Cooperative Formation of the Ligand-binding Site of the Inositol
1,4,5-Trisphosphate Receptor by Two Separable Domains*
Fumio
Yoshikawa
§,
Hirohide
Iwasaki
,
Takayuki
Michikawa
,
Teiichi
Furuichi
¶, and
Katsuhiko
Mikoshiba
From the
Department of Molecular Neurobiology,
Institute of Medical Science, University of Tokyo, Minato-ku,
Tokyo 108-8639 and the
Developmental Neurobiology Laboratory,
Brain Science Institute, Institute of Physical and Chemical Research
(RIKEN), Saitama 351-0198, Japan
 |
ABSTRACT |
Limited trypsin digestion of mouse cerebellar
membrane fractions leads to fragmentation of the type 1 inositol
1,4,5-trisphosphate receptor (IP3R1) into five major
components (Yoshikawa, F., Iwasaki, H., Michikawa, T., Furuichi, T.,
and Mikoshiba, K. (1999) J. Biol. Chem. 274, 316-327). Here we report that trypsin-fragmented mouse IP3R1 (mIP3R1) retains significant inositol
1,4,5-trisphosphate (IP3) binding activity that is
comparable to the intact receptor in affinity, capacity, and
specificity. This is despite the fact that the IP3-binding
core (residues 226-578), which is close to the minimum for high
affinity binding, is completely split into two tryptic fragments at the
Arg-343 and/or Arg-345, around the center of the core. Furthermore, we
have examined whether binding activity could be complemented in
vitro by mixing two distinct glutathione
S-transferase (GST) fusion proteins, which were
respectively composed of residues 1-343 and 341-604, almost
corresponding to two split binding components, and separately expressed
in Escherichia coli. The GST-fused residues 1-343 (GN)
showed no binding affinity for IP3, whereas the GST-fused
residues 341-604 (GC) displayed weak but definite activity with an
affinity >100-fold lower than that of the native receptor. Upon mixing
of both GN and GC, a high affinity site comparable to the native site
appeared. We suggest that the IP3-binding pocket consists
of two non-covalently but tightly associated structural domains each of
which has a discrete function: the C-terminal domain alone has low
affinity for IP3, whereas the N-terminal one alone is
incapable of binding but is capable of potentiating binding affinity.
 |
INTRODUCTION |
Inositol 1,4,5-trisphosphate
(IP3)1 is a
second messenger that mediates Ca2+ release from
intracellular stores by binding to the IP3 receptor (IP3R) which is a tetrameric IP3-gated
Ca2+ release channel (1, 2). Cerebellum has an
extraordinary density of specific IP3-binding sites (3) and
purified cerebellar IP3R protein binds IP3 in a
stoichiometric manner, namely one subunit for one IP3
(4-6). Ca2+ release experiments using various synthetic
inositol phosphates have suggested that molecular recognition of
IP3 is markedly stereospecific (7). Type 1 IP3R
(IP3R1) is the neuronal type and predominates in cerebellar
Purkinje cells (8-10). It is 2749 amino acids long (molecular mass
about 313 kDa) and is structurally divided into three parts as follows:
a large N-terminal cytoplasmic arm region (residues 1-2275), a
putative six membrane-spanning region clustered near the C terminus
(residues 2276-2589) which is thought to constitute an ion channel by
forming a tetramer, and a short C-terminal cytoplasmic tail region
(residues 2590-2749) (11). A series of deletion mutants showed that
the IP3R1 binds IP3 within the N-terminal 650 amino acids, independently of tetramer formation (12, 13).
We previously demonstrated the structural basis for molecular
recognition of IP3 by mouse IP3R1
(mIP3R1) (14). The minimum region for high affinity binding
has been localized within the 353 residues, 226-578, so that it
appears to be close to the binding "core." Within the core region,
we have identified 10 important basic amino acid residues all of which
are well conserved in all IP3R family proteins cloned to
date: three (Arg-265, Lys-508, and Arg-511) are critical and the other
seven are required for specific binding. Nahorski and Potter (7)
predicted that ionic interactions of positive charges on a binding site
with negative charges on the three phosphate groups of IP3
would make major contributions to specific recognition and binding.
