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(Received for publication, October 7, 1994; and in revised form, November 11, 1994) From the
Bacterially expressed synapse-specific clathrin assembly
protein, AP-3 (F1-20/AP180/NP185/pp155), bound with high affinity
both inositol hexakisphosphate (InsP InsP AP-3 was independently discovered in a variety of
contexts and has been known as pp155(12) , AP180(13) ,
NP185(14) , and F1-20(15, 16) . pp155,
AP180, and NP185 were shown to be the same protein and renamed
AP-3(17) . F1-20 and AP-3 were then shown to be
identical(11, 18) . Characterization of the
biochemical properties of AP-3 revealed that it is an unusually acidic (12, 13, 16) phosphoprotein (12, 16, 19, 20) and
glycoprotein(20) , which migrates anomalously on
SDS-PAGE(13, 16, 17, 21) . AP-3 has
the functional property that it binds to clathrin triskelia and
promotes their assembly into a homogeneous population of clathrin
cages(13, 21) . AP-3 was first cloned and sequenced in
1992 (16) . AP-3 was expressed in bacteria (11) and
shown to have full clathrin binding and assembly
properties(22) , establishing this as an ideal system in which
to pursue structure-function studies. AP-3 expression is neuronal
specific(14, 18, 23) . Within both the
peripheral and central nervous systems, AP-3 localizes to nerve
terminals(23, 24, 25) . The developmental
expression of AP-3 is coincident with synaptic
maturation(23, 24) . AP-3 is the only clathrin
assembly protein shown to be specific for synapses, which led to the
suggestion that it is involved in synaptic vesicle biogenesis and
recycling(11) . We considered that it would be of particular
significance if AP-3 bound InsP We now
report that AP-3 is indeed another member of this growing family of
vesicle trafficking proteins that bind InsP
Ins(1,2,4,5,6)P Ins(1,4,5)P
Any
background binding of [
Figure 1:
Scatchard analyses of
[
We measured the displacement of
[
Figure 2:
Displacement of
[
Figure 3:
The binding of specific inositol
polyphosphates to AP-3 inhibits clathrin assembly. Clathrin cages were
assembled by GST-AP-3 as described under ``Experimental
Procedures,'' in the presence of the indicated concentrations of
ligands and the % inhibition was calculated. Each data point was
derived from three independent experiments, and the error bars
represent the standard deviations. Because of the different scales,
inhibition of GST-AP-3-mediated clathrin assembly by InsP
Figure 4:
Scatchard analyses of
[
We have found that specific inositol polyphosphates bind to
AP-3 with high affinity and inhibit AP-3-mediated clathrin assembly.
The crucial finding that inositol hexasulfate has at least a 50-fold
lower affinity for AP-3 than InsP A key consequence of our
study with AP-3 is that inhibition of adaptin-mediated clathrin
assembly by specific inositol polyphosphates was previously only known
to be a characteristic of AP-2(7) . Our experiments have now
demonstrated that this is a more widespread phenomenon. It is of
further importance to understand the molecular basis for these effects.
For this task, our results highlight that AP-3 provides the simpler
model, since it is a single 91-kDa polypeptide (16) , whereas
AP-2 is a heterotetramer of 270 K We also think that it is particularly significant that
AP-3, as a new member of this family of inositol polyphosphate binding
proteins, is synaptically
localized(23, 24, 25) . It is also
fascinating to consider that an interaction of InsP PP-InsP
Volume 270,
Number 4,
Issue of January 27, 1995 pp. 1564-1568
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
) (K = 239 nM) and diphosphoinositol
pentakisphosphate (PP-InsP
) (K = 22 nM). The specificity of this ligand
binding was demonstrated by competitive displacement of bound
[
H]InsP. IC values were
as follows: PP-InsP
= 50 nM, InsP = 240 nM, inositol-1,2,4,5,6-pentakisphosphate
(Ins(1,2,4,5,6)P) = 2.2 µM,
inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P)
= 5 µM, inositol-1,3,4,5-tetrakisphosphate
(Ins(1,3,4,5)P
) > 10 µM,
inositol-1,4,5-trisphosphate (Ins(1,4,5)P) > 10
µM. Moreover, 10 µM inositol hexasulfate
(InsS) displaced only 15% of
[H]InsP. The physiological
significance of this binding is the ligand-specific inhibition of
clathrin assembly (PP-InsP > InsP >
Ins(1,2,4,5,6)P); Ins(1,3,4,5,6)P and
InsS did not inhibit clathrin assembly. We also observed
high affinity binding of InsP to purified bovine brain
AP-3. We separately expressed the 33-kDa amino terminus and the 58-kDa
carboxyl terminus, and it was the former that contained the high
affinity inositol polyphosphate binding site. These studies suggest
that specific inositol polyphosphates may play a role in the regulation
of synaptic function by interacting with the synapse-specific clathrin
assembly protein AP-3.
