From the Program in Molecular Medicine and Department of
Biochemistry and Molecular Biology and § Department of Cell
Biology, University of Massachusetts Medical Center, Worcester,
Massachusetts 01605 and the Department of Medicine,
Division of Signal Transduction, Beth Israel Deaconess Medical Center
and Department of Cell Biology, Harvard Medical School,
Boston, Massachusetts 02215
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ABSTRACT |
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Cellular levels of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) are rapidly elevated in response to activation of growth factor receptor tyrosine kinases. This polyphosphoinositide binds the pleckstrin homology (PH) domain of GRP1, a protein that also contains 200 residues with high sequence similarity to a segment of the yeast Sec7 protein that functions as an ADP ribosylation exchange factor (ARF) (Klarlund, J., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that dioctanoyl PtdIns(3,4,5)P3 binds the PH domain of GRP1 with a Kd = 0.5 µM, an affinity 2 orders of magnitude greater than dioctanoyl-PtdIns(4,5)P2. Further, the Sec7 domain of GRP1 is found to catalyze guanine nucleotide exchange of ARF1 and -5 but not ARF6. Importantly, PtdIns(3,4,5)P3, but not PtdIns(4,5)P2, markedly enhances the ARF exchange activity of GRP1 in a reaction mixture containing dimyristoylphosphatidylcholine micelles, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and a low concentration of sodium cholate. PtdIns(3,4,5)P3-mediated ARF nucleotide exchange through GRP1 is selectively blocked by 100 µM inositol 1,3,4,5-tetrakisphosphate, which also binds the PH domain of GRP1. Taken together, these data are consistent with the hypothesis that selective recruitment of GRP1 to PtdIns(3,4,5)P3 in membranes activates ARF1 and -5, known regulators of intracellular membrane trafficking.
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INTRODUCTION |
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The phosphoinositide 3-kinase (PI
3-kinase)1 enzymes represent
a major pathway by which biological signaling systems operate to
control cell functions (1-4). Many cell surface receptor tyrosine kinases (5, 6) as well as certain GTP-binding protein-linked receptors
(7, 8) acutely activate cellular PI 3-kinase activity, leading to the
generation of 3-polyphosphoinositides. Significant amounts of
phosphatidylinositol 3-phosphate (PtdIns(3)P) are present in
unstimulated cells, whereas phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol
3,4,5-trisphosphate (PtdIns(3,4,5)P3) are specifically
produced upon cell surface receptor activation (6). A large number of
biological processes have been implicated as targets of regulation by
this pathway, including membrane ruffling, chemotaxis, secretion,
insulin-stimulated glucose uptake, cell adhesion, cell growth, and
apoptosis as well as the regulation of early endosome structure
(7-13).
Recent work in this laboratory identified a potential effector protein,
GRP1, containing a PH domain that binds PtdIns(3,4,5)P3 but not Ptd(3,4)P2 or PtdIns(3)P (14). GRP1 also contains a 200-amino acid sequence with high similarity to a domain in the yeast
Sec7 protein, which is known to be required for transport of
polypeptides from the endoplasmatic reticulum to and through Golgi
membranes (15). A similar Sec7 domain in the mammalian proteins
cytohesin-1 and ARNO and in the yeast protein Gea1 were reported to
catalyze guanine nucleotide exchange on the small GTP-binding protein
ARF1 (16-18). ARF proteins in the GTP-bound form promote membrane
vesicle trafficking pathways by recruiting coat proteins to membranes,
causing membrane budding (19-21). A potential interaction between PI
3-kinase and ARF mutants had been suggested based on characteristic
changes of endosome morphology elicited by wortmannin in various
cultured cell lines (12). Taken together, these observations provide a
framework for the hypothesis that GRP1 or its homologs may mediate
cellular effects of 3-polyphosphoinositides that require ARF proteins.
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EXPERIMENTAL PROCEDURES |
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Recombinant GRP1 Protein--
To map the active regions of GRP1,
GST proteins fused to various domains of GRP1 were purified from
lysates of bacteria expressing the relevant pGEX5X-3 constructs as
described (14). For other experiments, GRP1 was cloned into pGEX-4T,
and the recombinant protein was purified. The GST-GRP1 fusion protein
was cleaved by incubation with 5 µg/ml thrombin in 200 µl of 20 mM Tris, pH 8.0, 2.5 mM CaCl2, 150 mM NaCl overnight at 4 °C. Complete cleavage was
verified by SDS-polyacrylamide gel electrophoresis. The proteins were
transferred to assay buffer (50 mM HEPES, pH 7.5, 1 mM MgCl2, 100 mM KCl, 1 mM dithiothreitol) using Centricon 30 microconcentrators (Amicon) and stored at 20 °C in 50% glycerol.