Thus, we have proposed that the IP3-binding core forms a
pocket with a positively charged inner surface lining of these basic
residues which recognizes and binds a negatively charged
IP3 ligand. Interestingly, all members of the
IP3R family share extensive homology in the core sequence,
except that the IP3R1 has an alternative splicing SI region
(15 residues, 318-332) (9, 15), adjacent to which ~30 residues form
the longest stretch of characteristic diversity within the family. An
internal deletion of residues 316-352 leads to loss of binding (13),
although neither the presence nor the absence of the SI segment
(mIP3R1SI+ nor mIP3R1SI
subtype,
respectively) significantly affects the
binding,2 suggesting that the
diversed stretch is not a prerequisite for binding but that its
boundary should be strictly defined.
Recently, we have shown that limited trypsin digestion of mouse
cerebellar membrane fractions causes fragmentation of the mIP3R1 into five major trypsin-resistant polypeptides and
that these five tryptic fragments I-V have tight structural-functional coupling because of the following: (i) co-sedimentation of all four
cytoplasmic peripheral fragments I-IV (40/37, 64, 76, and 40/36 kDa)
together with the membrane-spanning integral fragment V (91 kDa) by
centrifugation to pellet membrane proteins and immunoprecipitation with
C terminal-specific antibody, and (ii) retention of
IP3-induced Ca2+ release channel activity in
such fragmented mIP3R1. As a result of trypsinization, the
IP3-binding region is cleaved at the carboxyl side of
Arg-343 and/or Arg-345, around which is the alternative splicing SI
region, into two polypeptides: fragments I and II. These results have
demonstrated that the native IP3-binding region consists of
two major tightly folded structural components connected by one exposed
loop near the SI region; the latter loop is very susceptible to trypsin
attack, whereas the former two components are not. Similarly, fragments
III, IV, and V would be reflected by well folded conformation of the
native mIP3R1 channel. Thus, determination of the
structure-function relationships among these components might clarify
the molecular mechanism of ligand binding and channel opening of the
mIP3R1 channel.
In the present study, we describe the structure-function relationships
of the split tryptic IP3-binding region. We have
characterized the IP3 binding properties of
trypsin-fragmented mIP3R1, and we have demonstrated that
the tryptic mIP3R1, completely fragmented into five
polypeptides, still retains specific IP3 binding activity comparable to that of the intact receptor. To analyze further the split
IP3 binding components, we have separately synthesized two
glutathione S-transferase (GST) fusion proteins with these two components, and we have shown that high affinity binding site can
be reconstituted in vitro by complementation with both
distinct fusion proteins, each of which alone has no (N-terminal
component) or low affinity (C-terminal component) binding. From these
data, we propose that the functional structure of the
IP3-binding pocket consists of two well folded structural
domains that are non-covalently but tightly associated and a diverse
loop-like structure between these two domains.
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EXPERIMENTAL PROCEDURES |
Materials--
N-Tosyl-L-phenylalanyl
chloromethyl ketone-treated bovine pancreas trypsin, soybean trypsin
inhibitor, and
-globulin were purchased from Sigma;
[3H]IP3 was from NEN Life Science Products;
D-myo-inositol 1,4,5-trisphosphate was from
Dojindo; D-myo-inositol 2,4,5-trisphosphate and
D-myo-inositol 1,3,4,5-tetrakisphosphate were
from Calbiochem; polyethylene glycol (PEG) 6000 was from Wako Pure
Chemical; Solvable and Atomlight were from Packard; reduced glutathione
was from Boehringer Mannheim, and GST fusion vector pGEX-2T and
glutathione-Sepharose 4B were from Amersham Pharmacia Biotech.
Site-specific anti-mIP3R1 antibodies (polyclonal antibodies
N1, N3, anti-(1718-31), and 1ML1; monoclonal antibodies 10A6 and
18A10) have been described (18).