, (
)a near-ubiquitous constituent of
mammalian cells, is a particularly enigmatic polyphosphate. Even the
routes of its synthesis and metabolism remain incompletely
resolved(1) . Indeed, it was only recently that InsP was found not to be the metabolically lethargic compound that
most laboratories had assumed; now it is known that InsP and a diphosphoinositol derivative (PP-InsP)
participate in a rapid, ongoing cycle of phosphorylation and
dephosphorylation(2) . It is unclear how the cell is rewarded
by the considerable investment of ATP in this cycle. More uncertainty
surrounds the intracellular concentration and distribution of
InsP in mammalian cells; total cellular levels are
generally around 15 µM(3) , although there are
examples of cells with around 50 µM InsP(4) . Early suggestions that all of this
InsP was unlikely to be free in the cytosol arose from the
consideration that this polyphosphate would have a very limited
solubility in the cytosolic ionic environment (5) . Indeed,
there is now evidence that much of the cell's InsP could be nonspecifically bound to cellular membranes (6) . Against this puzzling background, the physiological
significance of InsP has also not been determined. However,
one promising line of enquiry follows from the demonstration of tight
binding of InsP to AP-2, an adaptor protein that promotes
the formation of clathrin-coated vesicles involved in receptor-mediated
endocytosis(7, 8, 9) . Binding of InsP inhibits AP-2-mediated clathrin assembly(7) . The idea
that this observation is of some fundamental importance has been
reinforced by the demonstration that InsP also binds with
high affinity to coatomer, a protein complex associated with vesicle
traffic between Golgi cisternae(10) . It is now an exciting
possibility that there may be a family of InsP-binding
proteins that are important to the process of vesicle trafficking. This
consideration has prompted us to pursue the observation that the
synapse-specific clathrin assembly protein AP-3 has some weak homology
with the polyphosphate-binding 
-adaptin domain of
AP-2(11) ., because it is in neuronal
cell types where there is the strongest evidence that levels of
InsP are acutely regulated by extracellular stimuli. For
example, [H]InsP levels in
[H]inositol-labeled N1E-115 neuroblastoma cells
were increased by up to 50% by either carbachol, elevated extracellular
[K], or prostaglandin E1(26) .
[
H]InsP levels in cultured rat
cerebellar granule cells respond in a similar manner to increases in
extracellular [K](27) .
. We also
describe the impact of ligand binding on the clathrin assembly
functions of AP-3. Furthermore, we report on the specificity of this
association, with particular reference to PP-InsP, since
coatomer was found to bind PP-InsP with even higher
affinity than that for InsP(10) .
