(Received for publication, May 30, 1995; and in revised form, August 3, 1995)
From the
Trimeric G proteins have emerged as important regulators of
membrane trafficking. To explore a role for G in endosome
fusion, we have taken advantage of
-adrenergic receptor kinase
(
ARK), an enzyme translocated to membranes by interaction with
G
. The COOH terminus of
ARK (
ARKct) has a
G
-binding domain which blocks some G
-mediated
processes. We found that
ARKct and peptide G, a peptide derived
from
ARKct, inhibit in vitro endosome fusion.
Interestingly, peptide G and ARF share sequence similarity. Peptide G
and
ARKct reversed ARF-mediated inhibition of endosome fusion and
blocked ARF binding to membranes. Using an ARF fusion protein, we show
that both G
and G
s interact with the small GTPase ARF,
an interaction that is regulated by nucleotide binding. We conclude
that G proteins may participate in the regulation of vesicular
trafficking by directly interacting with ARF, a cytosolic factor
required for transport.
Vesicular membrane trafficking among intracellular compartments
is now recognized to involve multiple small GTP-binding proteins
including members of the Ras-like superfamily such as Rab, ARF, and
Sar1 (reviewed by Goud and McCaffrey, 1991; Pryer et al.,
1992; Nuoffer and Balch, 1994). The ARF family, which includes several
distinct ARF proteins, seems to control the assembly of coat components
on transport vesicles. ARF (ADP-ribosylation factor) was originally
discovered as a cofactor required for the ADP-ribosylation by cholera
toxin of the heterotrimeric G protein G (Kahn and Gilman,
1984). The initial evidence for a role for ARF in vesicular transport
came from genetic studies in yeast where deletion of the ARF1 gene
resulted in a secretory defect (Stearns et al., 1990a, 1990b).
Using several in vitro assays that reconstitute transport
between different compartments, it has been shown that ARF is an
essential component required for transport (Balch et al.,
1992; Lenhard et al., 1992; Donaldson and Klausner, 1994). ARF
is also required for the assembly of the coat complex on
non-clathrin-coated vesicles (COP-coated vesicles) mediating transport
between Golgi compartments (reviewed by Rothman and Orci, 1992; Kreis
and Pepperkok, 1994; Donaldson and Klausner, 1994) and in the
association of AP-1 adaptor complex to Golgi membranes, raising the
possibility that ARF may also be required for the assembly of clathrin
coats at the trans-Golgi network (Stamnes and Rothman, 1993;
Traub et al., 1993).
A growing body of evidence indicates
that heterotrimeric GTP-binding proteins (G proteins) play a crucial
role in vesicular trafficking (reviewed by Bomsel and Mostov, 1992;
Barr et al., 1992; Burgoyne, 1992; Nuoffer and Balch, 1994).
Previous work from our laboratory indicates that fusion among endosomes
and between phagosomes and endosomes is controlled by G proteins
(Colombo et al., 1992a, 1994a; Beron et al., 1995).
Moreover, multiple G proteins seem to participate in different steps of
transport (Stow et al., 1991; Leyte et al., 1992;
Carter et al., 1993). We have reported that one of the G
proteins involved in endosomal fusion is G (Colombo et al., 1994b). The role of G
has also been
implicated in trafficking in polarized cells (Pimplikar and Simons,
1993; Bomsel and Mostov, 1993; Barroso and Sztul, 1994; Hansen and
Casanova, 1994) and in the secretory pathway (Leyte et al.,
1992). However, the actual mechanism by which these proteins regulate
traffic remains poorly understood.