Preparation of Membrane Fractions from Mouse Cerebellum--
ddY
mice (8-10 weeks old; Nippon SLC, Japan) were anesthetized and then
decapitated, and cerebella were quickly dissected. The cerebella were
homogenized by 10 strokes (850 rpm) in an ice-chilled glass Teflon
Potter homogenizer containing 9 volumes of 0.32 M sucrose,
1 mM EDTA, 100 µM phenylmethylsulfonyl
fluoride (PMSF), 10 µM pepstatin A, 10 µM
leupeptin, and 5 mM Tris-HCl, pH 7.4. The homogenate was
centrifuged at 1,000 × g for 15 min at 4 °C. The
supernatant was re-centrifuged under the same conditions to completely
remove P1 fraction. The second supernatant was centrifuged at
105,000 × g for 60 min at 2 °C. The precipitate
(crude microsome) was resuspended with binding buffer (1 mM
EDTA, 1 mM 2-mercaptoethanol, and 50 mM
Tris-HCl, pH 8.0 (at 4 °C)) to give a final concentration of about
15 mg/ml protein, frozen in liquid nitrogen, and stored at
80 °C
until use. Protein concentrations were determined by a Bio-Rad protein
assay kit using bovine serum albumin as a reference.
Trypsin Digestion--
Microsomal fraction (1 mg/ml protein) was
incubated with the desired concentration of trypsin at 35 °C in
trypsinizing buffer (120 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 20 mM Tris-HCl, pH 8.0). The
reaction was quenched with 10-fold weight excess of soybean trypsin
inhibitor and 0.1 mM PMSF.
IP3 Binding Assay--
IP3 binding was
carried out by incubating sample protein with 9.6 nM (or
4.8 nM for Scatchard analysis of microsomal fraction) [3H]IP3 in 100 µl of binding buffer for 10 min at 4 °C. The IP3/protein mixture was added to 4 µl
of
-globulin (50 mg/ml) and 100 µl of PEG precipitation buffer
(30% (w/v) PEG6000, 1 mM EDTA, and 50 mM
Tris-HCl, pH 8.0, at 4 °C) and incubated for 5 min at 4 °C. The
protein-PEG complexes were pelleted by centrifugation at 18,000 × g for 5 min at 4 °C. The pellet was dissolved in 180 µl
of Solvable and then neutralized by adding 18 µl of acetic acid. The
radioactivity of the neutralized samples was measured by mixing 5 ml of
Atomlight followed by counting in a liquid scintillation counter
(Beckman). The specific binding was defined as total binding minus
nonspecific binding that was measured in the presence of 2-100
µM cold IP3.
In Scatchard plots, lines were fit to the data by the least squares
method using either the single site relationship B/F =
B/Kd + Bmax/Kd or the two-site
relationship B/F = Bmax1/(Kd1 + F) + Bmax2/(Kd2 + F) as appropriate, where B is the specific
IP3 binding in pmol/mg protein, F is the
concentration of the free IP3, Kd is
dissociation constant for each separate site, and
Bmax represents the maximal number of binding
sites for each site (16).
