1 Laboratoire de la Dynamique de la Membrane et du Cytosquelette, UMR 144,
Centre National de la Recherche Scientifique, Institut Curie, Section
Recherche. 26 rue d'Ulm, 75241 Paris Cedex 5, France
2 Centre d'Immunologie INSERM/CNRS de Marseille-Luminy, Case 906, 13288
Marseille Cedex 9, France
3 Institut de Pharmacologie Moléculaire et Cellulaire, UPR411, CNRS, 660
route des Lucioles, Sophia-Antipolis, 06650 Valbonne, France
4 CNRS UMR 5539, Université Montpellier II, 34095 Montpellier Cedex 5,
France
* These authors contributed equally
Author for correspondence (e-mail:
philippe.chavrier{at}curie.fr
)
Accepted 1 May 2002
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Summary |
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Key words: ADP-ribosylation factor 6, Sec7 domain, Actin cytoskeleton, Endocytosis, Guanine nucleotide exchange factor
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Introduction |
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As with other Ras-related G proteins, ARFs operate as binary switches: ARF
with GDP is inactive, and replacement of bound GDP by GTP, which is catalysed
by guanine nucleotide exchange factors (GEFs), produces active GTP-ARF. We
recently reported the identification of EFA6 (Exchange factor for ARF6), a GEF
that activates ARF6 in vitro and, like ARF6, is involved in membrane recycling
and actin cytoskeleton remodelling (Franco
et al., 1999). EFA6, like all the ARF GEFs identified so far,
contains a 200 amino-acid domain, called the Sec7 domain, which is sufficient
for exchange factor activity. Structural and biochemical data have shown that
Sec7 domains harbour an
-helical structure forming a hydrophobic groove
in which the switch regions of ARF insert
(Béraud-Dufour et al.,
1998
; Cherfils et al.,
1998
; Goldberg,
1998
; Mossessova et al.,
1998
). In addition to EFA6, members of the ARNO/cytohesin
Sec7-domain-containing GEF family also activate ARF6, although they appear to
be more efficient for class I ARFs (Franco
et al., 1998
; Langille et al.,
1999
; Macia et al.,
2001
; Venkateswarlu and
Cullen, 2000
). Recently, a third ARF6 GEF has been identified,
termed ARF-GEP100, showing limited homology with the Sec7 domain of
EFA6 and ARNO (Someya et al.,
2001
). Although reasons for such diversity appear unclear, it
should be noticed that these GEFs differ in their expression profiles. EFA6
expression is restricted to the brain
(Perletti et al., 1997
),
whereas ARNO/cytohesin GEFs and ARF-GEP100 appear to be broadly
expressed (Kolanus et al.,
1996
; Someya et al.,
2001
).
ARF6 GEFs can also be distinguished at the level of their intracellular
distribution and regulation of membrane attachment. EFA6, ARNO and possibly
ARF-GEP100 contain a pleckstrin homology (PH) domain located
C-terminally to the Sec7 domain (Chardin et
al., 1996; Perletti et al.,
1997
; Someya et al.,
2001
). The PH domain is a protein module of
120 amino acids
found in many cytoskeletal and signaling proteins
(Rebecchi and Scarlata, 1998
).
In the case of EFA6 and ARNO family members, this PH domain is required for
localization to the plasma membrane
(Franco et al., 1999
;
Venkateswarlu et al., 1998
).
Overexpressed ARNO3/GRP1 is mostly cytosolic and translocates transiently to
the plasma membrane in response to cell activation with various agonists
(Gray et al., 1999
;
Venkateswarlu et al., 1998
).
It appears that the GRP1-PH domain [especially in its diglycine form
(Cullen and Chardin, 2000
;
Klarlund et al., 2000
)] has a
higher (650-fold) selectivity for phosphatidylinositol 3,4,5-triphosphate
[PtdIns(3,4,5)P3] over phosphatidylinositol
4,5-biphosphate [PtdIns(4,5)P2]. This specificity,
together with the fact that agonist-stimulated
PtdIns(3,4,5)P3 accumulation is rapid and transient, may
provide a molecular basis for the regulation of ARNO/cytohesin family GEF
association with the plasma membrane. In contrast to ARNO/cytohesin proteins,
overexpressed EFA6 remains mostly bound to the plasma membrane
(Franco et al., 1999
),
suggesting that the binding specificity of EFA6-PH domain is different.