Classically, trimeric G proteins
transduce extracellular signals to appropriate effector molecules
inside the cell. G proteins are comprised of three subunits, G,
G
, and G
. Binding of GTP causes the activation of the G
protein and the subsequent dissociation of G
from G
(Gilman, 1987). It is now widely accepted that signals by both G
and G
are physiologically relevant. Several recent reports
clearly demonstrate the prominent involvement of G
in several
transmembrane signaling systems. An increasing number of G
protein-coupled effectors which appear to be modulated by G
subunits have been identified (reviewed by Clapham and Neer, 1993;
Sternweis, 1994). On the other hand, G
specifically mediates
the translocation of cytosolic
-adrenergic receptor kinase
(
ARK), (
)one of the G protein-coupled receptor kinases,
to the plasma membrane. This translocation allows the phosphorylation
of activated receptors as part of the desensitization process (Inglese et al., 1993). A fragment of
ARK corresponding to the
last 222 C-terminal amino acids was found to contain the
``G
-binding domain'' (Pitcher et al.,
1992). A fusion protein corresponding to this G
-binding
domain blocks binding of
ARK to G
(Koch et al.,
1993) and prevents receptor phosphorylation. It has recently been shown
that this reagent interferes with multiple G
-mediated
processes such as G
-dependent activation of adenylyl cyclase
type II,
ARK2 regulated olfactory signal transduction, and atrial
K
channel activation (Reuveny et al., 1994;
Boekhoff et al., 1994; Inglese et al., 1994).
In
an attempt to study the possible role of G in the mechanism
or regulation of endosome fusion we used
ARK C-terminal fusion
protein and peptides derived from the G
-binding domain in a
cell-free assay that reconstitutes fusion between endosomes.
His6-
ARK fusion protein completely blocked endosome fusion while
His6-rhodopsin kinase had no effect. A single 28-amino acid peptide
(Peptide G) derived from the targeting domain of
ARK was also
found to inhibit fusion. Alignment of the cytosolic small GTP-binding
protein ARF and peptide G reveals that they share sequence similarity.
Our results suggest that
ARK COOH terminus and peptide G inhibit
endosome fusion by blocking the interaction of G
with ARF, a
cytosolic factor required for endosome fusion. In order to address this
provocative hypothesis, we constructed GST-ARF fusion proteins and
studied their direct interaction with purified G proteins. Our results
indicate that both G
and G
interact with the
small GTPase ARF. Activation of G
by either GTP
S
or aluminofluoride complexes completely blocked ARF-G
interaction,
indicating that the heterotrimer is the most likely candidate for ARF-G
protein interaction. Our results suggest that a direct collaboration
among heterotrimeric G proteins and ARF may regulate vesicular
transport.
Recombinant C-terminal half of ARF1 (ARF1ct)
protein was expressed as follows: the C-terminal half of the ARF1 cDNA
was amplified by polymerase chain reaction. The 5`-oligonucleotide
primer contained a BamHI linker followed by 14 nucleotide
residues downstream of nucleotide residue 307. The 3`-oligonucleotide
primer contained an EcoRI linker followed by 16 nucleotide
residues complementary to the carboxyl-terminal end of ARF1 cDNA. The
amplified cDNA was digested with the restriction enzymes BamHI
and EcoRI and then subcloned into the bacterial expression
vector pGEX-3T. The recombinant protein was expressed as fusion protein
in the E. coli strain JM109, with the NH-terminal
end fused to GST and induced with
isopropyl-1-thio-
-D-galactopyranoside to produce GST
fusion proteins.
The fusion proteins were purified by
glutathione-Sepharose either by standard techniques or using the
Sarkosyl method (Frangioni and Neel, 1993). The samples were dialyzed
against PBS and, if necessary, concentrated in a Centricon-10 (Amicon).
GST-ARK COOH-terminal and GST-RK COOH-terminal fusion proteins
were constructed and purified as described previously (Koch et
al., 1993). GST-Rab5 fusion protein, constructed and purified as
described (Barbieri et al., 1994), was kindly provided by Mary
K. Cullen (Washington University, St. Louis, MO).
To assess the possible
involvement of G in the mechanism of endosome fusion, the
COOH-terminal
ARK fusion protein was tested in the in vitro endosome fusion assay. Fig. 1A shows that a
6His-COOH-terminal
ARK1 fusion protein (
ARK1ct) completely
blocks fusion between endosomes (closed circles). The
inhibitory potency of
ARK1ct in the in vitro fusion assay
(EC
10-15 µM) was similar to the
inhibitory activity against G
activation of
ARK (Koch et al., 1993). Interestingly, the 6His-
ARK2ct
corresponding to the same region of
ARK2, another member of the G
protein-coupled kinase family, was a better inhibitor of endosome
fusion (triangles). In contrast, no effect was observed with
the COOH-terminal domain of rhodopsin kinase (open circles).