Construction, Expression, and Purification of GST Fusion
Protein--
Standard methods (PCR, cloning, and DNA sequencing) for
construction of the mIP3R1 cDNA-derived plasmids were
as described previously (14). DNA clones derived from PCR products were
all confirmed by sequencing. To generate fusion constructs between GST
and the IP3-binding region of mIP3R1, foreign
nucleotide fragments as described below were attached to the
mIP3R1 cDNA by PCR using appropriately designed
primers. A BamHI site (GGATCC) was attached at the 5'
terminus of the initiation codon (ATG) of the mIP3R1 for
the GST-fused residue 1-343 (GN and GN(R265Q)) and at the 5' terminus
of Arg-341 codon for the GST-fused residues 341-604 (GC), and
TAAGAATTC (stop codon + EcoRI site) was attached at the 3'
terminus of Arg-343 codon for the GN and GN(R265Q) and at the 3'
terminus of Arg-604 codon for the GC. A fragment including the R265Q
mutation was amplified using the R265Q mutant (14) as a template. Each
genetically worked fragment coding the residues 1-343 (for GN),
1-343(R265Q) (for GN(R265Q)) and 341-604 (for GC) was digested by
BamHI and EcoRI, and the resultant restriction fragment was cloned into the BamHI and EcoRI site
of the GST fusion vector pGEX-2T. Escherichia coli JM109
strain was transformed with these expression constructs, and
recombinant fusion proteins were expressed by the low temperature
method that was developed to increase solubility of expressed
IP3-binding proteins as described previously (14). Cells
were harvested, resuspended in phosphate-buffered saline, and disrupted
by sonication on ice. After centrifugation at 30,000 × g for 60 min at 2 °C, the supernatants were collected and
subjected to a GST purification step through glutathione-Sepharose 4B
column chromatography according to the manufacturer's protocol. The
purified GST fusion proteins were subjected to SDS-PAGE analysis and
IP3 binding assay.
 |
RESULTS |
Characterization of IP3 Binding Activity of the
Trypsin-fragmented mIP3R1--
We previously showed that
limited trypsin digestion of mouse cerebellar membrane fractions
fragmented the mIP3R1 into five major polypeptides
(fragments I-V), in which the IP3-binding region was split
into two fragments I and II (18). Intriguingly, these completely
fragmented mIP3R1 still retained strong activity for IP3-induced Ca2+ release comparable with that
of the intact receptor, suggesting that even the split
IP3-binding site was sufficient to couple with gating the
fragmented channel.
To characterize further the structural and functional properties of the
split IP3-binding site, we analyzed the mIP3R1
in trypsin-digested and undigested microsomal fractions of mouse cerebellum for specificity, affinity, and capacity of IP3
binding. Microsomal fractions were digested with 5 µg/ml trypsin for
4 min at 35 °C and then pelleted by centrifugation. Almost all of the tryptic fragments as well as the intact mIP3R1
collected into the pellet. The pellet was solubilized with 1% Triton
X-100 followed by centrifugation at 20,000 × g for 60 min at 2 °C to obtain the supernatant. The Triton extracts were then
subjected to Western blot analysis using six site-specific antibodies,
which specifically recognize the major tryptic fragments Ia/b (40/37
kDa), II (64 kDa), III (76 kDa), IVa/b (40/36 kDa), and V (91 kDa)
(18). As shown in Fig. 1A,
trypsinization resulted in complete fragmentation of the
mIP3R1 into five tryptic fragments that were detectable with the site-specific antibodies (Trypsin +), but when
pre-mixed with 50 µg/ml trypsin inhibitor and 0.1 mM PMSF
prior to the digestion, no apparent mobility change was observed in the
intact mIP3R1 band (Trypsin
). The IVa/b
fragments were only detected at very low levels, since the epitope for
anti-(1718-31) located in the alternative splicing SII region (40 residues 1692-1731) of these fragments is most labile to trypsinolysis
(18). The size difference between the Ia and Ib fragments has been
thought to be due to alternative splicing at the SI region (15 residues
318-332), the former derived from the SI+ subtype and the latter from
the SI
subtype (15, 18).

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Fig. 1.