Whether EFA6-PH domain interacts with phosphoinositide(s), protein(s) or both
is presently unknown. Finally, ARF-GEP100 colocalizes partially
with the early endosomal marker EEA-1, but in contrast to ARNO and EFA6,
ARF-GEP100 it could not be detected at the plasma membrane
(Someya et al., 2001
).
Therefore, different agonists acting through distinct signalling pathways
could promote ARF6 activation through the activity of EFA6, ARNO or
ARF-GEP100 and may lead to distinct cellular responses.
Understanding how ARF6 GEFs control ARF6 activity would help to clarify the
function of ARF6.
Expression of ARNO or EFA6 results in actin cytoskeleton reorganization,
probably as a consequence of ARF6 activation
(Franco et al., 1999;
Frank et al., 1998b
). However,
deletion of the EFA6 Sec7 domain or substitution of the conserved glutamate
residue (position 242) by a lysine, resulting in a catalytically inactive
protein, induces the formation of numerous long and thin actin-rich extensions
of the plasma membrane (Franco et al.,
1999
). Moreover, we found that the C-terminal region of EFA6
containing a putative coiled-coil motif is required for EFA6-mediated
cytoskeletal reorganization (Franco et
al., 1999
). These finding suggest that EFA6, besides activating
ARF6 through its Sec7 domain, may also exert a direct control over actin
cytoskeletal organization depending on its C-terminal region.
On the basis of the observation that EFA6 expression is restricted to the
brain, whereas ARF6 is expressed in a wide range of cell lines and tissues
(Cavenagh et al., 1996;
Tsuchiya et al., 1991
), we
tested the possibility that EFA6-like GEF(s) may regulate ARF6 activity
outside of the brain. Here, we describe a novel broadly distributed ARF6 GEF,
called EFA6B, containing a Sec7 domain highly related to EFA6, which will now
be referred as EFA6A. The adjacent PH and C-terminal domains of EFA6A and
EFA6B are also highly conserved. In cells, EFA6A and EFA6B colocalized with
ARF6 at the plasma membrane where they accumulate in subdomains. These
subdomains, which are enriched in actin filaments, correspond to
microvilli-like structures. The conserved C-terminal region of EFA6A/B GEFs,
when recruited to the membrane via the PH domain, triggered microvilli
lengthening. Our results suggest that EFA6-like GEFs, which are conserved in
evolution, are modular proteins consisting of an ARF6-specific Sec7 domain
linked to a membrane-anchoring PH domain and a C-terminal region involved in
actin cytoskeleton remodelling.
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Materials and Methods |
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cDNA cloning
EFA6B was cloned from a human placenta cDNA library (CLONTECH) using a 555
bp PvuII-PstI fragment corresponding to the Sec7 domain of
human EFA6A as a probe. Hybridisation was performed in 5xSSPE/10x
Denhardt/2% SDS for 18 hours at 65°C. Filters were washed with
2xSSC/O.5% SDS at 25°C for 30 minutes and 0.5xSSC/O.5% SDS at
55°C for 60 minutes, followed by autoradiography at -80°C. Eight
positive clones were isolated and sequenced from both ends. A 5' end
1.45 kb EcoRI fragment was derived from the longest clone
(
3.1 kb). A
2 kb cDNA was isolated and found to overlap over a
region of
1.3 kb at the 5' end of the 3.1 kb clone. The combined
3.8 kb sequence contained an open reading frame encoding a predicted
protein of 1056 amino acids. The cDNA clone reported here is present in the
EMBL database under accession number AJ459781. It is almost identical to a
sequence of Genbank/EMBL/DDBJ called TIC (accession no. U63127).
DNA constructs and transfection
EGFP-tagged EFA6A, EFA6A-PH (residues 351-494), -PHCTer (residues 349-645),
-CTer (residues 478-645), -PHCT1 (349-617), -PHCT2 (residues 349-587), -PHCT3
(residues 349-547), -PHCT4 (349-520), EFA6B, EFA6B-PHCTer (residues 752-1056),
and EFA6B-CTer (residues 879-1056) were generated by PCR with oligonucleotide
primers incorporating restriction sites. After digestion, the PCR products
were inserted into the multi-cloning sites of pEGFP-C3 (CLONTECH) to give
in-frame fusion with the N-terminal EGFP tag. All constructs were verified by
double-stranded DNA sequencing.
Cells were transfected using Fugene-6 (Roche) according to the manufacturer's instructions. Western blotting analysis of transfected cell extracts using anti-GFP antibodies showed that all proteins migrated at their expected size during SDS-PAGE (data not shown).
