytohesin-1, but the PH domain of cytohesin-1
was not. These data indicate that the unique diglycine motif in the
GRP1 PH domain, as opposed to the triglycine in ARNO and cytohesin-1,
directs its remarkable PtdIns(3,4,5)P3 binding selectivity.
Cell signaling processes are often initiated by the recruitment of
protein complexes to the cytoplasmic face of the plasma membrane, where
they act to elicit signaling events. Specialized regions or domains
within such signaling proteins function as adapters in the recruitment
process, linking these proteins to chemical motifs generated by
receptor activation. For example, specific membrane-bound protein
phosphotyrosine sites appear in response to activation of transmembrane
receptor tyrosine kinases by growth factors and other stimuli,
attracting Src homology (SH)1
2 domains within proteins that bind these sites (1). The proteins that
contain SH2 domains are enzymes, regulator proteins, or simply adapters
themselves that connect other proteins to the localized protein
phosphotyrosines. This paradigm has been extended to a large number of
protein domains and their respective ligands, and provides for an
effective means of mobilizing cellular signaling machines (2).
A particularly interesting membrane localization domain that has been
identified in over 100 proteins is the PH domain, which spans
approximately 120 residues and contains an invariant tryptophan in its
COOH-terminal region (3). Several PH domain structures have been solved
by NMR and by x-ray crystallography, giving rise to the concept that
their overall protein fold is formed from seven The polyphosphoinositides represent diverse membrane targeting sites
for PH domains because they include PtdIns(3,4)P2 and PtdIns(3,4,5)P3, which appear upon cell stimulation by
growth factors and other regulators of PI 3-kinases, as well as
PtdIns(4,5)P2, which is present in cells constitutively at
relatively high abundance (19). Recent studies have suggested that many
PH domains appear to show little or only modest ability to discriminate
among the polyphosphoinositides with different binding affinities,
while others show striking binding affinity preferences (15). Examples of the latter include the PH domain of phospholipase C Based on the above considerations, it appears that the diversity of
phosphoinositide binding properties characteristic of PH domains
provides multiple modes by which signaling proteins containing these
domains can be localized to membranes. A plausible hypothesis is that
PH domains within a family of proteins containing a second common
function may act to confer divergent membrane targeting modalities to
this function. The aim of the present studies was to test this
hypothesis with a family of proteins related to GRP1 (20). Like GRP1,
the proteins ARNO (27) and cytohesin-1 (28) contain a single PH domain
and a sec7 homology region that catalyzes GTP/GDP exchange on ARF
proteins. The PH domains of these three proteins show high sequence
similarity, but are not identical. We found that only GRP1 among these
proteins binds the PI 3-kinase product PtdIns(3,4,5)P3 with
high selectivity over PtdIns(4,5)P2, and much of this
difference in binding specificity could be accounted for by a single
additional glycine in the NH2-terminal regions of the ARNO
and cytohesin-1 PH domains. The high specificity of the GRP1 PH domain
for binding PtdIns(3,4,5)P3 over the more abundant
PtdIns(4,5)P2 species explains the highly sensitive
membrane localization response of GRP1 to PI 3-kinase activation in
intact cells.
Generation of GST PH Fusion Proteins--
GST fusion protein
containing the GRP1 sequence from residue 240 and through the remaining
COOH-terminal part of the molecule was generated as described
(20). The analogous constructs were made of cytohesin-1 and ARNO.
Site-directed mutagenesis was performed by the Stratagene
QuickChangeTM protocol. Plasmid preparations were screened for
the desired mutations, and all constructs were completely confirmed by
DNA sequencing.