ARF Exchange Assay--
Recombinant baculovirus encoding ARF1,
-5, or -6 (ARF cDNAs kindly provided by Dr. R. Klausner) fused at
the C terminus to a 9-amino acid sequence corresponding to the major
antigenic determinant of influenza virus hemagglutinin was constructed.
Sf9 cells were infected with the recombinant baculovirus, the
cells were harvested 3 days later by centrifugation for 5 min at 3000 rpm, and the pellets were stored at 70 °C. A cell pellet
corresponding to 50 ml of culture medium was dissolved in 1 ml of assay
buffer supplemented with 1% Triton X-100, 1 mM
benzamidine, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride, and 50 µM GDP. After
clarification by centrifugation at 20,000 rpm for 5 min, 50 µl of a
rabbit antiserum that had been produced by immunization with a peptide
(YPYDVPDYA) conjugated to hemocyanin was added and incubated overnight
on ice. The following day, 75 µl of Protein A conjugated to Sepharose
CL-4B (Sigma) was added and incubated on an end-over-end mixer
for 1 h. The beads were collected by centrifugation and washed
five times with 1 ml of assay buffer. An additional 500 µl of
Sepharose CL-4B was added as a carrier. Analysis of bound ARF from
Sf9 cells labeled with [3H]myristic acid by
Western blotting and autoradiography revealed that at least 90% of the
ARF molecules was modified by the lipid (22).
Lipid Binding and Competition Assays--
Binding assays were
performed as described (23). Briefly, GST fused to the PH region of
GRP1 (amino acids 239-399) was bound to glutathione immobilized on
agarose beads (Sigma). Synthetic 3H-labeled dioctanoyl
PtdIns(3,4,5)P3 (kindly provided by C.-S. Chen (24)) was
added, and after 1 h the beads were separated from the supernatant
by centrifugation. The amount of
[3H]PtdIns(3,4,5)P3 bound to the PH domain
was calculated by subtracting the amount of free 3H present
in supernatants from GST-GRP1 from the amount of free 3H in
supernatants from control GST. The data were fitted to the equation,
[bound] = Bmax × [free]/(KD + [free]), by least squares curve
fit. For the competition assays the beads containing the GST fusion
proteins were incubated with 2.5 µM
3H-labeled dioctanoyl PtdIns(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 with
buffer containing 0.5% Nonidet P-40 and counted in a scintillation
counter. The percentage of
[3H]PtdIns(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 n × L/(KI(app) + L),
where n is the percent 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 accurately reflect the ratios of the true dissociation
constants under the present experimental conditions (23).
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RESULTS |
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To test whether GRP1 functions as an ARF guanine nucleotide
exchange factor, hemagglutinin-tagged ARF1, -5, or -6 proteins produced
in Sf9 cells were incubated with recombinant GRP1 protein in the
presence of [35S]GTPS for various times prior to
determination of ARF-bound label. Fig. 1
shows a nearly linear rate of labeled guanine nucleotide binding to
these ARF proteins during this time course in the presence or absence
of GRP1. A 4-10-fold stimulation of ARF1 binding to [35S]GTP
S was observed in response to GRP1, whereas no
effect of GRP1 on ARF6 binding to nucleotide was detected. Up to 10 molecules of [35S]GTP
S could be bound to ARF1 per
molecule of GRP1 under our experimental conditions, showing that GRP1
acts catalytically. Guanine nucleotide exchange on ARF5 was also
significantly enhanced by GRP1, although to a lesser extent than that
for ARF1. Fig. 1D indicates that GRP1 maximally activates
labeled nucleotide binding to ARF1 and -5 when present at 2.5 µM under the conditions of this assay.
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Because the Sec7 homology domains of the related protein ARNO (16) have been shown to be sufficient for catalysis of guanine nucleotide exchange of ARF1, we analyzed the ARF1 exchange activity intrinsic to each of three segments of GRP1, including the Sec7 homology region (Fig. 2). GST fusion proteins of the N terminus and PH regions of GRP1 exhibited no effect on ARF1 binding to labeled nucleotide in our assay. In contrast, a GST fusion protein containing the Sec7 homology domain was as effective as the full-length GRP1 in catalyzing ARF1 exchange activity (Fig. 2).
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GRP1 was initially identified based on its high affinity binding to
PtdIns(3,4,5)P3 through its PH domain (14). We therefore questioned whether this polyphosphoinositide might regulate the ARF
exchange activity of GRP1. Fig. 2 shows that
PtdIns(3,4,5)P3 stimulated binding of
[35S]GTPS to ARF in a dose-dependent
manner. Half-maximal stimulation was seen at approximately 1 µM PtdIns(3,4,5)P3, and maximal activation was 3-4-fold. Importantly, PtdIns(3,4,5)P3 had no effect
on the ARF exchange activity catalyzed by the GRP1 Sec7 homology
domain, previously shown not to bind the polyphosphoinositide (14).