Northern blot analysis
A human multiple tissue Northern blot (CLONTECH) was hybridised with DNA
probes corresponding to a 1230 bp fragment of human EFA6A (corresponding to
residues 1-410) or a 364 bp fragment of human EFA6C (corresponding to the Sec7
domain) or a 215 bp fragment from human EFA6B (residues 637-708) or a human
ß-actin cDNA probe as a control. The DNA probes were labeled with
[-32P]dCTP (3000 mCi/mol) using Random Primer labelling kit
(Roche Molecular Biochemicals), and hybridisation was performed at 68°C in
5xSSPE/2% SDS/10x Denhardt. The membrane was washed with
0.5°SSC/0.1% SDS at 65°C for 60 minutes, followed by autoradiography
at -80°C. The expression profile quantified by RT-PCR ELISA concerning
clone KIAA0942 (EFA6D) is available at
http://www.kazusa.or.jp/huge/gfpage/KIAA0942/
.
Analysis of EFA6B expression by immunoblotting
Jurkat or BHK (25x106) cells, or BHK cells transfected
with pSR-VSVG-EFA6B (2x106 cells) were lysed in RIPA
buffer (1% NP40, 0.5% Sodium Deoxycholate, 0.1% SDS, 50 mM Tris pH7.5, 100 mM
NaCl, 1 mM EDTA, protease inhibitors). After clearing DNA by centrifugation,
lysates were first incubated with Pansorbin (Calbiochem) and then 5 µg/ml
rabbit affinity-purified anti-EFA6B, mouse monoclonal anti-VSV-G epitope
antibodies (clone P5D4) or irrelevant (anti-Sec10) antibodies were added for 2
hours at 4°C. Protein G Sepharose (Amersham Pharmacia Biotech) was added
for 1 hour at 4°C, and Sepharose beads were finally washed once in RIPA
buffer and twice in PBS. Proteins were separated by SDS-PAGE on 8%
polycacrylamide gel and transferred on PVDF membrane. For immunoblotting
analysis, anti-EFA6B or anti-VSV-G antibodies (1 µg/ml) were used, followed
by ECL procedure (Amersham Pharmacia Biotech).
GTPS binding assay
Myristoylated ARF1 and ARF6 were produced and purified as described
previously (Chavrier and Franco,
2001). A portion of EFA6B coding sequence starting at an internal
methionine residue (position 245) and including the Sec7 domain was subcloned
into pET3a. pET3aEFA6A (Franco et al.,
1999
) and -EFA6B(245-1056) were transformed into Escherichia
coli BL21 (DE3) strain, and protein expression was induced by addition of
0.5 mM isopropyl ß-D-thiogalactoside to a 0.21 culture for 2 hours at
37°C. Then, bacteria were lysed using a French press, and the lysate
produced was centrifuged at 20,000 g. EFA6B(245-1056) was
recovered in the insoluble fraction. Pellets containing EFA6A or
EFA6B(245-1056) were solubilized in urea buffer (Tris/HCl 50 mM pH 8.0; 1 mM
MgCl2; 1 mM DTT; 10 M urea) and ultracentrifuged (350,000
g for 20 minutes in TL100.3 Beckman rotor). The urea
concentration of the supernatants was decreased by successive dialysis in
order to obtain soluble EFA6A and EFA6B(245-1056) proteins in the absence of
urea. After dialysis, samples were ultracentrifuged (350,000 g
for 20 minutes in TL100.3 Beckman rotor), and supernatants were used as source
of GEFs in the nucleotide exchange experiments. The concentration of GEFs was
determined by scanning Coomassie-stained SDS gel.
[35S]GTPS binding measurements were performed as
previously described (Franco et al.,
1999
). Briefly, myrARF1 or myrARF6 (1 µM) were incubated at
30°C in 50 mM Hepes/NaOH pH7.5, 1 mM MgCl2, 1 mM DTT, 100 mM
KCl, 10 µM [32S]GTP
S (1000 cpm/pmol, NEN) supplemented
with 1.5 mg/ml azolectin vesicles (Sigma). EFA6A or EFA6B(245-1056) were added
to a final estimated concentration of 200 nM. At indicated times, aliquots of
25 µl were measured for radioactivity.
Immunofluorescence and time-lapse video microscopy
For immunofluorescence analysis, transfected cells were fixed in 3%
paraformaldehyde 36 hours after transfection. Cells were processed for
immunofluorescence microscopy as described previously
(Guillemot et al., 1997).