Materials
Proteins
The proteins GST-AP-3
(AS15AS108
)(11) ,
GST-NH
-33kDa (22) , and GST, were all expressed
exactly as described previously (22) and purified on
glutathione-Sepharose, as described previously(22) . Bovine
brain clathrin and AP-3 were purified from bovine brain clathrin-coated
vesicles as described previously(22) , based on subtle
modifications of (13) . SDS-PAGE analysis of the purified
clathrin revealed three silver-stained bands, corresponding in apparent
molecular weight to clathrin heavy chain, and to the two clathrin light
chains. Clathrin was determined to be free of detectable contaminating
AP-3 by Western blot analysis with the F1-20 monoclonal antibody
utilizing the ECL detection system as described
previously(11) . One cycle of assembly-disassembly was carried
out as described previously (22) . Protein concentrations were
determined spectrophotometrically using extinction coefficients which
were calculated from the reported amino acid sequences of clathrin (29, 30, 31) and AP-3(16) , according
to the relation = number of tryptophan
residues(5690) + number of tyrosine residues(1280)(32) ,
and found to be as follows: AP-3, 39,970, GST-AP-3, 80,650;
GST-AP-3-58kDa-COOH, 63,440; GST-AP-3-33kDa-NH
,
57,890; GST, 40,680. Bovine AP-3 was shown to be free of detectable
contaminating clathrin by Western blot analysis with anti-clathrin
heavy chain monoclonal antibody F21-5 utilizing the ECL detection
system. Monoclonal antibody F21-5 was generated by fusion of spleens
from mice immunized with clathrin which had been purified as described (22) and checked to be free of contaminating AP-3 by Western
blot analysis. The monoclonal antibody was found to be very specific
for clathrin heavy chain and did not display detectable
cross-reactivity with bacterially expressed or bovine AP-3, clathrin
light chains, AP-1 or AP-2. ()Monoclonal antibody
F1-20, has been shown to be specific for
AP-3(11, 15, 16, 23) . A construct
expressing the carboxyl-terminal 58 kDa of AP-3 was constructed by
polymerase chain reaction using two primers: 58k5`,
5`-AGCAGGATCCCCGGGTCTTCTCCAGCCACAACTGTTACA-3` and SZ405,
5`-CTCAGGTTAGTTTTTTCCTATTCAGTCACA-3` and plasmid pGEX3X-F1-20
(AS15) as the template. The polymerase chain reaction
product was digested with XmaI and StuI and the
1.3-kilobase fragment was gel-purified. The XmaI-StuI
fragment (2.2 kilobase) in plasmid pGEX3X-F1-20
(AS15
) was replaced by the 1.3-kilobase fragment. The
resulting construct expresses the carboxyl 58-kDa portion of AP-3 (from
amino acid 304 to the stop codon) fused with the 26-kDa GST fragment in
the amino terminus. This new plasmid is called
pGEX3X-F1-20-COOH-58kDa and was introduced into Escherichia
coli BL21. GST-58-kDa COOH terminus of AP-3 was expressed and
purified under the same conditions as described previously for GST-AP-3 (22) .
Inositol Polyphosphates
Defined mass amounts of
PP-InsP were prepared by phosphorylation of InsP (purchased from Calbiochem and repurified by us on
HPLC(2) ) using kinase activity present in homogenates of the
AR4-2J pancreatoma(2) : 12-ml incubations contained 25 mM HEPES (pH 7.2), 20 mM phosphocreatine, 7 mM MgSO, 5 mM NaATP, 1 mM NaEDTA, 1 mM dithiothreitol, 0.2 mg/ml
phosphocreatine kinase, 5 µM [H]InsP (5000 dpm/nmol) plus 0.3
mg of AR4-2J homogenate protein/ml. After 90 min, the reaction was
quenched with 4 volumes of ice-cold buffer containing 2 mM EDTA, 10 mM triethylamine, 0.1 M Tris-HCl (pH
7.7 at 25 °C). Next, the quenched reaction was divided into 10-ml
aliquots, each of which was applied to an NENSORB Preparative
deproteinizing cartridge. The flow-throughs were saved. Each column was
washed with 2 
4-ml aliquots of the quenching buffer, and all
the flow-throughs were combined and loaded onto a Partisphere SAX HPLC
column. The HPLC gradient was as described previously(28) ,
except that buffers A and B were each supplemented with 1 mM NaEDTA. The PP-InsP peak was saved and
desalted(2) . was prepared by
dephosphorylation of InsP using Aspergillus ficuum phytase (Sigma) which we further purified as described
previously(33) : 0.001 unit (as defined in (33) ) of
phytase were incubated for 25 min at 37 °C in a 3-ml incubation
containing 50 mM BisTris (pH 6), 1 mM EDTA, 0.5
mM EGTA, 0.5% (w/v) bovine serum albumin, 2 mM [H]InsP (30 dpm/nmol). Reactions
were quenched with perchloric acid, neutralized, and chromatographed on
an Adsorbosphere SAX HPLC column(2) . Fractions containing
Ins(1,2,4,5,6)P were saved and desalted and then
rechromatographed on a Partisphere SAX HPLC column(28) . The
Ins(1,2,4,5,6)P was again saved and desalted. was obtained from LC Services, Woburn, MA.
