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INTRODUCTION |
Many G protein-coupled receptors
(GPCRs),1 such as the
2-adrenergic receptor (
2-AR), utilize
classical clathrin-coated vesicle pathways for internalization. Several
lines of evidence indicate that phosphorylation of the receptor by a G
protein-coupled receptor kinase (GRK) followed by
-arrestin binding
are crucial steps in this process (reviewed in Refs. 1-3). The role of
GRK-mediated receptor phosphorylation in the internalization of
receptors is to facilitate the binding of
-arrestins.
-Arrestins
serve a dual role in the regulation of GPCRs. First, they mediate rapid desensitization by binding to GRK-phosphorylated receptors, sterically interdicting signaling to the G protein (4). Second, they target receptors for internalization via clathrin-coated pits, allowing subsequent dephosphorylation and resensitization of the receptors (5).
Thus, a mutated form of the
2-AR (Y326A) that is a poor substrate for the GRKs fails to undergo agonist-dependent
internalization. However, receptor internalization can be rescued by
overexpressing GRKs (6). Similarly, overexpressing
-arrestin1 with
the Y326A mutant also rescues receptor internalization (7). Further
confirming the essential role of
-arrestins in the internalization
process is the finding that dominant negative forms of
-arrestin1,
namely V53D (8) and
arr1S412D (5), dramatically impair
agonist-stimulated receptor sequestration.
It has been previously demonstrated that
-arrestin1 and 2 bind
directly and stoichiometrically to clathrin (9) and that the function
of
-arrestin1 in GPCR internalization is regulated by
phosphorylation/dephosphorylation of the
-arrestin1 molecule (5).
Cytoplasmic
-arrestin1 is constitutively phosphorylated on a
carboxyl-terminal serine (Ser-412) and is recruited to the plasma
membrane upon agonist stimulation of the receptor, where it becomes
rapidly dephosphorylated. Dephosphorylation of
-arrestin1 is
required for clathrin binding and the subsequent targeting of receptors
to clathrin-coated pits but not for receptor binding and receptor
desensitization. It is interesting to note that other members of the
arrestin family (visual arrestin,
-arrestin2, and splice variants)
do not possess the Ser-412 residue and are therefore not subject to the
same regulation as
-arrestin1. Whether the ability of
-arrestin1
to promote GPCR internalization arises solely as a consequence of its
ability to bind clathrin or whether additional
-arrestin1-binding
proteins are involved in this process remains to be determined. In an
attempt to further define the role of
-arrestin1 in the regulation
of GPCR function, we used the yeast two-hybrid system to screen for
novel
-arrestin1-binding proteins.
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EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Screen--
The full cDNA of rat
-arrestin1 was subcloned into the yeast expression vector pGBT9
using EcoRI and BamHI. Following amplification in
Escherichia coli, the pGBT9
-arrestin1 plasmid was
transformed into yeast strain HF7c, and a yeast two-hybrid screen was
carried out essentially according to the CLONTECH
MATCHMAKER protocol using a GAL4 activation domain fusion library in
pGAD10 (MATCHMAKER rat whole brain cDNA library,
CLONTECH). Interacting proteins were isolated by
growth selection on Trp, His, and Leu dropout plates and subsequently
by filter
-galactosidase assays. Isolated library clones were
analyzed by DNA sequencing using an ABI automated DNA sequencer (Howard
Hughes Nucleic Acid Facility, Duke University).
Cell Culture and Transfection--
The plasmids of interest were
transfected into either HEK 293 cells (for sequestration assays) or COS
cells (cellular coimmunoprecipitation) using LipofectAMINE according to
the manufacturer's instructions (Life Technologies, Inc). To determine
the association of
-arrestin1 and NSF, COS cells were cotransfected
with either Flag-tagged
-arrestin1 (or Flag-tagged
arr1S412A and
arr1S412D) and/or His6-NSF-c-Myc, pcDNA3 empty vector
was used as a control. Cells were harvested in lysis buffer (LB; 50 mM HEPES, pH 7.4, 0.5% Nonidet P-40, 250 mM
NaCl, 10% glycerol, 2 mM EDTA, 0.5 mM ATP, 1 mM dithiothreitol, 1 µM phenylmethylsulfonyl
fluoride 25 µg/ml apoprotin, and 1 µM leupeptin). The
-arrestins were immunoprecipitated using a polyclonal M2 antibody
specific to the Flag epitope. The proteins were resolved by SDS-PAGE.