This result is consistent with earlier observations showing that the
COOH-terminal domain of RK (RKct) does not bind to G
. RKct
lacks the G
-binding domain and consequently does not interact
with G
subunits (Pitcher et al., 1992). The
differential effect observed with
ARKct and RKct fusion proteins
appears to rule out any nonspecific effect of these polypeptides.
Figure 1:
A, the carboxyl-terminal
domain of ARK inhibits in vitro endosome fusion.
Endosomes containing fusion probes were mixed in fusion buffer with
gel-filtered cytosol (0.2 mg/ml) containing 20 µM GTP
S. Increasing concentrations of 6His-
ARK1 COOH
terminus (closed circles), 6His
ARK2 COOH terminus (triangles), or 6His RK COOH terminus (open circles)
were added to the fusion mixture. B, peptides from the
G
-binding domain of
ARK inhibit endosome fusion.
Increasing concentrations of peptide G1, a 28-amino acid
peptide from the G
-binding domain of the
ARK1 (closed circles). Triangles, peptide G2, a
peptide corresponding to the same region of
ARK2. Open
circles: peptide G
, a nonactive
ARK1 peptide,
which is actually the first 15 amino acid residues of peptide
G
, used as a control. The samples were incubated for 45 min
at 37 °C to allow fusion to occur. Fusion was stopped by cooling at
4 °C and assessed as described under ``Experimental
Procedures.'' Values are expressed as percentage of the control
fusion without any addition. Data represent one of three similar
experiments.
In
order to identify the critical regions involved in ARK binding to
G
, Koch and collaborators(1993) synthesized several peptides
corresponding to the targeting domain. A single 28-amino acid peptide
(Peptide G
) derived from the targeting domain of
ARK1
was found to inhibit G
activation of
ARK with an
IC
of 76 µM. In contrast, peptide
G`
, containing only the first 15 amino acid residues of
peptide G
, was inactive. Fig. 1B shows that
peptide G
was also inhibitory of endosome fusion with a
similar EC
(closed circles). No inhibitory effect
was observed with peptide G`
(open circles). As
observed with
ARK2, peptide G
corresponding to the
same region of
ARK
was a more potent inhibitor of
endosome fusion (triangles).
Since the COOH-terminal domain
of ARK selectively binds to G
, the inhibitory effect
observed with the fusion protein and with peptide G suggests that a
G
-mediated process is involved in in vitro endosome
fusion. The results further suggest that
ARKct and peptide G are
likely blocking the interaction of G
subunits with a
factor(s) required for in vitro endosome fusion.
Figure 2:
Homology of peptides G and
G
to ARFs. Peptide G
, a 28-amino acid peptide
corresponding to
ARK1 residues Trp
to Ser
and peptide G
corresponding to the same region of
ARK2 were aligned with the COOH-terminal half of members of the
ARF family using the J. Hein method with PAM250 residue weight table. A
segment of five amino acids (ELRDA) from peptide G
,
identical to a fragment corresponding to amino acids 115-119 of
ARF1, is boxed. Equivalent sequence similarity was observed
with other members of the ARF family. Sequence positions for the
rightmost residue of each polypeptide are given in the right-hand
column.
Figure 3:
Peptide G reverses GTP
S-
and ARF-mediated inhibition of fusion by inhibiting ARF binding to the
membranes. A, endosome fusion was tested in the presence of
0.8 mg/ml cytosol supplemented with 20 µM GTP
S to
inhibit fusion. The inhibitory effect of GTP
S was reversed by
addition of increasing concentrations of peptide G
.