Ins(1,3,4,5)P was obtained from the University of Rhode
Island Foundation (Kingston, RI). Ins(1,3,4,5,6)P was
purchased from Boehringer Mannheim. [H]InsP (NET 1023) and PP-[H]InsP (NET
1093) were obtained from DuPont NEN.Methods
Inositol Polyphosphate Binding Assay
The binding
of inositol polyphosphates to AP-3 was determined by a slight
modification of a polyethylene glycol precipitation
procedure(10) . AP-3 (preparation indicated in the figure
legend) was incubated at 4 °C in 50 µl of a solution containing
25 mM Tris-HCl (pH 7.5 at 25 °C), 5 mg/ml bovine
-globulin, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, [
H]InsP (approximately 20,000 dpm) or
PP-[H]InsP (approximately 3000 dpm).
Other additions are given in the figure legends. After 20 min, protein
was precipitated by addition of 35 µl of ice-cold 30% (w/v)
polyethylene glycol, followed by immediate vortexing. After a further
10 min on ice, samples were centrifuged for 10 min at 10,000 g at 4 °C. The supernatants were carefully aspirated and
the pellets were quickly washed with 1 ml of the incubation buffer
without immunoglobulin. The final pellet was dissolved in 1 ml of 1%
SDS, followed by 8 ml of Monoflow scintillation fluid, and was then
counted for H. Immunoblots with a monoclonal antibody
against AP-3 (F1-20(15) ) indicated that the majority of
the AP-3 protein was precipitated by polyethylene glycol.H]inositol polyphosphates
to -globulin itself (<5% of total) was subtracted from the
total binding observed in the presence of AP-3. We also subtracted
nonspecific binding which was measured in the presence of excess (10
µM) InsP
; this was less than 5% of the total
binding. As a control for the AP-3 preparations that were fusions with
GST, we carried out binding studies with GST, and found that GST by
itself did not bind any of the inositol polyphosphates. The parameters
for the Scatchard plots were calculated using the ``LIGAND''
program developed by the Analytical and Biostatistical Section,
Division of Computer Research and Technology, National Institutes of
Health, Bethesda, MD. In all experiments, data could only be fitted to
a single binding site.Clathrin Assembly Assays
GST-AP-3, and bovine
brain clathrin triskelia, were dialyzed into isolation buffer (pH 6.7).
1.4 mg/ml clathrin triskelia were incubated with either 1.2 mg/ml
GST-AP-3 or 1.2 mg/ml GST (negative control) in ice-cold isolation
buffer for 4 h. Clathrin cage assembly was evaluated by the
sedimentation method (18, 34) . The pellet and
supernatant fractions were analyzed by 10-15% gradient SDS-PAGE.
Clathrin heavy chain was visualized by ECL-Western blotting using a
clathrin heavy chain-specific monoclonal antibody F21-5. The
distribution of clathrin heavy chain between the pellet and supernatant
fractions was quantitated using the Millipore BioImage system with 3cx
scanner. Assembly percentage was calculated as [pellet/(pellet
+ supernatant)] 100. Background sedimentation was
subtracted from the negative control (clathrin triskelia incubated with
GST protein). Under these conditions, 55% of the clathrin triskelia
were assembled into cages by GST-AP-3.
AP-3 Binds to Inositol Polyphosphates with High
Affinity and Specificity
Even in highly purified preparations of
AP-3 from brain, it is difficult to exclude the possibility of
contaminants being present, which even in small quantities might still
contribute significantly to the overall ligand binding parameters.