Western blot analysis was performed using a monoclonal antibody
specific for NSF (6E6). NSF was visualized using either ECL or
quantitated using alkaline phosphatase-conjugated secondary antibody
(Amersham) followed by phosphoimager analysis. The nitrocellulose was
stripped of immunoglobulin, and
-arrestin1 was visualized or
quantitated using an polyclonal antibody specific for
-arrestin1
(19) to confirm equal expression of the
-arrestins.
Determination of Nucleotide Dependence of the
-Arrestin1-NSF
Interaction--
Cellular coimmunoprecipitations were carried out as
described above. COS-1 cells were cotransfected with plasmids encoding either empty vector, Flag-
-arrestin1, NSF, or Flag-
-arrestin1 plus NSF. Flag-
-arrestin1 was immunoprecipitated using an M2 monoclonal antibody specific for the Flag epitope. Immunoprecipitates were washed three times with LB buffer, then equally divided, and
transferred to fresh tubes. One-half of each immunoprecipitate was
given a final wash with LB buffer containing 0.5 mM ATP and 2 mM EDTA (ATP/EDTA), and the other half was washed with LB
buffer containing 2 mM EDTA plus 10 mM
MgCl2 (ATP/Mg2+) to allow ATP hydrolysis on the
immunoprecipitated NSF. Immunoprecipitates were resolved by SDS-PAGE
and transferred to nitrocellulose membranes. NSF was visualized using
the 6E6 monoclonal antibody and quantitated by phosphoimager analysis.
Immunoblots were stripped of IgG and reprobed for
-arrestin1 using
an antibody specific to
-arrestin1, such that the amount of NSF
immunoprecipitated under nonhydrolyzable versus hydrolyzable
conditions could be normalized to the amount of
-arrestin1 immunoprecipitated.
2-AR Sequestration as Determined by Flow
Cytometry--
Agonist-induced
2-AR internalization was
determined by immunofluorescence flow cytometry analysis as has been
described previously (31). HEK 293 cells were transiently transfected
with the plasmid encoding a Flag-tagged
2-AR and either
a pcDNA3 control vector, a
-arrestin1 expression plasmid
(
-arrestin1 or
arr1S412D), an NSF expression vector, or NSF with
arr1S412D. Receptor fluorescence was defined as signal above the
fluorescence of untransfected cells. Sequestration is defined as the
fraction of total cell surface receptors that are removed from the
plasma membrane (and thus are not accessible to antibodies added to the
cells) following agonist treatment.
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RESULTS AND DISCUSSION |
The Yeast Two-hybrid System Identifies NSF as a
-Arrestin1-binding Protein--
To identify novel proteins that
interact with
-arrestin1 we screened a rat brain cDNA library
using a full-length
-arrestin1 cDNA as bait in the yeast
two-hybrid system screen (10). Several clones positive for
His
cell growth and
-galactosidase expression were
isolated including a cDNA clone encoding a portion of the
amino-terminal region of NSF
(N-ethylmaleimide-sensitive
fusion protein (NSF) (Fig.
1). This clone was found to require the
Gal4 DNA-binding domain-
-arrestin1 fusion for activity. NSF is an
ATPase whose hydrolytic activity has previously been demonstrated to
play an essential role in intracellular membrane trafficking (11).
Through its interaction with soluble NSF attachment proteins (SNAPs), NSF binds to a group of membrane receptors referred to as SNAP receptors (SNAREs) to form a complex known as the "20 S particle" (12). The ATPase activity of NSF disassembles the 20 S complex. NSF
forms a homohexameric protein complex (13, 14) wherein each subunit
contains three primary domains: an amino-terminal domain (N-domain)
required for SNAP/SNARE binding and two ATPase domains, referred to as
D1 and D2, required for fusion complex disassembly and hexamer
formation respectively (Fig. 1) (15, 16). The region of NSF found to
bind
-arrestin1 lies within the N-domain, identified as the binding
domain for other proteins known to interact with NSF (16-18).

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Fig. 1.
Partial amino acid sequence of rat NSF.
The amino acid sequence of the yeast two-hybrid cDNA clone isolated
as a potential binding partner for -arrestin1 and its location
within the NSF molecule is indicated. Each NSF subunit comprises three
domains, the N-domain, essential for SNAP/SNARE binding, and two
ATP-binding domains (D1 and D2), required for fusion complex
disassembly and hexamer formation, respectively.