Endosome fusion was measured as described under ``Experimental
Procedures.'' Fusion is expressed in relative units. B,
endosomal vesicles were resuspended in cytosol (0.2 mg/ml) containing
20 µM GTP
S. Fusion was assessed in the presence (closed circles) or the absence (open circles) of 15
µg/ml purified recombinant myristolated ARF1. The inhibitory effect
of ARF was reversed by addition of increasing concentrations of peptide
G
. The results are representative data of a experiment
performed three times. C, enriched endosomal fraction
(10-20 µg of total protein) was resuspended in fusion buffer,
containing 1 mg/ml cytosolic proteins. Samples were incubated for 5 min
at 37 °C in the presence of: lane a, no additions; lane b, 50 µM peptide G
; lane
c, 25 µM peptide G
; lane d, 50
µM peptide G
(control peptide). After
preincubation, 20 µM GTP
S was added, and the samples
were incubated for additional 20 min at 37 °C. After incubation,
the samples were washed with 1 ml of homogenization buffer containing
20 µM GTP
S and 1 mM MgCl
, and
the membranes were recovered by centrifugation for 5 min at 50,000
g. The membrane proteins were resolved by
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose,
and probed with anti-ARF antibodies. Data represent one of three
similar experiments.
As another approach to directly show that peptide G was
competing with ARF for interaction with membranes, we studied the
binding of ARF to endosomal membranes by Western blot assay. Fig. 3C shows that incubation of enriched endosomal
membranes with cytosol in the presence of 20 µM GTPS
resulted in binding of ARF (lane a). Preincubation of the
membranes for 5 min at 37 °C before the addition of GTP
S with
peptide G
or G
(lanes b and c) inhibited the binding of ARF to crude endosomal membranes.
As expected no inhibition of ARF binding was observed with the control
peptide G
` (lane d).
Taken together our results indicate that peptide G interferes with ARF function by blocking the interaction of this protein with the membranes.
Figure 4:
Diagrammatic representation of GST-ARF
fusion proteins. Each GST fusion protein was constructed, expressed,
and purified as described under ``Experimental Procedures.''
The GST protein is shown shaded. ARF4, wild type
ARF4. ARF4( 1-17), a mutant ARF4 with the first 17
amino acids deleted. ARF1ct, a mutant ARF1 corresponding to
the COOH-terminal half of ARF1. Peptide G is shown to indicate the
regions that display homology with ARF.
Figure 5:
A, binding of G to GST-ARF
fusion proteins in the presence or absence of G
subunits. Purified
bovine brain G
subunits (300 nM) were incubated with
different GST fusion proteins (600-700 nM) for 30 min on
ice in PBS containing 0.01% lubrol. The binding of the proteins to
glutathione-Sepharose beads and the detection were performed as
described under ``Experimental Procedures.'' Western blot
analysis showing G
binding to GST fusion proteins: lane
1, GST-
ARK1ct, positive control; lane 2, GST; lane 3, GST-RKct; lane 4, GST-ARF4 (
1-17); lane 5, GST-ARF1ct; lane 6, GST-rab5; lane
7, GST-ARF4 (
1-17) + 1.2 µM purified
recombinant G
; lane 8, GST-ARF4
(
1-17) + 1.2 µM purified recombinant
G
. GST, GST-RKct, and GST-rab5 were used as negative
controls. The results are representative data of a experiment performed
four times. B, binding of G
to
GST-ARF(
1-17) is competed by purified ARF. Purified bovine
brain G
subunits were incubated with GST-ARF(
1-17)
as described. Control, no additions; +ARF4, 7
µM purified recombinant ARF4; +ARF1, 3
µM purified recombinant ARF1; + BSA, 7
µM BSA (control). The binding and the detection were
performed as described under ``Experimental Procedures.''
Data represent one of two similar
experiments.
G binding to ARF-GST was specifically competed by purified
recombinant ARF1 and ARF4 (Fig. 5B), but not by BSA
indicating the specificity of the ARF-G
association.