Images were collected on a Leica DMRB microscope equipped with a cooled CCD
camera (MicroMAX 5 MHz, Princeton Instruments, inc) with a 100x/1.4 PL
Apo lens and Metaview software (Universal Imaging). For confocal microscopy,
optical sections were taken with a Leica TCS SP2 confocal microscope.
Scanning electron microscopy
BHK cells were transfected with EGFP-EFA6A (or EFA6A-PHCTer) together with
CD25/FKBP2, a chimeric surface protein comprising the extracellular
and transmembrane regions of human CD25
(Castellano et al., 1999).
Before fixation, cells were incubated with biotinylated anti-CD25 monoclonal
antibody (5 µg/ml in complete medium, clone B1.49.9, Beckman Coulter) for
30 minutes at 4°C. Cells were washed in complete medium and incubated for
30 minutes at 4°C with 2.5 µm diameter streptavidin-coated latex beads
(Bangs Laboratories, Inc.) after centrifugation at 400 g for 4
minutes to sediment the beads. After extensive washing in complete medium, and
a final wash in PBS, cells were fixed and processed for scanning electron
microscope (SEM) analysis as described previously
(Guillemot et al., 1997
).
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Results |
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Human EFA6-like Sec7 domains were aligned with various other Sec7 domains
using Clustal W (Thompson et al.,
1994), and on the basis of the alignment a phylogenetic-type tree
was calculated using NJplot (Perriere and
Gouy, 1996
) (for space reasons a subset of the complete clustal
alignment is shown in Fig. 1B).
Human EFA6 Sec7 domains could be grouped into a family that is clearly
distinct from the ARNO/cytohesin/GRP-1 family
(Fig. 1C). Interestingly, the
Sec7 domains of Caenorhabditis elegans Y55D9A.1 (accession no.
AL032649, 816 amino acids) and Drosophila melanogaster CG6941 (900
amino acids) proteins were also grouped together with human EFA6-like domains,
indicating that this subgroup has been conserved throughout evolution (see
below). The conservation between EFA6 family Sec7 domains is clearly apparent
in the clustal alignment shown in Fig.
1B. Besides residues that are conserved in all Sec7 domains
(invariant residues are shown in green and conserved residues in yellow,
respectively), some positions are conserved only within the EFA6 family
(highlighted in purple in Fig.
1B). Moreover, the homology between EFA6-related proteins was not
limited to the Sec7 domain. Significant homology was observed in a pleckstrin
homology (PH) domain adjacent to the Sec7 domain (see schematic representation
of EFA6-family protein structure in Fig.
9A and Fig. 9B for
an alignment of EFA6 family PH domains) and in the C-terminal region
containing a predicted coiled-coil motif (aligned in
Fig. 9C). By contrast, no
significant similarity was found in the N-terminal domains of EFA6 family
proteins. Altogether, these observations indicate that the overall
organisation of a module consisting of the Sec7 and PH domains, as well as the
C-terminal region, is conserved in EFA6 family proteins throughout
evolution.
|
Expression of the EFA6 family genes in human tissues was analysed by
northern blotting. As previously reported, mRNA for EFA6A was mostly detected
in the brain (Perletti et al.,
1997). In addition, a smaller message was detected in the small
intestine and colon (Fig. 2A).
EFA6C expression was also restricted to the brain
(Fig. 2C). By contrast, EFA6B
had a broad expression, which mirrored that of EFA6A. The highest EFA6B levels
were found in placenta, pancreas, spleen, thymus and peripheral blood. The
message was also detected in lung, liver, kidney, prostates but was barely
detectable in heart, brain, ovary, small intestine and colon, whereas a
smaller transcript was detected in testis
(Fig. 2B). The multiple size
EFA6A and EFA6B mRNAs detected by northern blot analysis may represent
differently spliced forms. EFA6D expression profile available from the HUGE
Database (clone KIAA0942, see Materials and Methods section) revealed that
this member of the EFA6 family is mostly expressed in the brain and, to lower
extent in liver, the kidney, testis and ovary.