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-Arrestin1 Binds NSF in Vitro and in Intact Cells--
Because
the yeast two-hybrid system can detect weak interactions that may not
occur under physiological conditions, we sought to confirm the yeast
interaction data both in vitro and in a mammalian expression
system. Recombinant
-arrestin1 and recombinant NSF (both purified
from E. coli) were incubated in the presence of ATP/EDTA
(nonhydrolyzable conditions).
-Arrestin1 was immunoprecipitated using a polyclonal antibody directed against
-arrestin1 (19). The
immunoprecipitates were subsequently probed for NSF using a monoclonal
antibody directed against this protein (Fig.
2A, upper panel). A
similar experiment was performed whereby NSF was immunoprecipitated and
the immunoprecipitates were immunoblotted for
-arrestin1 (Fig.
2A, lower panel).
-Arrestin1 and NSF formed a
complex in vitro in a manner that was dependent upon the
concentration of the two protein components.

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Fig. 2.
-Arrestin1 binds NSF in
vitro and in intact cells. A, NSF (50 pmol)
was incubated with increasing concentrations of -arrestin1 (0-50
pmol) in the presence of ATP/EDTA. -Arrestin1 was immunoprecipitated
(IP) using a -arrestin1-specific polyclonal antibody. The
immunoprecipitates were resolved by SDS-PAGE and immunoblotted
(IB) for NSF using a monoclonal antibody specific for NSF.
The upper panel shows that as increasing amounts of
-arrestin1 are immunoprecipitated, increasing amounts of NSF can be
visualized. Similarly, if -arrestin1 (50 pmol) is incubated with
increasing amounts of NSF (0-50 pmol) and NSF is immunoprecipitated,
increasing amounts of -arrestin1 are recovered (lower
panel). B, when NSF and Flag-tagged -arrestin1 (wild
type, arr1S412A, or arr1S412D) are coexpressed in COS cells and
the -arrestins immunoprecipitated using an antibody specific to the
Flag epitope, equal amounts of NSF are coimmunoprecipitated. Results
are representative of four independent experiments.
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To determine whether NSF and
-arrestin1 associate in intact cells, a
cDNA encoding NSF was cotransfected into COS cells with cDNA
encoding Flag-tagged
-arrestin1. The association of NSF with
-arrestin1 was examined by immunoprecipitating
-arrestin1 and
analyzing the isolated immunoprecipitates for NSF content. As shown in
Fig. 2B, in the absence of NSF overexpression, a complex of
-arrestin1 and endogenous cellular NSF is detected (Fig.
2B, compare no
arr1 with
arr1 only). Moreover,
overexpression of NSF increases the amount of
-arrestin1-NSF complex
immunoprecipitated (Fig. 2B, compare
arr1 only and
arr1 + NSF). These results demonstrate that
-arrestin1 and NSF
interact in cells. Coupled with the data showing an association between
these proteins in vitro, this suggests that the
-arrestin1-NSF interaction may be of physiological relevance.
It has previously been shown that the phosphorylation state of
-arrestin1 regulates its endocytic function; the dephosphorylated form of
-arrestin1 preferentially binds to clathrin and promotes GPCR internalization (5). The binding of
-arrestin1 to the
2-AR, however, is independent of its phosphorylation
state (5). Therefore, we investigated the effect of
-arrestin1
phosphorylation on NSF binding. Two
-arrestin1 phosphorylation
mutants, where Ser-412 has been replaced with either alanine
(
arr1S412A) or aspartate (
arr1S412D) have been described
previously (5). The
arr1S412A mutant has been shown to simulate the
dephosphorylated form of
-arrestin1, whereas the
arr1S412D mutant
has been shown to mimic its phosphorylated form. The association of NSF
with wild type
-arrestin1,
arr1S412A, or
arr1S412D was
comparable (Fig. 2B). The observation that
-arrestin1-NSF
complex formation is not dependent on the phosphorylation state of
-arrestin1 suggests that NSF could potentially interact with
-arrestin1 either in the cytosol (phosphorylated
-arrestin1)
and/or at the plasma membrane (dephosphorylated
-arrestin1).
-Arrestin1 has been shown to translocate from the cytosol to the
plasma membrane and colocalize with clathrin-coated vesicles following
agonist activation of the
2-AR (8). Thus, interaction of
-arrestin1 with NSF may serve to target NSF to the clathrin-coated
vesicles following GPCR activation.