Effects of trypsinization on IP3
binding of the mIP3R1. Mouse cerebellar microsomal
fractions (1.5 mg of protein) in 1.5 ml of the trypsinizing buffer were
digested with 5 µg/ml trypsin for 4 min at 35 °C in the presence
(Trypsin ) and the absence (Trypsin +) of 50 µg/ml trypsin inhibitor and 0.1 mM PMSF. The digested
samples were centrifuged at 105,000 ×g for 60 min at
2 °C to collect the insoluble membrane fractions. The pellet was
resuspended in 0.5 ml of the binding buffer, solubilized by the
addition of 10% (w/v) Triton X-100 to give a final detergent
concentration of 1%, and rotated for 30 min at 4 °C. The
Triton-treated mixtures were centrifuged at 20,000 × g
for 60 min at 2 °C to obtain the supernatants (Triton extracts). We
confirmed by Western blotting that almost all of the tryptic fragments
and the intact receptor were collected in these extracts (data not
shown). The Triton extracts were subjected to Western blotting and
IP3 binding assay. A, the Triton X-100 extracts
(2.5 µg of protein) were separated by 8% SDS-PAGE and probed with
N1, N3, 10A6,
anti-(1718-31), 1ML1 and 18A10
antibodies which specifically recognize the Ia/b, II, III, IVa/b and V
major tryptic fragments, respectively. Intact IP3R and
major tryptic fragments are indicated by an arrowhead and
arrows, respectively. B, specific
[3H]IP3 (9.6 nM) binding to the
Triton extracts (45 µg of protein). Nonspecific binding was measured
in the presence of 2 µM cold IP3. Values are
the mean ± S.D. of four experiments.
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By using the Triton extracts containing these tryptic fragments, the
following [3H]IP3-binding experiments were
carried out. Although the IP3-binding core was completely
separated into two fragments I and II, which were recognized by the N1
and N3 antibodies, respectively (Fig. 1A), the fragmented
mIP3R1 (Trypsin +) still retained specific IP3 binding activity equivalent to that of the intact
receptor (Trypsin
) (Fig. 1B). In
[3H]IP3 binding competition experiments with
various inositol phosphates, the fragmented mIP3R1
(Trypsin +) showed comparable ligand binding specificity to
the intact receptor (Trypsin
) in the order of (1,4,5)IP3 > (2,4,5)IP3 > (1,3,4,5)IP4 (Fig.
2A). Scatchard analysis showed
that the capacity for IP3 was unaffected by trypsin
digestion; the Bmax (pmol/mg protein) was 23 for
the fragmented mIP3R1 (Trypsin +) and 22 for the
intact receptor (Trypsin
) (Fig. 2B). In
addition, the affinity for IP3 (Kd
value) was also comparable between the fragmented mIP3R1
(13 nM) and the intact receptor (19 nM). These
results suggested that a rigid folded conformation of the tetrameric
mIP3R1 complex in the native membrane might preserve its
inherent IP3-binding pocket from trypsin digestion, even
though the IP3-binding core was split into two near the
center.

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Fig. 2.
Binding characteristics of the fragmented
mIP3R1 with limited trypsin digestion. All samples
used (Trypsin ( ) and Trypsin (+)) were the same
as those in Fig. 1. A, competition of specific
[3H]IP3 (9.6 nM) binding to the
Triton extracts (45 µg of protein) by various inositol phosphates:
(1,4,5)IP3 (filled circles),
(2,4,5)IP3 (open triangles), and
(1,3,4,5)IP4 (filled squares). Values are
normalized to 100% of controls measured without competitor.
Nonspecific binding was measured in the presence of 2 µM
IP3. Each point is the mean of two experiments.
B, Scatchard plots of inhibition of specific
[3H]IP3 (4.8 nM) binding to the
Triton extracts (45 µg of protein) by cold (1,4,5)IP3.
Nonspecific binding was measured in the presence of 10 µM
IP3. The results of a typical experiment are shown.
Kd and Bmax of each sample
shown are the means of two experiments.
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Release of IP3 Binding Activity from the Insoluble to
Soluble Fraction by Prolonged Trypsin Digestion--
Most
IP3 binding activity and fragments I and II were collected
in an insoluble membrane fraction upon mild trypsin digestion, whereas
a proportion of these fragments became soluble upon more extensive
digestion. We next examined the temporal profile of IP3
binding activity in relation to increasing time of digestion (Fig.