|
Expression of EFA6B at the protein level was assessed by
immunoprecipitation using specific antibodies
(Fig. 2D). Antibodies directed
against a C-terminal peptide derived from the human EFA6B polypeptide sequence
(see Materials and Methods) detected a protein migrating with an apparent
molecular weight of 180 kDa in lysates of Jurkat human T lymphocytes and
BHK cells (Fig. 2D, lane 1 and
2, respectively). Noticeably, the nucleotide sequence of EFA6B cDNA predicts a
protein of molecular weight 116.34 kDa. Therefore, EFA6B that was N-terminally
tagged with VSVG was overexpressed in BHK cells as a control. The
overexpressed protein was immunoprecipated with anti-EFA6B (lane 3) or
anti-VSV-G antibodies (lane 4) and detected by immunoblotting with the
corresponding antibodies revealing a protein migrating with an apparent
molecular weight similar to the endogenous polypeptide. In contrast, the 180
kDa species was not detected when irrelevant antibodies were used for
immunoprecipitation (lanes 5 to 7). Altogether, these results indicate that
EFA6B is expressed in Jurkat and BHK cells and migrate with a
180 kDa
apparent molecular weight in SDS-PAGE.
EFA6 and EFA6B are ARF6-specific GEFs associated with dynamic
plasmalemmal subdomains
Given the strong similarity between the Sec7 domain of EFA6A and EFA6B, we
assessed the ability of EFA6B to catalyse nucleotide exchange by measuring
guanine 5'-[-thio]-triphosphate binding on recombinant
myristoylated ARF6 (mARF6) or mARF1. The data presented in
Fig. 3A show that like EFA6A
(open square), recombinant EFA6B (with the N-terminal region deleted, i.e.
EFA6B/245-1056) stimulated nucleotide exchange on mARF in vitro (open
diamond). In the same conditions, EFA6A and EFA6B/245-1056 had very low
guanine nucleotide exchange activity on mARF1
(Fig. 3B).
|
The intracellular distribution of EGFP-tagged EFA6A and EFA6B was then
compared with that of their substrate ARF6 using immunofluorescence microscopy
analysis. As previously shown (Franco et
al., 1999), overexpressed ARF6 and EFA6A were found at the
plasmalemma. In addition to a homogenous distribution at the cell surface,
both proteins appeared to accumulate in subdomains of the dorsal cell surface
and cell periphery at specific sites corresponding to microvilli-like
protrusions and membrane ruffles, respectively
(Fig. 4A,B). A very similar
distribution was also observed in the case of EGFP-EFA6B
(Fig. 4C,D). The punctuate
localisation of EFA6A/EFA6B at the plasma membrane coincided with that of
cell-surface glycoproteins stained with wheat germ agglutinin, confirming the
surface distribution of the ARF6 GEFs (data not shown). When live BHK cells
expressing EGFP-tagged EFA6A were imaged by high-resolution fluorescence
video-microscopy, we could observe bright fluorescent ribbon-like structures
on the dorsal cell-surface that corresponded to membrane ruffles developing
and moving laterally in the plane of the membrane. These structures were
highly dynamic, as new ruffles continuously formed and collapsed
(Fig. 5). Smaller fluorescent
structures corresponding to microvilli-like protrusions were also visible on
the cell's dorsal surface. Treatment of the cells with latrunculin D totally
abolished movement of all these structures, suggesting that actin filament
dynamics are essential for movement of EFA6A-enriched microvilli and membrane
ruffles (data not shown). Altogether, these findings indicate that both EFA6A
and EFA6B GEFs colocalized with their ARF6 substrate at the plasmalemma and
suggest that these proteins are enriched in specific subdomains corresponding
to membrane ruffle-like structures and dorsal microvilli-like protrusions.
|
|
We then compared the distribution of EFA6A and EFA6B with that of F-actin
as cortical cytoskeleton is known as a major organizer of epithelial-cell
microvilli and plasmalemmal protrusions of non-epithelial cells
(Bretscher, 1999). Staining
with phalloidin to label filamentous actin and confocal sectioning through the
dorsal cell surface demonstrated that EFA6A and EFA6B colocalized with F-actin
in membrane ruffles and dorsal microvilli-like protrusions in transfected BHK
cells (Fig. 6A-C,
Fig. 6M-O, respectively). We
next sought to identify the regions of EFA6A required for the localization in
microvilli-like structures. We had previously shown that deletion of the PH
domain prevented membrane localization of EFA6A
(Franco et al., 1999
).