Figure 6:
A, binding of G to
GST-ARF. Purified bovine brain G
(300 nM) with or
without purified recombinant G
(700 nM) was
incubated with GST-ARF4(
1-17) as described in Fig. 5. B, G
interacts with ARF in the presence or
absence of G
. GST-ARF4(
1-17) or GST alone was
incubated with purified bovine brain G
(300 nM),
purified recombinant G
(500 nM) or both as
described. C, activation of G
by AlF blocks
G
-ARF association. GST-ARF4(
1-17) was
incubated with 300 nM of purified bovine G
subunits
and/or with 700 nM recombinant G
for 30 min
at 30 °C in PBS containing 0.01% lubrol and 10 mM MgCl
in the presence or the absence of AlF (100
µM AlNH
(SO
)
+ 10
mM KF). D, activation of ARF by GTP
S inhibits
G
-ARF association. GST-ARF4(
1-17) was preincubated
for 90 min at 37 °C in 50 mM HEPES-K, pH 7.5, containing
0.01% lubrol, 1 mM DTT, and 10 mM MgCl
in
the presence of 50 µM GTP
S, 50 µM
GDP
S or no additions. Nucleotide exchange on ARF was stopped by
cooling at 4 °C. Subsequently, 300 nM of purified bovine
G
subunits were added, and the samples were incubated for
additional 30 min at 4 °C. The binding of the proteins to
glutathione-Sepharose and the detection were performed as described
under ``Experimental Procedures.'' Western blot analysis
showing G
and/or G
binding to GST fusion
proteins. Data represent one of three similar
experiments.
It is known that GTPases
function as molecular switches changing their conformation when they
are activated. Aluminum fluoride (AlF) is a classical activator of
heterotrimeric G proteins but does not activate members of the small
GTPase family such as ARF (Kahn et al., 1992). Therefore, in
order to independently activate the heterotrimeric G protein, the
effect of AlF was tested in the binding assay. As shown in Fig. 6C, activation of G by AlF
completely blocked ARF-G
association both in the
presence or the absence of G
. As expected, AlF did not affect
ARF-G
association.
Given that both ARF and heterotrimeric
G proteins are regulated by nucleotide binding we next studied the
effect of either GTPS or GDP
S. Similar to the effect observed
with AlF, GTP
S almost completely blocked G
-ARF
association (data not shown); essentially no effect was observed with
GDP
S. Our results clearly indicate that G
in the
GDP-bound form associates with ARF either in the presence or the
absence of G
subunits. Activation of G
by
either GTP
S or AlF completely blocked ARF-G
interaction,
indicating that the heterotrimer is the most likely candidate for ARF-G
protein interaction. GTP
S inhibited ARF-G
association (Fig. 6D) suggesting that ARF interacts with
G
in the GDP-bound state.
The subunits of heterotrimeric G proteins modulate
the activity of several signal-transducing effector molecules such as
phospholipase C, phospholipase A2, certain isoforms of adenylate
cyclase and cardiac muscarinic potassium channels (reviewd by Clapham
and Neer, 1993). G
also mediates the membrane translocation
of the
-adrenergic receptor kinases (
ARK1 and
ARK2)
where they phosphorylate activated receptors (Inglese et al.,
1993). The COOH-terminal domain of
ARK (
ARKct) contains the
targeting domain for binding to G
(Pitcher et al.,
1992), and a fusion protein corresponding to this targeting domain
blocks the binding of
ARK to G
(Koch et al.,
1993). Moreover,
ARKct appears to act as a general G
antagonist, inhibiting G
-mediated signals other than
ARK
translocation such as G
-dependent activation of adenylyl
cyclase type II,
ARK2-regulated olfactory signal transduction, and
atrial K
channel activation (Reuveny et al.,
1994; Boekhoff et al., 1994; Inglese et al., 1994;
Koch et al., 1994).