Lipid Binding and Competition Assays--
Binding assays were
performed exactly as described (14). Briefly, GST/PH domain fusion
proteins were bound to glutathione immobilized on agarose beads
(Sigma). Synthetic
[3H]C8PtdIns(3,4,5)P3 (kindly
provided by C.-S. Chen, University of Kentucky) were added, incubated
with constant agitation, and the beads were separated from the
supernatant by centrifugation after 1 h. The amounts of
[3H]C8PtdIns(3,4,5)P3 bound to
the PH domains were calculated by subtracting the amount of free
3H present in supernatants from beads bound to the GST/PH
domains from the amount of free 3H in supernatants from
beads containing only GST. To calculate apparent
KD values, the data were fitted to the equation,
[bound] = Bmax × [free]/(KD + [free]), by a least squares
curve fit. For the competition assays, the beads containing the GST
fusion proteins were incubated with 2.5 µM
[3H]C8PtdIns(3,4,5)P3 in the
presence of different concentrations of unlabeled lipids. After 1 h of incubation at room temperature, the beads were washed and counted
in a scintillation counter. The percentage of
[3H]C8PtdIns(3,4,5)P3 bound was
calculated based on the amount of 3H bound to the beads in
the absence of competitor. The total 3H bound to the GST
control was less than 0.5% of the total 3H bound to GRP1
PH domain. The data were fitted to the equation, % bound = 100 Immunofluorescence--
1 × 105 CHO-T cells
were seeded on 18-mm coverslips in 12-well tissue culture plates. The
next day, the cells were transfected with 5 ng of the indicated
constructs and 0.5 µg of empty PCMV5 vector (to assure low levels of
expression) using LipofectAMINE (Life Technologies, Inc.) under
serum-free conditions. The constructs were tagged at the COOH terminus
with either the HA (YPYDVPDYA) or Myc (AEEQKLISEEDLLK) epitope tags,
and they were detected using either an anti-HA epitope antiserum
produced in this laboratory (23) or supernatants from the 9E10.2
hybridoma (American Type Culture Collection). Cells were stimulated
with 0.1 µM insulin for 5 min or left untreated, fixed,
and processed for immunofluorescence the following day as described
(23).
In order to characterize the polyphosphoinositide binding
properties of the PH domains of GRP1 family proteins, GST fusion constructs were made for each of the GRP1, ARNO, and cytohesin-1 PH
domains. Binding of each of these GST-PH domain fusion proteins to the
PI 3-kinase product PtdIns(3,4,5)P3 was estimated by
incubation of these proteins with various concentrations of
water-soluble[3H]C8
PtdIns(3,4,5)P3. As depicted in Fig.
1, each of the PH domains studied here
bound the labeled 3'-polyphosphoinositide in a
concentration-dependent manner. Binding of
C8PtdIns(3,4,5)P3 to each of the PH domains also showed saturation kinetics, with similar stoichiometries of
association at high concentrations. Assuming a single binding site for
the polyphosphoinositide on each PH domain, the highest apparent
affinity for the labeled C8PtdIns(3,4,5)P3 was
exhibited by the GRP1 PH domain (apparent KD = 0.05 µM). However, the PH domains of ARNO and cytohesin-1
also bound this lipid species with relatively high affinity (apparent
KD = 1.6 and 1.0 µM,
respectively).
High affinity for PtdIns(3,4,5)P3 alone may not be
sufficient to assure regulated membrane localization of a PH domain in response to generation of this lipid species in intact cells through the action of PI 3-kinase. This is because the abundance of
PtdIns(4,5)P2, which is present constitutively in cell
membranes, is much higher than the 3'-polyphosphoinositides even in
stimulated cells. We thus evaluated the relative apparent affinities of
the GRP1, ARNO, and cytohesin-1 PH domains for
PtdIns(4,5)P2 by using this lipid as a competitive
inhibitor of labeled C8PtdIns(3,4,5)P3 binding. Fig. 2 depicts this type of experiment
and shows a striking difference in behavior between the GRP1 PH domain
versus the PH domains of ARNO and cytohesin-1. For the
latter two PH domains, C8PtdIns(4,5)P2 competed for binding of labeled
C8PtdIns(3,4,5)P3 almost as well as unlabeled
C8PtdIns(3,4,5)P3 itself. The concentration of
PtdIns(4,5)P2 that half-maximally inhibits labeled
PtdIns(3,4,5)P3 binding to these two PH domains is within a
few -fold of the half-maximal inhibitory concentration measured for
PtdIns(3,4,5)P3. In contrast, the GRP1 PH domain binding
profile exhibits a striking difference between these lipids in
respect to their ability to compete with labeled
C8PtdIns(3,4,5)P3. In the case of the
GRP1 PH domain, C8PtdIns(4,5)P2 is about 650 times less effective in competing for binding of labeled
C8PtdIns(3,4,5)P3 as is
C8PtdIns(3,4,5)P3.
