(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) (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.
InsP, (
)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) .
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, 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 now
report that AP-3 is indeed another member of this growing family of
vesicle trafficking proteins that bind InsP. 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) .
Ins(1,2,4,5,6)P 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.
Ins(1,4,5)P 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.
Any
background binding of [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.
Figure 1:
Scatchard analyses of
[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 10
M 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.
We measured the displacement of
[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.
Figure 2:
Displacement of
[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.
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 (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.
Figure 4:
Scatchard analyses of
[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 10
M 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.
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 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.
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(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%. (
)
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 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.
PP-InsP 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.