-Arrestin1-NSF Complex Formation Is
ATP-dependent--
The association of NSF with its
previously identified binding partners (SNAPs, SNAREs, and more
recently the GluR2 subunit of the AMPA receptor) (17, 18) exhibits an
ATP dependence. NSF has been shown to bind stably to SNARE complexes
and does not dissociate from them when locked in the ATP state (13). To
determine whether
-arrestin1 binding to NSF is also modulated by the
nucleotide status of NSF, immunoprecipitations from cells were
performed either in the presence of ATP/EDTA or ATP/Mg2+
(ATP nonhydrolyzable and hydrolyzable conditions, respectively). As
seen in Fig. 3. NSF preferentially
coimmunoprecipitated with
-arrestin1 in the presence of ATP/EDTA.
The binding of NSF and
-arrestin1 was significantly impaired under
conditions in which ATP is hydrolyzed (ATP/Mg2+). Notably,
a mutant NSF capable of binding but not hydrolyzing ATP (E329Q;
described in Ref. 15) showed no significant difference in
-arrestin1
binding in the presence of ATP/EDTA or ATP/Mg2+ (data not
shown).

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Fig. 3.
The -arrestin1-NSF
complex formation is ATP-dependent. COS cells were
transiently transfected with plasmids encoding Flag-tagged
-arrestin1 (5 µg) or NSF (5 µg) as controls or Flag-tagged
-arrestin1 and NSF (5 µg of each). Cells were harvested in lysis
buffer (containing either 0.5 mM ATP/2 mM EDTA
or 0.5 mM ATP/4 mM MgCl2) 48 h
post-transfection. -Arrestin1 was immunoprecipitated using an
antibody specific to the Flag epitope. The precipitates were resolved
by SDS-PAGE and immunoblotted for NSF. The amount of NSF bound was
normalized to the amount of -arrestin1 immunoprecipitated. Results
represent the means ± S.E. for seven independent
experiments.
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Overexpression of NSF Potentiates
2-AR
Internalization--
-Arrestin1 has been shown to promote
internalization (also referred to as sequestration) of agonist-occupied
2-AR (7). If the
-arrestin1-NSF interaction is
important in regulating GPCR function, then given the well
characterized role of NSF in vesicle trafficking, overexpression of NSF
might also regulate
2-AR internalization. Thus, HEK 293 cells were transfected with Flag-tagged
2-AR alone or
with wild type
-arrestin1,
arr1S412D, NSF, or NSF and
arr1S412D. Consistent with previously published results (20),
overexpression of
2-AR with
-arrestin1 in HEK 293 cells results in a modest enhancement of
2-AR
sequestration, and overexpression of the
arr1S412D (5) mutant
results in a reduction of
2-AR sequestration compared
with cells transfected with
2-AR only (Fig.
4). Dramatically, NSF overexpression
enhances
2-AR sequestration approximately 2-fold.
Furthermore, coexpression of NSF and the
arr1S412D mutant in HEK 293 cells appears to rescue the dominant negative effect of
arr1S412D,
such that sequestration was found to be comparable with that in cells
expressing
2-AR only (Fig. 4). Coexpression of wild type
-arrestin1 with NSF did not further enhance NSF-stimulated
2-AR sequestration (data not shown).

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Fig. 4.
Overexpression of NSF potentiates
2-AR internalization. Plasmid DNA
encoding Flag tagged 2-ARs (1 µg) was transiently
transfected into HEK 293 cells together with 10 µg of empty vector
pCMV5 (control) or -arrestin1 and pCMV5 (5 µg of each) or
arr1S412D and pCMV5 (5 µg each) or NSF and pCMV5 (5 µg of each)
or arr1S412D and NSF (5 µg each). Sequestration was determined by
flow cytometry analysis as described previously (27). Receptor
sequestration is expressed as the percentage of loss of cell surface
immunofluorescence following a 20-min exposure to 10 µM
isoproterenol at 37 °C. Results are the means ± S.E. of three
to five independent experiments.