3). Microsomal fractions were treated
with 10 µg/ml trypsin for 0, 5, 10, 20, and 40 min and were separated
in insoluble and soluble fractions by centrifugation at 105,000 × g for 60 min. The pelleted insoluble fractions were
solubilized with 1% Triton X-100 to obtain Triton extract. An
equivalent volume of the soluble fraction (sup) and the
Triton extract of insoluble fraction (ppt) were subjected to
[3H]IP3 binding (Fig. 3A) and
Western blotting assay (Fig. 3B). As the digestion time with
trypsin was extended to 10 min, both the insoluble and soluble fraction
showed a sharp difference in temporal changes of IP3
binding activity; the former exhibited gradual attenuation, whereas the
latter showed a gradual increase. When the digestion time was extended
to beyond 20 min, no more marked changes were observed. These temporal
profiles appear to parallel the immunoblotting patterns (Fig.
3B); as the digestion time was extended to 10 min, the
levels of the Ia/b and II fragments, containing the epitopes for the N1
and N3, respectively, were decreased in the insoluble fraction but
increased in the soluble fraction, and thereafter (at 20 and 40 min) no
apparent change was seen in both fractions. However, more extensive
digestion caused loss of binding activity and of these immunoreactive
fragments (data not shown). These data indicated that the two tryptic
Ia/b and II fragments were tightly associated with the tryptic
mIP3R1-membrane complex and that some portions released by
the prolonged digestions appeared to retain conformation for specific
binding by interaction between fragments.

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Fig. 3.
Temporal profiles of IP3 binding
activity of fragments I and II in soluble and insoluble membrane
fractions upon prolonged trypsin digestion. A, mouse
cerebellar microsomal fractions (1 mg/ml protein) were incubated with
10 µg/ml trypsin for 0, 5, 10, 20, and 40 min at 35 °C in
trypsinizing buffer (closed). The reactions were
quenched with 100 µg/ml trypsin inhibitor and 0.1 mM
PMSF. As a control (open symbols), microsomal fraction was
supplemented with trypsin inhibitor and PMSF followed by the addition
of trypsin and then incubated for 40 min at 35 °C. The insoluble and
soluble fractions were separated by centrifugation at 105,000 × g for 60 min at 4 °C. The pellet was then solubilized in
1% Triton X-100 solution in the same way as described in Fig. 1.
Equivalent volumes of soluble fractions (circles) and Triton
extract of insoluble fraction (squares) were subjected to
the [3H]IP3 binding assay. Nonspecific
binding was measured in the presence of 2 µM
IP3. B, equivalent volumes of soluble fraction
(sup) and Triton extract of insoluble fraction
(ppt) prepared as described above were subjected to
immunoblotting with the N1 and N3 antibody. An asterisk
shows a nonspecific band.
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Reconstitution of IP3 Binding Activity by
Complementation between Two Recombinant Binding Components--
To
examine the functional interaction between the two tryptic fragments I
and II containing the split IP3-binding core, we tried to
express recombinant proteins corresponding to these fragments in an
E. coli system. However, it was difficult to obtain
sufficient amounts of soluble proteins, especially for the fragment II
(data not shown). To solve this, we constructed GST fusion proteins as
shown in Fig. 4. The GN was composed of
GST fused to the N-terminal residues 1-343, corresponding to almost
the entire fragment I. We previously showed that Arg-265 within the
IP3-binding core is one of three basic amino acid residues
for which Gln substitutions (R265Q) caused a complete loss of binding
activity (14). The GN(R265Q) had this R265Q mutation in the GN
construct. The GC consisted of a GST fusion protein with residues
341-604, corresponding to almost all of the N-terminal 29-kDa
subfragment derived from fragment II, which took place upon extensive
trypsinolysis (18). These GST fusion proteins were expressed in
E. coli and were purified through a glutathione column (Fig.
5A).
[3H]IP3 binding assay showed that neither GN
nor GN(R265Q) alone had any significant binding activity, whereas the
GC alone showed some traces of activity (Fig. 5B).

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Fig. 4.
Constructs of GST fusion proteins carrying
the IP3-binding domains of mIP3R1. A
schematic representation of the N-terminal region of the
mIP3R1 is shown at the top, where the
IP3-binding core (residues 226-578; solid line)
and the alternative splicing SI segment (residues 318-332; open
box) are indicated. Boxes represents GST fusion
proteins. All constructs were made in pGEX-2T; GN, GST + residues
1-343; GN(R265Q), R265Q mutant of GN; GC, GST + residues 341-604;
GST, pGEX-2T vector alone as a control.