In this report we present evidence that
the COOH-terminal portion of ARK (
ARKct) and peptides
corresponding to the G
-targeting domain of
ARK inhibit in vitro endosome fusion. The results suggest that a
G
-mediated signal is involved in either the mechanism or the
regulation of endosome fusion. Indeed, our results suggest that
ARKct and peptides from the G
-binding domain (peptides
G) block the interaction of G
with a factor(s) required for
endosome fusion. We believe that one of these factors is ARF for the
following reasons: (i) peptide G and ARF share sequence homology, (ii)
peptide G reverses GTP
S- and ARF-mediated inhibition of endosome
fusion, (iii) peptide G inhibits ARF binding to membranes. Supporting
evidence for a direct interaction between ARF and G
was
provided by an in vitro binding assay using ARF-GST fusion
proteins. Our study establishes that G
binds to ARF and that
this interaction is specifically competed by purified recombinant ARF
and enhanced by G
.
While the binding of G to
immobilized ARF is specific, only small amounts of the available
G
subunits bound to GST-ARF. However, the binding was
increased by the addition of G
. A trivial explanation is that most
of the G
has been simply denatured during its preparation.
Another possibility is that ARF
binds only to a specific
subset of the G
combinations comprising the heterogeneous
preparation isolated from bovine brain. An interesting possibility is
that G
may require interaction with another protein to be in
the right conformation for binding. The G
-binding domain of
ARK shares homology with the novel pleckstrin homology domain (PH
domain). This domain is found in a variety of signaling molecules such
Ras-GAP, Ras-GRF, SOS, and others (Shaw, 1993; Musacchio et
al., 1993). Recently, it has been shown that proteins with PH
domains bind to G
in vitro (Touhara et al.,
1994). Protein-protein interactions between proteins containing a PH
domain and G
may play a significant role in cellular
signaling. Although the presence of a PH domain has not been described
for ARF, it is tempting to speculate that putative ARF accessory
proteins such as ARF-GAP or ARF-GRF may indeed contain such a domain
and that they may regulate ARF activity in conjunction with
G
. Current models for the interaction between ARF and target
membranes propose that activation of ARF by a protease- and brefeldin
A-sensitive membrane-bound nucleotide-exchange factor (Helms and
Rothman, 1992; Donaldson et al., 1992b; Randazzo et
al., 1993) results in association of ARF-GTP with the lipid
bilayer. Our results indicating that ARF in the GDP form interacts with
G
suggest that these proteins may form a multimeric complex
that allows the interaction of ARF with its nucleotide exchange factor
resulting in ARF activation.
The results presented in this report
are the first direct evidence indicating that both G and G
associates directly with ARF. There is a
precedent for this connection in that ARF is the co-factor necessary
for the ADP-ribosylation of G
by cholera toxin and a
possible interaction with G
has been previously
suggested (Kahn and Gilman, 1984, 1986). Interestingly, during the
purification of ARF from bovine brain, ARF eluted in two peaks, one
coincidental with G
. Addition of AlF was necessary to
obtain a single peak of ARF activity (Kahn and Gilman, 1984, 1986). The
more likely target for AlF is the GDP-form of G
. In agreement with
the results of Kahn and Gilman, our data indicate that G
in the GDP-bound conformation associates with ARF since
activation of G
by either GTP
S or AlF completely
blocked ARF-G
interaction. Recently, Finazzi and
collaborators(1994) have shown that AlF plus GTP stabilizes the active
state of ARF by preventing the rapid hydrolysis of the GTP loaded onto
ARF. These authors have postulated that an AlF-sensitive target may
lead to a persistent activation of ARF by inhibiting an ARF GAP or by
making the ARF-GTP either insensitive or inaccessible to ARF GAP. While
the exact role and mechanism of action of G
remains to be defined,
our results of complete inhibition of G
-ARF association by AlF
suggest the intriguing possibility that G
in the GDP-bound form
may regulate ARF GTPase activity.