Therefore, we assessed the binding of bacterially expressed GST-AP-3 to
InsP This protein preparation has been characterized
previously and was shown to bind and assemble clathrin as well as AP-3
purified from bovine brain(22) . We found the average K
for the binding of InsP to GST-AP-3
to be 239 nM, with an average B
of 0.16
mol/mol of protein. A representative Scatchard plot is shown (Fig. 1A). The finding that the B value is less than one may reflect some proteolysis. Because the
protein under analysis was expressed as a fusion with GST, we also
examined the binding of GST to InsP. We found no
significant binding, at concentrations of InsP up to 10
µM. We purified AP-3 from bovine brain cerebral cortex and
found that it bound to InsP with a similarly high affinity
as the bacterially expressed protein (data not shown). InsP was not the ligand with the highest affinity for AP-3. Rather,
PP-InsP, a recently discovered metabolite of
InsP(2) , binds to bacterially expressed GST-AP-3
with an average K of 22 nM, and an
average B of 0.20 mol/mol of protein (Fig. 1B).
H]InsP and
PP-[H]InsP binding to AP-3. GST-AP-3
(0.5-2 µg) was incubated with the indicated concentrations of
InsP or PP-InsP, and the proportions of bound
and free ligand were estimated as described under ``Experimental
Procedures'' (after subtraction of nonspecific binding, which was
determined with 10M ligand, see insets). For InsP
(A), the calculated K is 190 nM, and the B
is 0.17 mol/mol of protein. Three further
experiments gave K values of 170, 400,
and 195 nM with corresponding B
values
of 0.074, 0.18, and 0.2 mol/mol of protein. For PP-InsP (B), the calculated K is
22 nM, and the B
is 0.21 mol/mol of
protein. Two further experiments gave K values of 26 and 17 nM with corresponding B
values of 0.21 and 0.17 mol/mol of
protein.
H]InsP from GST-AP-3 by the
following: PP-InsP, InsP,
Ins(1,2,4,5,6)P, Ins(1,3,4,5,6)P,
Ins(1,3,4,5)P, and Ins(1,4,5)P. As a control
for specificity, we also examined the displacement of
[H]InsP from GST-AP-3 by
InsS. Displacement curves (Fig. 2), and IC values (Table 1) indicate that the relative affinities were
PP-InsP
> InsP > Ins(1,2,4,5,6)P > Ins(1,3,4,5,6)P > Ins(1,3,4,5)P > Ins(1,4,5)P >> InsS. We
conclude that AP-3 has a high affinity binding site for specific
inositol polyphosphates.
H]InsP from AP-3 by competing
ligands. GST-AP-3 (1.3-2.0 µg) was incubated with 5 nM [H]InsP and indicated
concentrations of one of the following competing ligands (from left to right): PP-InsP (inverted
triangles), InsP (closed circles),
Ins(1, 2, 4, 5, 6) P (open circles),
Ins(1, 3, 4, 5, 6) P (triangles), Ins(1,3,4,5)P (closed
squares), and InsS (open squares).
[H]InsP binding, as a percentage of
total [H]InsP, was determined as
described under ``Experimental Procedures.'' All data points
are means of duplicate determinations. Each curve is representative of
two or more experiments.
The Binding of Inositol Polyphosphates to AP-3 Inhibits
Clathrin Assembly
In order to asses the functional consequences
of inositol polyphosphates binding to AP-3, we examined the effects of
inositol polyphosphates on AP-3-mediated clathrin assembly, using a
modification of the quantitative clathrin assembly assay (18, 34) (Fig. 3). The modification was to
avoid dialysis, since we found that if we carried out the quantitative
clathrin assembly assay exactly as described(18, 34) ,
the concentration of inositol polyphosphates changed as a function of
time due to movement through the dialysis membrane. We found strong
inhibition of clathrin assembly by PP-InsP > InsP > Ins(1,2,4,5,6)P. Note that the rank order of
potency for inhibition parallels the relative binding affinities of
these ligands. For each polyphosphate, the values for the K were lower than the concentrations that
inhibited clathrin assembly, but this is not surprising, since the two
assays were, out of necessity, performed under different experimental
conditions. There may be marginal inhibition of assembly by
Ins(1,3,4,5,6)P, and there was no measurable inhibition by
InsS at concentrations up to 150 µM (Fig. 3). Thus, we conclude that the binding of specific
inositol polyphosphates to AP-3 inhibits clathrin assembly.