The data depicted in Fig. 2 suggest the hypothesis that receptor signaling through PI 3-kinases, which generates PtdIns(3,4,5)P3, might specifically regulate ARF proteins through GRP1. To further test this concept, the relative binding affinities of PtdIns(3,4,5)P3 versus PtdIns(4,5)P2, which is constitutively present in cell membranes, for the PH domain of GRP1 were determined. A GST fusion protein of the PH domain of GRP1 was incubated with various concentrations of synthetic [3H]dioctanoyl PtdIns(3,4,5)P3, and binding was determined (Fig. 3A). These experiments show high affinity binding of this polyphosphoinositide, with a calculated Kd = 0.5 µM, assuming one binding site per GRP1 molecule. Fig. 3B reveals that PtdIns(3,4,5)P3 competes for [3H]dioctanoyl PtdIns(3,4,5)P3 binding to the PH domain of GRP1 with a KI(app) that is 10-20-fold lower than for PtdIns(4,5)P2. Comparison of the competition profiles for these two polyphosphoinositides containing identical fatty acyl side chains (dioctanoyl) showed that PtdIns(3,4,5)P3 had a 50-100-fold higher affinity when compared with PtdIns(4,5)P2. PtdIns(3,4)P2 had also a 50-fold lower affinity than PtdIns(3,4,5)P3 (Fig. 3B).
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The data depicted in Fig. 3 suggested that high specificity of
PtdIns(3,4,5)P3-mediated activation of ARF exchange may
characterize GRP1 function. We tested the specificity of
GRP1-stimulated ARF exchange by these phosphoinositides under
conditions of various charge densities in detergent-phosphatidylcholine
micelles (Fig. 4). Interestingly, when a
relatively high concentration of negative charge is present in
the micelles (0.1% cholate), PtdIns(3,4,5)P3, PtdIns(4,5)P2, and PtdIns(3,4)P2 all stimulated
ARF1 exchange activity in the presence of GRP1. In the absence of
charge (0.1% CHAPS, no cholate), none of these phosphoinositides were
able to enhance the ARF exchange activity of GRP1 (Fig. 4). In
contrast, PtdIns(3,4,5)P3 selectively stimulated
[35S]GTPS binding to ARF1 in the presence of GRP1 when
a low level of negative charge (0.05% cholate) was present with the
phosphatidylcholine in our assay. Under these conditions
PtdIns(4,5)P2 and PtdIns(3,4)P2 had
significantly less effect. These experiments establish an in vitro assay system that reveals selective regulation of
GRP1-catalyzed ARF1 guanine nucleotide exchange activity by
PtdIns(3,4,5)P3.
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Previous results (16, 25, 26) and data in Fig. 4 suggest that guanine nucleotide exchange of ARF proteins require an interface of ARF, exchange factor, and negatively charged phospholipid, indicating that recruitment of GRP1 to the phospholipid membrane may be a key element of the regulation of ARF guanine nucleotide exchange. Thus we reasoned that Ins(1,3,4,5)P4, the polar head group of PtdIns(3,4,5)P3, should compete for binding and recruitment of GRP1 to PtdIns(3,4,5)P3 in micelles, thus blocking GRP1-mediated ARF1 exchange. Fig. 5 shows results that are consistent with this prediction. Addition of 100 µM Ins(1,3,4,5)P4 to our assay virtually ablated the increased ARF1 guanine nucleotide exchange activity catalyzed by GRP1 in response to PtdIns(3,4,5)P3. In concert with data in Fig. 4 indicating high binding specificity of the GRP1 PH domain, other polar head groups tested, including Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,6)P4, and Ins(1,2,5,6)P4, failed to inhibit GRP1-mediated ARF1 guanine nucleotide exchange (Fig. 5).
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DISCUSSION |
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The results presented here demonstrate the specific binding and regulation of GRP1-catalyzed ARF1 guanine nucleotide exchange by PtdIns(3,4,5)P3, a product of receptor-regulated PI 3-kinase activity (Figs. 2, 4, and 5). These experiments (Fig. 2) also localize ARF exchange activity to an approximately 200-residue segment of the GRP1 protein that exhibits high sequence similarity to a region of the Saccharomyces cerevisiae Sec7 protein (27). GRP1 was found to catalyze nucleotide exchange on ARF1 and -5 but not ARF6 (Fig. 1). Interestingly, ARF5 was reported not to be a substrate for cytohesin-1 (17), but a detailed description of the specificities of the various isoforms of GRP1 is still lacking.