An interesting outcome of our
experiments relates to the role of the amino-terminal domain of ARF in
mediating ARF function. It has been reported that the amino terminus of
ARF is critical for function since deletion of this domain results in a
global reduction of ARF activities (Kahn et al., 1992). A
synthetic peptide derived from the amino terminus of ARF inhibits ARF
activity including cholera toxin activation, as well as intra-Golgi
transport (Kahn et al., 1992) and fusion between endosomes
(Lenhard et al., 1992). Moreover, the amino-terminal 13
residues of ARF1 are required for cofactor activity in the
ADP-ribosylation by cholera toxin when G is the substrate
(Randazzo et al., 1994). However, Vaughan and collaborators
(Hong et al., 1994) have shown that the amino terminus of ARF
is not necessary for in vitro activation of cholera toxin
using as a substrate agmatine. Although the basis for this disparity is
not clear, this latest result suggests that other domains, besides the
amino terminus, are likely involved in the interaction of ARF with the
toxin. Our results indicate that the ARF domain involved in
ARF-G
interaction does not require the amino-terminal 17
amino acids since G
binds to GST-ARF4 and to the truncated
ARF mutants (ARF4
1-17 and ARF1ct) to a comparable extent.
However, we cannot rule out the possibility that the presence of GST at
the amino terminus may interfere with the proper folding and binding
capacity of this domain.
It has been demonstrated that ARF plays an
essential role in regulating coatomer binding (Donaldson et
al., 1992a; Palmer et al., 1993) and AP-1 recruitment
onto Golgi membranes (Traub, et al., 1993; Stamnes et
al., 1993). Moreover, a number of studies have provided evidence
for the involvement of heterotrimeric G proteins in coat assembly
(Donaldson et al., 1991; Ktistakis et al., 1992).
Association of ARF and -COP with Golgi membranes is sensitive to a
number of reagents that modulate heterotrimeric G protein function
(Donaldson et al., 1991; Ktistakis et al., 1992). In
addition to GTP
S, AlF, known to specifically activate trimeric G
proteins (Kahn, 1991), enhances the binding of
-COP to Golgi
membranes (Serafini et al., 1991). These findings and the
observation that G
inhibits both ARF and
-COP binding
(Donaldson et al., 1991) suggest that G proteins regulate coat
protein binding. We have also recently shown that both heterotrimeric G
proteins and ARF regulate priming of endosomal membranes for fusion
(Lenhard et al., 1994). Addition of G
resulted in
inhibition of GTP
S-mediated priming of endosomes. In contrast,
addition of ARF to the assay enhanced priming in the presence of
cytosol. These observations suggest that ARF enhances binding of
cytosolic factors required for fusion onto the endosomal membrane.
Although the linkage between ARF binding and coat assembly with
heterotrimeric G proteins has been proposed based on the data
summarized above, to date no direct evidence for the interaction
between ARF and trimeric G proteins has been presented. Our data would
support a model in which heterotrimeric G proteins regulate binding of
essential proteins at least in part, by directly interacting with ARF.
Finally, several recent observations implicate a signal transduction
mechanism in the regulation of vesicular traffic. The findings from
Bomsel and Mostov(1993) indicating that binding of dIgA to the pIgR
stimulates the formation of transcytotic vesicles suggest that ligand
binding generates a signal that is transduced to the intracellular
sorting machinery. Interestingly, in Chinese hamster ovary cells
transfected with muscarinic receptors, endosomal trafficking was
inhibited by carbachol (Haraguchi and Rodbell, 1991). More
specifically, antigen-induced activation of the IgE receptor and
activation of protein kinase C regulate the GTP-dependent binding of
ARF and -COP to Golgi membranes (De Matteis et al.,
1993). Furthermore, the recent identification of phospholipase D as an
effector of ARF (Brown et al., 1993; Kahn et al.,
1993) raises the possibility that a novel signal transduction pathway
may regulate intracellular membrane traffic. Our results of a direct
interaction between ARF and trimeric G proteins suggest that ARF may be
a nexus linking heterotrimeric G proteins and downstream effectors (i.e. PLD). Given the enormous potential for specificity with
24 possible combinations of G
and several ARFs, our present
observations, together with those of others, provide a novel prospect
by which trimeric G proteins and ARF provide fine control of vesicular
traffic and its response to extracellular signals.
Note Added in Proof-While this paper was under review, we became aware of a paper reporting similar results (Franco, M., Paris, S. and Chabre, M.(1995) FEBS Lett.362, 286-290).