Therefore, we expressed the isolated EGFP-tagged EFA6A-PH domain (residues
351-494; Fig. 9A,B), and its
localization was analysed by confocal microscopy of fixed BHK cells stained
for F-actin. Similar to the EFA6A distribution, the EFA6A PH domain
demonstrated accumulation in scattered actin-rich microvilli-like protrusions
at the plasma membrane (compare Fig. 6A-C
with 6D-F). Therefore, the PH domain makes a major contribution to
the localization of EFA6A to plasma membrane subdomains. Interestingly, the
distribution of the isolated PH domain from phospholipase C
(PLC
) was similar to EFA6A, whereas the GRP1/ARN03 PH domain was mostly
cytosolic in these conditions (data not shown). Given the fact that the PH
domain of PLC
has a high affinity for PtdIns(4,5)P2
and has been characterized as a marker for membrane-associated
PtdIns(4,5)P2 (Varnai
and Balla, 1998
), whereas the GRP1 PH domain has a much higher
affinity for PtdIns(3,4,5)P3 than for
PtdIns(4,5)P2 (Kavran
et al., 1998
), our results suggest that binding of
PtdIns(4,5)P2 to the conserved PH domain of EFA6A is
probably a major determinant of membrane attachment and microvilli-like
distribution of ARF6 GEFs.
|
The conserved C-terminal coiled-coil motif of EFA6-family GEFs
triggers microvilli lengthening
The C-terminal region distal to the PH domain is conserved in all EFA6
family GEFs (Fig. 9A,C; see
Discussion), suggesting an important function for this domain. Furthermore, we
previously found that this region is required for induction of membrane
protrusion upon EFA6A overexpression
(Franco et al., 1999).
Therefore, constructs corresponding to the EFA6A C-terminal region fused to
the PH domain (EFA6A-PHCTer, residues 349-645), or alone (EFA6A-CTer, residues
478-645) were expressed in BHK cells as EGFP fusion proteins, and their
distribution was compared with F-actin. EFA6A-PHCTer accumulated in long
finger-like extensions that were induced at the dorsal surface of transfected
cells (Fig. 6G, arrows). Most
of these extensions were clearly stained for F-actin
(Fig. 6H,I, arrows). By
contrast, the distribution of EFA6A-CTer was diffuse and excluded from
intracellular vacuole profiles, suggesting that this construct was mostly
cytosolic (Fig. 6J-L). In
addition, EFA6A-CTer did not induce any morphological change in the
transfected cells. These results indicate that the C-terminal region of EFA6A,
when bound to the membrane via the PH domain, is able to trigger the formation
of finger-like extensions of the plasma membrane. Similarly, a construct
corresponding to the isolated PH domain and C-terminal region (PHCTer) of
EFA6B (residues 752-1056) induced the formation of finger-like extensions and
was found within these structures (Fig.
6P-R, arrows). Cells expressing the EFA6B-CTer construct with the
PH domain deleted (residues 879-1056) exhibited diffuse cytosolic staining
without morphological change (data not shown). We also noticed that
EFA6B-PHCTer demonstrated a strong nuclear accumulation
(Fig. 6P), whereas neither
full-length EFA6B nor EFA6BCter showed such a distribution
(Fig. 6M) (data not shown). A
possible explanation is that a cryptic nuclear targeting motif may become
exposed in EFA6B-PHCTer. Altogether, these findings suggest a role for the
conserved C-terminal regions of EFA6A and EFA6B in mediating morphological
changes at the plasma membrane when bound to the membrane via the adjacent PH
domain in the absence of Sec7 domain activity.
The morphology of these structures was further characterized by examining the surface of EFA6A and EFA6A-PHCTer-expressing cells by scanning electron microscopy. Transfected cells were identified using antibody-coupled beads recognizing the extracellular region of a transmembrane protein that was co-expressed with the EFA6A constructs (see the Materials and Methods section). In general, EFA6A-expressing cells had more microvilli-like protrusions on their dorsal surface than control cells expressing the transmembrane protein alone, but these structures were of similar size (compare Fig. 7A,B with 7C,D). Strikingly, expression of EFA6A-PHCTer triggered the appearance of a `hedgehog-like' morphology with the cell surface being covered by long (1-3 µm length) finger-like membrane extensions. Longer extensions, which may have been flattened during the fixation procedure, were laid down on the cell surface (Fig. 7E,F, arrows), whereas smaller structures were stood up (double-head arrows). The diameter of the longer structures was similar to microvilli-like protrusions of control cells or cells overexpressing native EFA6A, suggesting that EFA6A-PHCTer increased the length of pre-existing microvilli-like structures (arrows). Altogether, these findings indicate that overexpressed EFA6A accumulates in subdomains of the plasma membrane where it promotes formation of actin-rich plasmalemmal microvilli-like protrusions. In addition, they suggest that membrane attachment of the C-terminal region of EFA6A in these subdomains via the PH domain induces a dramatic remodelling of the plasma membrane by promoting lengthening of these microvilli.