Distinct Polyphosphoinositide Binding Selectivities for
Pleckstrin Homology Domains of GRP1-like Proteins Based on Diglycine
Versus Triglycine Motifs*
,,
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sheets with
connecting loops that form a ligand binding scaffold (4-10). The PH
domain fold is similar to that of several other ligand binding protein
domains that differ substantially in amino acid sequence, and has
therefore been denoted as the prototype of a superfamily or superfold
(11). The major class of ligands that bind most PH domains are the
polyphosphoinositides (12-15), although some PH domains do not appear
to bind these lipids (11). Other ligands have been reported to bind
some PH domains as well, including /
subunits of trimeric G
proteins (16), actin (17), and protein kinase C (18). Results from the
several PH domain structures that have been solved in association with a phosphoinositide headgroup show that the ligand binding site can vary
in location. Thus, the spectrin and phospholipase C delta PH domains
both bind inositol 1,4,5-triphosphate, but in different regions
flanking the 1/2 loop (4, 5).
, which shows
high selectivity for PtdIns(4,5)P2, and the PH domain of the ARF guanine nucleotide exchange factor GRP1, which binds
PtdIns(3,4,5)P3 with much higher affinity than
PtdIns(3,4)P2 or PtdIns(4,5)P2 (15, 20, 21).
Consistent with these binding characteristics, the phospholipase C
delta PH domain targets to cell plasma membranes under basal conditions
(22), while the GRP1 PH domain is recruited to plasma membranes only
upon PI 3-kinase stimulation (23-25). Another example of such
regulated recruitment is the activation of the protein kinases PDK1 and
Akt/protein kinase B through their membrane localization in response to
generation of 3'-phosphoinositides by PI 3-kinases (26). These results
reinforce the concept that a major mechanism for signaling through the
PI 3-kinase products PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 is the recruitment of signaling proteins to
cell membranes via binding of their PH domains.
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n × L/(KI(app) + L), where
n is the percentage of specific binding, L is the
concentration of unlabeled lipid added, and
KI(app) is the apparent competitive dissociation constant. The ratios of the apparent dissociation constants equal the
ratios of the true dissociation constants under these experimental conditions (14).
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View larger version (18K):
[in a new window]
Fig. 1.
Binding of
[3H]C8PtdIns(3,4,5)P3 to the PH
domains of GRP1, ARNO, and cytohesin-1. Approximately 150 pmol of
GST fusion proteins of the PH domains were bound to
glutathione-Sepharose and incubated with various concentrations of
[3H]C8PtdIns(3,4,5)P3, and
binding was determined (21). The measurements were fitted to the curve
[bound] = Bmax × [free](KD + [free]) using the method of
least squares. This equation assumes single binding sites. The values
are means of duplicate measurements, and the error
bars represent the range of the measurements. Values on the
ordinate are represented as % of maximal binding. The data are from
one of four experiments with essentially identical results.
View larger version (20K):
[in a new window]
Fig. 2.
Competition of binding
[3H]C8PtdIns(3,4,5)P3 by
unlabeled dioctanoyl phosphoinositides. GST fusion proteins of the
PH domains immobilized on glutathione-Sepharose were incubated with
various concentrations of unlabeled
C8PtdIns(4,5)P2 or
C8PtdIns(3,4,5)P3 together with the
radioligand. The measurements were fitted to the equation % bound = 100 n × L(KI(app)) + L), where
n is the percentage of specific binding, L is the
concentration of unlabeled lipid added, and
KI(app) is the apparent competitive dissociation
constant. The values are means of duplicate determinations, and the
error bars represent the range of the
measurements. The data are from one of three experiments with
essentially identical results.
These data for the GRP1 PH domain are consistent with those reported by Kavran et al. (15), who showed a difference of greater than 2 orders of magnitude for its binding to the head group of PtdIns(3,4,5)P3 versus that for PtdIns(4,5)P2. Taken together with our results, we can conclude that the properties of the PH domains of the GRP1 family proteins are strikingly divergent in respect to their affinities for PtdIns(4,5)P2, while their affinities for the PI 3-kinase product PtdIns(3,4,5)P3 are reasonably similar. These data raise two interesting questions. First, what is the structural basis for the diverse binding properties of the GRP1 versus ARNO and cytohesin-1 PH domains? Second, is there a functional difference in the activities of these PH domains that can be observed in intact cells, based on the high abundance of PtdIns(4,5)P2 relative to PtdIns(3,4,5)P3?