The identification of GRP1 based on its binding to
PtdIns(3,4,5)P3 in an expression cDNA library screen
suggested a particularly high affinity for this polyphosphoinositide
(14). Our present results confirm this expectation, revealing a
Kd = 0.5 µM for binding
[3H]dioctanoyl PtdIns(3,4,5)P3 (Fig. 2).
Importantly, the PH domain of GRP1 exhibits an apparent affinity for
dioctanoyl PtdIns(4,5)P2 that is about 2 orders of
magnitude lower than that for dioctanoyl PtdIns(3,4,5)P3
(Fig. 2). This extraordinary specificity for the 3-polyphosphoinositide is a distinctive characteristic of the GRP1
PH domain among the many PH domains that have been characterized for
phosphoinositide binding (28). Furthermore, this degree of specificity
is consistent with that required of a protein regulated in intact cells
by PtdIns(3,4,5)P3 signaling, given that
PtdIns(4,5)P2 is much more abundant in cell membranes (6).
Taken together, the data in Figs. 2-5 provide strong support for the
hypothesis that GRP1 functions in intact cells in conjunction with
membrane-localized PtdIns(3,4,5)P3 generated by
receptor-activated PI 3-kinase activity.
Particular attention in this study was focused on the relationship
between the specificity of PtdIns(3,4,5)P3 binding to GRP1 and the specificity of PtdIns(3,4,5)P3-mediated activation
of GRP1 exchange activity for ARF1 (Figs. 2 and 4). Previous work has
emphasized the requirements for ARF myristoylation and phospholipid to
obtain optimal nucleotide exchange rates for ARF proteins, suggesting
exchange occurs at the membrane surface (26). In the case of ARNO,
PtdIns(4,5)P2 was found to be sufficient for its
recruitment to membranes as well as activation of ARF exchange (16),
suggesting specificity of its PH domain for binding this phosphoinositide or a nonspecific effect of PtdIns(4,5)P2
to recruit ARNO to membranes based on a charge effect, or both. All
three polyphosphoinositides tested were effective in stimulating
GRP1-mediated exchange activity when a high charge density was present
on micelles in the assay (Fig. 4), indicating that GRP1 can be
nonspecifically bound to membranes under these conditions. Selectivity
of PtdIns(3,4,5)P3 in stimulating ARF1 nucleotide exchange
by GRP1 over that observed for PtdIns(4,5)P2 or
PtdIns(3,4)P2 was revealed at lower charge density (Fig.
4). These data are consistent with the hypothesis that binding of GRP1
to membranes containing ARF may include two components: specific
interaction with PtdIns(3,4,5)P3 through its PH domain and
interactions with acidic phospholipids through one or more
clusters of its basic amino acid residues. At concentrations of GRP1
that yield maximal [35S]GTPS loading of ARF1, no
stimulation by PIP3 was observed, consistent with the
concept that activation of GRP1 exchange activity results from
recruitment to membranes (data not shown).
The high activity observed for ARF1 guanine nucleotide exchange catalyzed by the GRP1 Sec7 domain suggests that ARF1 function may be closely related to the physiological role of GRP1 (Fig. 1). ARF1 has been implicated in vesicle transport related to Golgi membrane function as well as in secretory and exocytosis pathways (19, 21). It is noteworthy that several targets of receptor-activated PI 3-kinase signaling involve intracellular membrane-trafficking systems, including mast cell secretion and insulin-sensitive GLUT4 glucose transporter translocation to the plasma membrane in muscle and adipocytes (9, 11, 29). Thus it is tempting to hypothesize a role for GRP1 in regulating such processes in response to localized synthesis of PtdIns(3,4,5)P3.
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ACKNOWLEDGEMENTS |
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Numerous helpful discussions with Dr. David Lambright are greatly appreciated. We thank Kristin Kwan for excellent technical assistance and Jane Erickson for expert assistance in the preparation of this manuscript. Dr. Andrew Cherniack provided expert assistance in preparation of the figures.
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FOOTNOTES |
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* This work was supported by Grants KD30898 (to M. P. C.), DK30648 (to M. P. C.), and DK40330 (to S. C.) from the National Institutes of Health.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.
¶ To whom correspondence should be addressed. Tel.: 508-856-2254; Fax: 508-856-1617; E-mail: Michael.Czech{at}banyan.ummed.edu.
1
The abbreviations used are: PI 3-kinase,
phosphoinositide 3-kinase; PtdIns, phosphatidylinositol; PH, pleckstrin
homology; ARF, ADP ribosylation factor; GST, glutathione
S-transferase; GTPS, guanosine
5
-O-(thiotriphosphate); CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PIP3, phosphatidylinositol trisphosphate.
2 J. K. Klarlund and M. P. Czech, unpublished observations.
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REFERENCES |
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