(open triangle), InsS (closed
triangle),
Ins(1, 2, 4, 5, 6) P (open circle), and Ins(1,3,4,5,6)P (closed circle) is shown in A, and inhibition
by PP-InsP is shown in B.
Mapping of the High Affinity Inositol Polyphosphate
Binding Site to the 33-kDa Amino Terminus of AP3
The
amino-terminal one-third of AP-3 is relatively neutral, with an amino
acid composition typical of a globular
structure(11, 16) . The carboxyl-terminal two-thirds
of AP-3 is extraordinarily acidic, with an unusually high amount of
proline, serine, threonine, and alanine, and a self-repeating
structure(11, 16) . It has been shown that while the
33-kDa amino terminus of AP-3 has the ability to bind to clathrin
triskelia(17, 22) , it cannot assemble clathrin
triskelia into clathrin cages(17, 22) , or bind to
preassembled clathrin cages(22) . Here we have examined the
ability of the bacterially expressed 33-kDa amino terminus of
AP-3(11) , and 58-kDa carboxyl terminus of AP-3, to bind to
inositol polyphosphates. There was no measurable binding of
GST-58kDa-COOH-AP-3 to InsP in the range 5 nM to
10 µM. In contrast, Scatchard analyses revealed that
GST-33kDa-NH-AP-3 binds to InsP with an average K of 173 nM and an average B of 0.77 mol/mol of protein (Fig. 4A) and to PP-InsP with an average K of 76 nM and an average B of 0.60 mol/mol of protein (Fig. 4B). We conclude that the high affinity inositol
polyphosphate binding site is located in the 33-kDa amino terminus of
AP-3.
H]InsP and
PP-[H]InsP binding to the 33-kDa
amino terminus of AP-3. GST-33-kDa NH terminus of AP-3 (0.3
µg) was incubated with the indicated concentrations of InsP or PP-InsP, and the proportions of bound and free
ligand were estimated as described under ``Experimental
Procedures'' (after subtraction of nonspecific binding, which was
determined with 10M ligand, see insets). For InsP
(A), the calculated K is 165 nM, and the B
is 0.86 mol/mol of protein. One further
experiment gave a K value of 180 nM with a corresponding B
value of 0.68
mol/mol of protein. For PP-InsP (B), the
calculated K is 75 nM, and the B
is 0.65 mol/mol of protein. One further
experiment gave a K value of 77 nM with a corresponding B
value of 0.55
mol/mol of protein.
testifies to the protein
being specific for certain polyphosphates of inositol, rather than
simply negative charge density. Furthermore, our observation that
PP-InsP, InsP, and Ins(1,2,4,5,6)P were all more potent ligands than Ins(1,3,4,5,6)P confirms that AP-3 has a distinct preference for a specific
configuration of phosphate groups. Although these findings have
provided useful information on the specificity for
Ins(1,2,4,5,6)P, which had only an 8-fold lower affinity
for AP-3 than InsP, this observation has a limited
physiological bearing, since there is approximately 50-fold less
Ins(1,2,4,5,6)P in cells than InsP. The finding
that the strength of inhibition of clathrin assembly by the various
ligands paralleled their binding affinity for AP-3 provides a
functional as well as structural measure of the specificity. In
addition to investigating the consequences of changes in ligand
concentration upon AP-3 in vivo, another important line of
enquiry will be to determine if alterations in the degree of
glycosylation of the protein(20) , its phosphorylation
state(12, 16, 19, 20) , or
alternative RNA splicing(11, 16, 18) , will
prove to be regulatory processes that act by altering the affinity of
the ligands to modulate clathrin assembly.(35) .