|
In order to establish the domain(s) of the C-terminal region required for microvilli lengthening, we assessed the effect of constructs derived from EFA6A-PHCTer-presenting serial truncations from the C-terminus. These constructs expressed as GFP-fusion protein (PHCT1-3, starting at position 349 and ending at position 617, 587, and 547, respectively) were able to induce dorsal finger-like extensions (Fig. 8A-C), except for the shortest one (PHCT4, residues 349-520), which had the putative coil-coiled motif deleted (Fig. 8D), indicating that the conserved C-terminal coil-coiled region is essential for microvilli lengthening.
|
![]() |
Discussion |
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EFA6 family members contain a 200 amino-acid Sec7 domain positioned
right in the centre of the protein (Fig.
9A). The Sec7 domain is found in all ARF GEFs described so far.
Human EFA6 Sec7 domains are 60-70% identical to each other and only
30-35% identical to ARNO/cytohesin Sec7 domains
(Fig. 1B,C;
Table 1). As we previously
reported in the case of EFA6A (Franco et
al., 1999
; Macia et al.,
2001
), EFA6B exhibits GEF activity in vitro only with ARF6 (class
III ARF) and not with ARF1 (class I ARF), raising the question of how EFA6
GEFs discriminate between two ARF proteins that share 67% sequence identity.
Determination of the three-dimensional structure of a complex between yeast
Gea2 Sec7 domain and human ARF1 revealed that residues from switch-1 and -2
regions of ARF1 insert in a hydrophobic groove within the Sec7 domain
(Béraud-Dufour et al.,
1998
; Goldberg,
1998
; Mossessova et al.,
1998
). This groove, formed by a loop between
helices F and
G (motif 1; Fig. 1B) and a
central portion of helix H (motif 2)
(Cherfils et al., 1998
;
Mossessova et al., 1998
) is
conserved in all known Sec7 domains (Fig.
1B; invariant residues are highlighted in green, and conserved
residues in yellow). The invariant glutamate residue in motif 1 (in blue in
Fig. 1B) is critical for
guanine nucleotide exchange, and it acts by inserting directly in the ARF
nucleotide-binding pocket and thereby promoting nucleotide dissociation
(Béraud-Dufour et al.,
1998
; Goldberg,
1998
; Mossessova et al.,
1998
). By contrast, some residues that are identical in
EFA6-family Sec7 domains (highlighted in purple in
Fig. 1B) differ in other
Sec7-domain-containing GEFs. These residues may contribute to ARF substrate
specificity. In addition, although the amino-acid sequence of the switch-1/2
regions of ARF1 and ARF6 are very similar
(Tsuchiya et al., 1991
), the
recent determination of the three-dimensional structure of GDP-bound ARF6 has
revealed differences from the structure of GDP-ARF1 that may explain its
specificity (Ménétrey et
al., 2000
).
Adjacent to the Sec7 domain, there is a PH domain whose sequence is highly
conserved amongst EFA6 family members. (The EFA6A PH domain is 70%
identical to EFA6C/EFA6D,
50% identical to EFA6B/CG6941 and
40%
identical to Y55D9A.1.) This domain is most similar to the human ßIV
spectrin PH domain (Berghs et al.,
2000
) (34% identity; Fig.
9B). The well characterized PH domain of ß-spectrin binds to
membrane PtdIns(4,5)P2 with high affinity, and residues
known to form critical contacts with the PtdIns(4,5)P2
head group in ß-spectrin and PLC
PH domains
(Hyvonen et al., 1995
;
Rameh et al., 1997
) are
strikingly conserved in all EFA6 family PH domains
(Fig. 9B, bold characters). In
fact, several observations suggest that the PH domain of EFA6 GEFs supports
membrane association by interacting with PtdIns(4,5)P2.
First, we previously reported that deletion of a region of EFA6A comprising
its PH domain prevented membrane localization
(Franco et al., 1999
). Second,
we have now observed that overexpressed EFA6A- and PLC
-PH domains have
similar a distribution in BHK cells (data not shown). Third, we found that
EFA6A and EFA6B (as well as their isolated PH domain) accumulate in dynamic
actin-rich membrane ruffles and microvilli-like structures. Similarly, the
GFP-tagged PLC
-PH domain, which was used to follow the dynamics of
PtdIns(4,5)P2 in living cells
(Varnai and Balla, 1998
),
revealed non-uniform fluorescence in distinct structures identified as ruffles
and microvilli-like protrusions from the dorsal cell surface
(Tall et al., 2000
).