We addressed the structural basis of the PtdIns(4,5)P2
binding affinity differences revealed in Fig. 2 by examining the PH domain amino acid sequences derived from mouse GRP1 (20), human ARNO
(29) obtained from the ATCC and sequenced by us, and mouse cytohesin-1
(30). Interestingly, while there are 18 locations containing residues
that differ in at least one of the three PH domains, only one of these
changes occurs within the first 60 residues of the
NH2-terminal region (Fig. 3).
This one change is a deletion of a single glycine in the GRP1 PH domain
relative to the two others, and it occurs near the predicted 1/2
loop region known to bind polyphosphoinositide in other PH domain
structures. A mutant GRP1 PH domain cDNA was thus engineered that
contains an additional glycine in position 274, converting the normal
diglycine at that position to triglycine. Conversely, a mutant ARNO PH
domain was engineered in which the corresponding glycine was deleted, converting the triglycine motif to diglycine. Fig.
4 (left panels) shows the consequences of these mutations in the GRP1 and ARNO PH
domains with respect to binding
[3H]C8PtdIns(3,4,5)P3. Mutant,
triglycine GRP1 PH domain exhibits a severalfold lower affinity for the
labeled 3'-polyphosphoinositide compared with native GRP1 PH domain,
whereas mutant, diglycine ARNO PH domain shows an enhanced affinity
compared with the native construct. In the case of the mutant GRP1 PH
domain, the binding profile appears to be converted almost exactly to
that characteristic of ARNO and cytohesin-1 (compare Figs. 1 and 4).
These data are consistent with the hypothesis that the unique diglycine
motif in GRP1 directs its higher affinity binding to
PtdIns(3,4,5)P3 as compared with that observed for ARNO and
cytohesin-1, which display a third glycine in this position.
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Additional experiments were conducted to evaluate the role of the diglycine in the GRP1 PH domain relative to its unique low affinity for PtdIns(4,5)P2 (i.e. high selectivity for PtdIns(3,4,5)P3). Fig. 4 (upper right panel) reveals that the mutant, triglycine GRP1 PH domain displays an apparent increase in its affinity for PtdIns(4,5)P2 compared with the native protein. This conclusion is based on the enhanced ability of this lipid to compete with labeled PtdIns(3,4,5)P3 for binding to the mutant protein compared with the native GRP1 PH domain. Thus, the relative apparent KI values for PtdIns(3,4,5)P3 versus PtdIns(4,5)P2 in these experiments with mutant GRP1 PH domain are different by only about 34-fold versus about a 650-fold difference for the native protein. Conversely, deletion of a glycine from the triglycine motif of ARNO PH domain leads to a much enhanced selectivity for binding PtdIns(3,4,5)P3, due to a 34-fold decrease in affinity for PtdIns(4,5)P2 (Fig. 4, lower right panel). Thus, this mutation dramatically increases the divergence between apparent KI values for PtdIns(3,4,5)P3 versus PtdIns(4,5)P2 (2.3-fold for native protein, 43-fold for mutant). The apparent KI values obtained in these experiments are presented in Table I. Taken together, these results indicate that the presence of two versus three glycines in the PH domains of these proteins contributes greatly to the divergent selectivity of these domains for PtdIns(3,4,5)P3. Two glycines positioned in the native GRP1 PH domain (compared with the three in native ARNO or cytohesin-1) would appear to promote a structure that both slightly enhances affinity for PtdIns(3,4,5)P3 and to a larger extent decreases affinity for PtdIns(4,5)P2. Based on solved structures of PH domains associated with PtdIns(4,5)P2 or PtdIns(3,4,5)P3, it is difficult to predict what structural elements might be positioned to enhance binding of a triphosphate while at the same time decreasing affinity for the corresponding diphosphate. Nonetheless, this is what the structure of the GRP1 PH domain achieves when compared with the ARNO and cytohesin-1 PH domain structures. Understanding the molecular basis for this effect will necessarily await solving the three-dimensional structure of the GRP1 PH domain.