Indeed, our mapping of the high affinity polyphosphate binding site to
the 33-kDa amino terminus of this 91-kDa polypeptide further narrows
the search for the amino acids that comprise a polyphosphate binding
domain. The latter may even provide a general motif for
polyphosphate-dependent influences upon vesicle traffic, which may be
mediated not only by AP-2 and AP-3, but also by coatomer (10) and perhaps other proteins. It is of particular interest
that the 33-kDa amino terminus of AP-3 has been well conserved in
evolution. Cloning of the Xenopus homologue of AP-3 revealed
that while the overall identity between mouse and Xenopus AP-3
was 77%, the identity in the 33-kDa amino terminus was 97%. () with
AP-3 may be at the heart of the provocative finding that injection of
this polyphosphate into the nucleus tractus solitarius of rat brain
resulted in a decrease in both arterial blood pressure and heart
rate(36, 37) . An acceptable molecular basis for these
putative ``neurotransmitter-like'' effects has never been
developed previously. Our data now raise the novel possibility that
extracellularly applied InsP might gain access to synaptic
adaptor proteins such as AP-3, likely through endocytosis, and thereby
perturb synaptic signaling. In this respect, it is notable that
InsP was more potent than Ins(1,3,4,5,6)P at
inducing hypotension and bradycardia (36) ; this is the same
rank order of affinity of these ligands for AP-3. had a 5-10 fold higher affinity for AP-3 compared with
InsP. Therefore our experiments should provide further
impetus to the goal of defining the intracellular distribution of both
of these inositol polyphosphates. Total cellular InsP is
around 15 µM(3) , but it has often been considered
likely that much of this material does not have immediate access to the
cytosol, but instead is sequestered inside an organelle, or bound to
cellular membranes(5, 6) . Indeed, the only enzyme
known to dephosphorylate InsP inside mammalian cells is
itself restricted to the interior of endoplasmic reticulum (33) . However, levels of InsP in N1E-115
neuroblastoma cells and cerebellar granule cells have been reported to
change rapidly in response to extracellular stimuli(26) . Total
cellular levels of PP-InsP are about 5% those of
InsP(2, 28) , but it is also not known
where in the cell this particular compound may be located.
Nevertheless, levels of PP-InsP are regulated by changes in
intracellular Ca(28) , which opens up another
potential molecular basis for the regulation of AP-3 function and
synaptic vesicular traffic by extracellular stimuli.
), inositol hexakisphosphate; PP-InsP,
diphosphoinositol pentakisphosphate; Ins(1,2,4,5,6)P,
inositol-1,2,4,5,6-pentakisphosphate; Ins(1,3,4,5,6)P,
inositol-1,3,4,5,6-pentakisphosphate; Ins(1,3,4,5)P,
inositol-1,3,4,5-tetrakisphosphate; Ins(1,4,5)P,
inositol-1,4,5-trisphosphate; InsS, inositol hexasulfate;
GST, glutathione S-transferase; BSA, bovine serum albumin.))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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A. Chakraborty, M. A. Koldobskiy, K. M. Sixt, K. R. Juluri, A. K. Mustafa, A. M. Snowman, D. B. van Rossum, R. L. Patterson, and S. H. Snyder From the Cover: HSP90 regulates cell survival via inositol hexakisphosphate kinase-2 PNAS, January 29, 2008; 105(4): 1134 - 1139. [Abstract] [Full Text] [PDF]
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 Y.-S. Lee, S. Mulugu, J. D. York, and E. K. O'Shea Regulation of a Cyclin-CDK-CDK Inhibitor Complex by Inositol Pyrophosphates Science, April 6, 2007; 316(5821): 109 - 112. [Abstract] [Full Text] [PDF]