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NSF exists in both cytosolic and membrane-associated forms and was
originally purified on the basis of its ability to complement in
vitro intra-Golgi transport reactions that had been blocked by
treatment with N-ethylmaleimide (21). NSF has a general role in vesicular transport and functions at most if not all vesicular transport events by virtue of its action on SNARE proteins. SNARE proteins have been classified into two families known as v-SNAREs (vesicle membrane receptor) and t-SNAREs (target membrane receptor) (12). The cognate pairing of a v-SNARE with a t-SNARE leads to the
formation of a docking complex for membrane fusion to occur. The
formation of this complex, termed the 20 S particle, is
ATP-dependent. ATP hydrolysis by NSF disassembles the 20 S
complex releasing the v-SNARE and t-SNAREs, thereby recycling these
components for future fusion events (reviewed in Ref. 11). The
observation that the neuronal SNAREs, syntaxin (v-SNARE) and
synaptobrevin (t-SNARE), no longer interact with NSF following one
round of ATP hydrolysis indicates that some structural modification
occurs in one or both of these NSF-binding proteins (22). Taken
together, these observations suggest that NSF promotes conformational
changes in proteins with which it interacts in an
ATP-dependent manner.
That the function of NSF is not restricted to vesicular transport is
suggested by the observation that NSF binds to the AMPA receptor (17,
18, 23). AMPA receptors are a class of ionotropic glutamate receptors
that mediate fast synaptic transmission and are composed of subunits
GluR1-GluR4. Notably, NSF can bind directly to the GluR2 subunit of the
AMPA receptor, providing the first example of an NSF cell surface
receptor interaction (17, 18, 23). NSF and GluR2 exist in a complex in
brain extracts not only with each other but also with SNAPs (18, 23).
The GluR2-SNAP-NSF complex resembles the SNARE-SNAP-NSF complex in
being disassembled by ATP hydrolysis (18). However, in contrast to the
classical 20 S particle, NSF can bind directly to GluR2 independently
of SNAPs. The binding site for NSF maps to a short region of the cytoplasmic tail of GluR2 distinct from the very carboxyl-terminal domain that interacts with the PDZ protein, GRIP (24). GRIP is believed
to serve as a molecular scaffold on which a large complex of proteins
is constructed, including AMPA receptors. It has been suggested that
ATP hydrolysis by NSF may disassemble AMPA receptor interaction with
GRIP leading to mobilization, insertion or internalization of the
receptor (reviewed in Ref. 25).
Identification of an interaction between
-arrestin1 and NSF brings
together two proteins previously found to be associated with
clathrin-coated vesicles.
-Arrestin1 has been shown to interact with
clathrin and target receptors to clathrin-coated pits for internalization (9). NSF has been shown to be associated with membranes
derived from clathrin-coated vesicles (26). Purified clathrin-coated
vesicles not only contain significant amounts of NSF but are enriched
in the ability to support formation of 20 S fusion complexes. The
current model for
2-AR internalization requires
agonist-activated receptor to recruit
-arrestin to the plasma
membrane, where it binds receptor. Following receptor binding,
-arrestin1 targets the receptor to clathrin and promotes receptor internalization.
By analogy with the many proposed roles of NSF in vesicular transport,
one can envisage several possible functions of NSF binding to
-arrestin1 that might involve the NSF-driven
association/disassociation of
-arrestin1 with/from receptor and/or
other components of the clathrin-coated vesicle. One potential role for
the interaction of NSF with
-arrestin1 may be to alter the
conformation of
-arrestin1, thereby regulating the interaction of
this protein with other components of the endocytic pathway. One
-arrestin1-binding partner whose interaction with
-arrestin1
could potentially be facilitated by NSF is clathrin. This may provide a
potential explanation for the observation that overexpression of
NSF is able to rescue the dominant negative effect of the
arr1S412D mutant.
arr1S412D is believed to behave as a dominant
negative with regards to
2-AR internalization due to a
reduced ability to bind clathrin (5). Because NSF binds equally well to
arr1S412D and wild type
-arrestin1, NSF could potentially induce
conformational changes in
arr1S412D, increasing its affinity for
clathrin, thus allowing internalization to proceed. Whether or not NSF
has effects on regulating the interaction of
-arrestin with clathrin
(and potentially other coat components) remains to be determined. A
possible role of NSF in
2-AR internalization by
mediating vesicle fusion during endocytosis is presently unclear, because any potential SNAREs for this process have yet to be identified.
An alternative role for the NSF-
-arrestin1 interaction, by analogy
to the proposed role of NSF in the AMPA-GRIP interaction, is that NSF
binding to
-arrestin1 might involve the NSF-driven dissociation of
the
2-AR-
-arrestin1 complex from elements of the
cytoskeleton. NSF ATP hydrolysis might possibly release the receptor
from some constraining influence, leading to mobilization and
internalization. A detailed investigation of the function of
-arrestin1-NSF complex formation may reveal hitherto unsuspected roles for NSF in clathrin coat-mediated endocytosis.