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It is noteworthy that a previous study has claimed that the ARNO PH domain, like that of GRP1, is highly selective in its binding to PtdIns(3,4,5)P3 versus PtdIns(4,5)P2 (31). This may relate to a difference in amino acid sequence in the ARNO PH domain used in that work compared with the present study. In this regard, human ARNO has been reported to contain either the diglycine (27) or triglycine (29) motifs, based on its independent isolation and sequencing by different laboratory groups. As shown in Fig. 3, we have employed a native ARNO PH domain construct that displays the triglycine motif. Interestingly, mouse cytohesin-1 has also been reported to contain either a diglycine (32) or triglycine (30) by different laboratory groups. Even more remarkable is the recent observation by Vaughan and colleagues (33) that cDNA from the same sample of human brain contained sequences coding for both the di- and triglycine motifs of ARNO, cytohesin-1 and GRP1. The ratios varied such that approximately 80% of GRP1 was found as the diglycine variant, whereas the others were predominantly in the triglycine forms. It is possible that different splicing variants or different alleles of these proteins are expressed in human and mouse tissues that differ in the presence or absence of this single glycine residue in the PH domain. This would provide heterogeneity of phosphoinositide binding to otherwise identical proteins.
We next addressed the second question posed above related to the
functional consequences of differential selectivity of binding PtdIns(3,4,5)P2 and PtdIns(4,5)P2 by the PH
domains of GRP1 versus ARNO and cytohesin-1. First,
experimental conditions were developed in which membrane localization
of the GRP1 PH domain in intact cells could be demonstrated to depend
on its ability to bind PtdIns(3,4,5)P3. These studies are
based on previous work by others (24, 25) as well as our laboratory
(23) showing recruitment of either full-length GRP1 or its PH domain to
ruffles in plasma membranes of cells stimulated with insulin or growth
factors. Ruffles appear to be sites in the plasma membrane containing
the machinery for production of particular high levels of
PtdIns(4,5)P2 and PtdIns(3,4,5)P3 (19).
Such recruitment is dependent on PI 3-kinase and is abolished by
inhibitors of the enzyme. In the course of evaluating several mutant
GRP1 PH domain constructs, we discovered that substituting alanine for
lysine 273 virtually eliminated binding to
[3H]C8PtdIns(3,4,5)P3 (Fig.
5A). HA epitope-tagged native
and mutant K273A GRP1 PH domains were expressed in CHO-T cells and the
cells incubated without or with insulin. The cells were then evaluated by immunofluorescence microscopy following staining with primary polyclonal anti-HA antibody and secondary fluorescein
isothiocyanate-labeled anti-rabbit antibody. Fig. 5B shows a
cytoplasmic distribution of native HA-tagged GRP1 PH domain in control
cells and a clear recruitment of this protein to membrane ruffles upon
treatment of the cells with insulin. As found previously (23-25),
membrane localization of the GRP1 PH domain in response to insulin was abolished by the PI 3-kinase inhibitor wortmannin (data not shown). The
mutant K273A PH domain also displays a cytoplasmic disposition under
basal conditions, but fails to respond to insulin (Fig. 5, A
and B). These data demonstrate that under these experimental conditions the ability of the GRP1 PH domain to localize to plasma membranes requires the ability to bind PtdIns(3,4,5)P3.
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Comparative studies of this type were then conducted using HA-tagged
constructs of the PH domains of GRP1, which shows high selectivity for
binding PtdIns(3,4,5)P3, and of Myc epitope-tagged cytohesin-1, which exhibits reasonably high affinity for both PtdIns(3,4,5)P3 and PtdIns(4,5)P2. Fig.
6 shows that, in these experiments, the
PH domain of cytohesin-1 displays a cytoplasmic disposition whether or
not insulin is added to CHO-T cells. This failure of the cytohesin-1 PH
domain to localize to membranes occurs under conditions identical to
those in which the GRP1 PH domain is recruited to membrane ruffles in
response to insulin (Figs. 5 and 6). Interestingly, under these
same conditions, full-length native cytohesin-1 also presents a
cytoplasmic localization in the absence of insulin, but is markedly
translocated to membrane ruffles in response to the hormone as is
full-length GRP1 (Fig. 6). Recruitment of full-length cytohesin-1 to
membrane ruffles in response to insulin is fully inhibited by
wortmannin (data not shown), indicating this recruitment requires one
or more lipid products of PI 3-kinase.