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 I. Stavrou and T. J. O'Halloran The Monomeric Clathrin Assembly Protein, AP180, Regulates Contractile Vacuole Size in Dictyostelium discoideum Mol. Biol. Cell, December 1, 2006; 17(12): 5381 - 5389. [Abstract] [Full Text] [PDF]
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 S. Kim, H. Kim, B. Chang, N. Ahn, S. Hwang, G. Di Paolo, and S. Chang Regulation of transferrin recycling kinetics by PtdIns[4,5]P2 availability FASEB J, November 1, 2006; 20(13): 2399 - 2401. [Abstract] [Full Text] [PDF]
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 C. Auesukaree, H. Tochio, M. Shirakawa, Y. Kaneko, and S. Harashima Plc1p, Arg82p, and Kcs1p, Enzymes Involved in Inositol Pyrophosphate Synthesis, Are Essential for Phosphate Regulation and Polyphosphate Accumulation in Saccharomyces cerevisiae J. Biol. Chem., July 1, 2005; 280(26): 25127 - 25133. [Abstract] [Full Text] [PDF]
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J. W. Verbsky, S.-C. Chang, M. P. Wilson, Y. Mochizuki, and P. W. Majerus The Pathway for the Production of Inositol Hexakisphosphate in Human Cells J. Biol. Chem., January 21, 2005; 280(3): 1911 - 1920. [Abstract] [Full Text] [PDF]
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M. Fujii and J. D. York A Role for Rat Inositol Polyphosphate Kinases rIPK2 and rIPK1 in Inositol Pentakisphosphate and Inositol Hexakisphosphate Production in Rat-1 Cells J. Biol. Chem., January 14, 2005; 280(2): 1156 - 1164. [Abstract] [Full Text] [PDF]
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E. Nagata, H. R. Luo, A. Saiardi, B.-I. Bae, N. Suzuki, and S. H. Snyder Inositol Hexakisphosphate Kinase-2, a Physiologic Mediator of Cell Death J. Biol. Chem., January 14, 2005; 280(2): 1634 - 1640. [Abstract] [Full Text] [PDF]
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 S. T. Safrany Protocols for Regulation and Study of Diphosphoinositol Polyphosphates Mol. Pharmacol., December 1, 2004; 66(6): 1585 - 1591. [Abstract] [Full Text] [PDF]
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X. Pesesse, K. Choi, T. Zhang, and S. B. Shears Signaling by Higher Inositol Polyphosphates: SYNTHESIS OF BISDIPHOSPHOINOSITOL TETRAKISPHOSPHATE ("InsP8") IS SELECTIVELY ACTIVATED BY HYPEROSMOTIC STRESS J. Biol. Chem., October 15, 2004; 279(42): 43378 - 43381. [Abstract] [Full Text] [PDF]
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 M. G. Roth Phosphoinositides in Constitutive Membrane Traffic Physiol Rev, July 1, 2004; 84(3): 699 - 730. [Abstract] [Full Text] [PDF]
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A. Jha, N. R. Agostinelli, S. K. Mishra, P. A. Keyel, M. J. Hawryluk, and L. M. Traub A Novel AP-2 Adaptor Interaction Motif Initially Identified in the Long-splice Isoform of Synaptojanin 1, SJ170 J. Biol. Chem., January 16, 2004; 279(3): 2281 - 2290. [Abstract] [Full Text] [PDF]
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E. A. Orchiston, D. Bennett, N. R. Leslie, R. G. Clarke, L. Winward, C. P. Downes, and S. T. Safrany PTEN M-CBR3, a Versatile and Selective Regulator of Inositol 1,3,4,5,6-Pentakisphosphate (Ins(1,3,4,5,6)P5): EVIDENCE FOR Ins(1,3,4,5,6)P5 AS A PROLIFERATIVE SIGNAL J. Biol. Chem., January 9, 2004; 279(2): 1116 - 1122. [Abstract] [Full Text] [PDF]
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D. I. Fisher, S. T. Safrany, P. Strike, A. G. McLennan, and J. L. Cartwright Nudix Hydrolases That Degrade Dinucleoside and Diphosphoinositol Polyphosphates Also Have 5-Phosphoribosyl 1-Pyrophosphate (PRPP) Pyrophosphatase Activity That Generates the Glycolytic Activator Ribose 1,5-Bisphosphate J. Biol. Chem., November 27, 2002; 277(49): 47313 - 47317. |
ABSTRACT