Altogether, our findings indicate that EFA6A and EFA6B localize to subdomains
of the plasma membrane, probably through interaction of their PH domain with
membrane-bound PtdIns(4,5)P2. This localization may
reflect a non-uniform distribution of PtdIns(4,5)P2, which
appears to be enriched in highly dynamic regions of the plasma membrane
(Tall et al., 2000
). Finally,
it is also quite remarkable that the PH domain of ARNO/cytohesin GEFs
(specially in its diglycine form) (Cullen
and Chardin, 2000
; Klarlund et
al., 2000
) is highly selective for
PtdIns(3,4,5)P3. Accordingly, membrane recruitment of
ARNO/cytohesin GEFs was found to require phosphoinositide 3-kinase activation
(Gray et al., 1999
;
Venkateswarlu et al., 1998
).
Given the fact that ARNO has been shown to activate ARF6 to some extent
(Frank et al., 1998a
), these
and our findings, which point to distinct regulatory mechanisms for membrane
association and activation for EFA6 versus ARNO/cytohesin family members,
suggest that various signalling pathways may operate through distinct GEF
families resulting in ARF6 activation.
In addition to the PH domain, all EFA6 family GEFs share a conserved
150 amino-acids C-terminal region, which includes a
30 amino-acid motif
with a predicted coiled-coil structure
(Fig. 9A,C). This conservation,
combined with our initial observation that deletion of this region abolished
EFA6-induced actin cytoskeleton reorganization
(Franco et al., 1999
), points
to an essential function for the C-terminus. In agreement with this
assumption, we found that overexpression of the C-terminal region of EFA6A/B
fused to the adjacent PH domain triggers lengthening of microvilli-like
structures at the plasma membrane, and this requires the integrity of the
putative coiled-coil motif. By contrast, the C-terminal region of EFA6A (or
EFA6B, data not shown), expressed without the PH domain, accumulates in the
cytosol and is ineffective in inducing the `hedgehog-like' morphology,
indicating that membrane targeting of the C-terminal region is required for
microvilli lengthening. The possibility that microvilli lengthening results
from dominant inhibition of EFA6A (or EFA6B)-GEF activity, and hence
inhibition of ARF6 activation by the overexpressed C-terminus is unlikely, as
expression of a dominant inhibitory mutant form of ARF6 (ARF6T27N), which also
results in ARF6 inactivation, does not induce the `hedgehog-like' phenotype
(data not shown). In addition, microvilli lengthening is abolished when the PH
domain/C-terminal module of EFA6A (PHCter) is expressed together with a
constitutively activated mutant of ARF6 (ARF6Q67L; data not shown).
Altogether, our data are consistent with a dual function for EFA6 GEFs as
shown by ARF6 activation by the Sec7 domain and the direct effect of the
C-terminus on the actin cytoskeleton in microvilli-like structures. Uncoupling
these two functions either by deletion of the C-terminal region or by
deletion/mutagenesis of the Sec7 domain, prevents membrane ruffling formation
or triggers microvilli lengthening, respectively
[(Franco et al., 1999
), this
study]. Although the mechanism underlying the regulation of actin cytoskeleton
reorganization by the conserved C-terminal region is presently unknown, the
fact that it requires the integrity of the coiled-coil motif is remarkable.
Coiled coils are
helical structures involved in homo- or
heterooligomeric association of helices
(Lupas, 1996
). So far, we have
not been able to detect EFA6 oligomers (C.C. and P.C., unpublished), and we
are actively searching for proteins interacting with this region of EFA6 that
could control the morphology of microvilli-like structures.
This study suggests that EFA6 family proteins could influence the
composition and organization of membrane subdomains at the cell cortex by
coordinating phosphoinositide signalling and ARF6-mediated events on membrane
remodelling. Our results should help clarify the function of EFA6 family GEFs
in cellular processes requiring membrane movement to the cell surface and
actin cytoskeleton remodeling, such as regulated exocytosis or phagocytosis,
which are all known to depend on ARF6 activity
(Bajno et al., 2000;
Galas et al., 1997
;
Zhang et al., 1998
).
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References |
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