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Taken together, these data indicate that, in the case of GRP1, its PH domain is sufficient to explain regulated membrane localization, whereas the PH domain cannot account for this property of cytohesin-1. Another domain of cytohesin-1 such as its coiled coil region or its sec7 domain must direct its regulated membrane targeting. This may occur either directly or indirectly through binding to another cellular component which is responsive to PI 3-kinase activation. It is likely that the same is the case for ARNO, which like cytohesin-1 also binds both PtdIns(3,4,5)P3 and PtdIns(4,5)P2 (Fig. 2).
It is important to note that the conditions of these experiments
depicted in Figs. 5 and 6 are critical to obtaining the results described. Most important is that the HA-tagged constructs expressed in
the CHO-T cells are present at low levels, which is achieved by using
low concentrations of expression vector during transfection of cells.
At high expression levels, significant membrane targeting of all
constructs shown in Fig. 6 is observed (data not shown). This is
presumably due to the action of lower affinity interactions that are
promoted by the high protein concentrations. A report (34) showing
regulated membrane targeting of the cytohesin-1 PH domain in intact
cells may be due to higher expression levels or the different cell type
used. In addition, whereas localization in the present study was
determined after fixation using PH constructs with short sequence tags,
the previous report employed GFP fusion proteins in live cells. It is
interesting that the PH domain of cytohesin-1 does not display a
definitive plasma membrane disposition in unstimulated cells (Fig. 6),
given its reasonable affinity for PtdIns(4,5)P2 (Fig. 2)
and the high abundance of this lipid in plasma membranes. In this
regard, the cytohesin-1 PH domain is unlike that of phospholipase C,
which does localize to plasma membranes of unstimulated cells (22). The
cytohesin-1 PH domain is thus similar to some other PH domains,
including those in -adrenergic receptor kinase 1 and pleckstrin
itself, which do not discriminate greatly between binding
PtdIns(4,5)P2 and PtdIns(3,4,5)P3, but also do
not localize to plasma membranes in intact cells (15). This is likely
due to the somewhat lower affinity that these three PH domains exhibit
for PtdIns(4,5)P2 compared with the PH domain of
phospholipase C.
The data presented here show that the PH domains of the GRP1 family
proteins GRP1, ARNO, and cytohesin-1 are heterogeneous with respect to
their relative binding affinities for the polyphosphoinositides PtdIns(3,4,5)P3 and PtdIns(4,5)P2. The results
are consistent with the hypothesis that this heterogeneity functions to
confer diverse membrane localization mechanisms to these ARF exchange factors. Thus, the PH domain of GRP1 is sufficient to respond to the
presence of the PI 3-kinase product PtdIns(3,4,5)P3 in cell
membranes, while the PH domains of ARNO and cytohesin-1 are not. These
latter proteins may employ their PH domains in conjunction with other
membrane localization motifs to affect recruitment to cell membranes.
Thus, these PH domains may be necessary but not sufficient for membrane
targeting. It is also possible that the GRP1 versus ARNO and
cytohesin-1 PH domains function in the localization of these proteins
to distinct plasma membrane microdomains not resolved by the microscopy
methodology employed here or in other studies to date. Further
refinement of imaging techniques will be required to test this idea. In
any case, our results indicate that a single glycine in the amino acid
sequences of the ARNO and cytohesin-1 PH domains, not present in GRP1,
contributes greatly to the heterogeneity of phosphoinositide binding
and thus to the divergent membrane localization properties of these proteins.
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ACKNOWLEDGEMENTS |
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We thank Dr. David Lambright for helpful discussions and critical reading of this manuscript. We appreciate the excellent assistance of Jane Erickson in preparation of the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK30648 (to M. P. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St., Worcester, MA 01605. Tel.: 508-856-2254; Fax: 508-856-1617; E-mail: michael.czech@umassmed.edu.
Published, JBC Papers in Press, July 25, 2000, DOI 10.1074/jbc.M002435200
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ABBREVIATIONS |
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The abbreviations used are: SH, Src homology; PH, pleckstrin homology; PtdIns(3, 4)P2, phosphatidylinositol 3,4-diphosphate; PtdIns(4, 5)P2, phosphatidylinositol 4,5-diphosphate; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-triphosphate; CHO-T, Chinese hamster ovary cells expressing human insulin receptors; HA, hemagglutinin; GST, glutathione S-transferase; PI, phosphatidylinositol.
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