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Fig. 5.
Complementation of the IP3
binding activity by two separately expressed GST-fused
IP3-binding domains. A, SDS-PAGE analysis
of recombinant GST-fused IP3-binding domains. Two µg of
GST fusion proteins, GN, GN(R265Q) and GC, and GST (see Fig. 4) were
separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue
R-250. Sizes of molecular makers in kDa are shown in the
right. B, specific
[3H]IP3 (9.6 nM) binding to the
GST fusion proteins. IP3 binding of GST fusion proteins (7 µg) were individually analyzed: GST, GN,
GN(R265Q), and GC. Two GST fusion proteins were
mixed (7 + 7 µg), and then the IP3 binding activity of
the mixtures was analyzed: GN+GC, GN(R265Q)+GC,
GST+GC, GN+GST, and GN(R265Q)+GST.
Nonspecific binding was measured in the presence of 2 µM
IP3. Values are the mean ± S.D. of three
experiments.
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To examine whether the two split components worked cooperatively to
bind IP3, we performed a complementation test by which two
of these separately synthesized proteins were simply mixed in
vitro (right half of Fig. 5B). The
combinations of GN + GST, GC + GST, GN(R265Q) + GST, and GN(R265Q) + GC
had no significant change in [3H]IP3 binding.
In marked contrast, the combination of GN + GC displayed a dramatic
increase in the specific binding activity, suggesting that the GN and
the GC complemented each other to retrieve IP3 binding
activity, even though both have a large extra GST moiety at the N terminus.
To characterize further this structural and functional reconstitution
of an IP3-binding site by mixing two separately synthesized GN and GC, we carried out Scatchard analysis as shown in Fig. 6. The GN alone exhibited no binding
activity as the control GST. On the other hand, the GC alone could form
a very low affinity site with Kd value of 4.7 µM. It was of particular interest that the mixture of GN + GC displayed a plot fitted by two binding sites; the low affinity
site appeared to be consistent with that of the GC alone, and the high
affinity site had a Kd value of 11 nM,
about 400-fold lower than that of the low affinity site and comparable
to that of the intact mIP3R1. Judging from the
Bmax value of 40 pmol/mg protein, it was
estimated that about 2% of the total GC (about 1800 pmol/mg of
protein) was involved in reconstitution of this high affinity site by
complementation with the GN.

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Fig. 6.
Scatchard analysis of the specific
IP3 binding to the GST fusion proteins. Scatchard
plots of inhibition of specific [3H]IP3 (9.6 nM) binding to the GST fusion proteins by cold
(1,4,5)IP3. Each protein (7 µg) was analyzed
individually: GC, GN, and GST. The
mixture of GN (7 µg) and GC (7 µg) was analyzed: GN+GC.
In the plot of GN+GC, the quantity of protein is tentatively
expressed as that of the GC regardless of the GN, since the GN alone
has no significant activity. Nonspecific binding was measured in the
presence of 200 µM IP3. An inset
in the GN + GC plot shows an enlargement of a plot for a high affinity
site.
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 |
DISCUSSION |
Mild trypsin digestion of a cerebellar membrane fraction generates
five major tryptic fragments, including the N-terminal two fragments
Ia/b (40/37 kDa) and II (64 kDa), that share the entire
IP3-binding site and are tightly associated with the
insoluble membrane fraction (18). In the present study, we have shown that the trypsinized IP3-binding site of mIP3R1
retains significant affinity, specificity, and binding capacity for
IP3, comparable to those of the intact one. These results
indicate that the trypsinized IP3-binding site may retain
functional tertiary structure. Prolonged trypsinization caused
concomitant loss of IP3 binding activity and release of
fragments I and II from the insoluble mIP3R1-membrane complex to the soluble fractions. These results demonstrate that the
folded conformation of the IP3-binding site is stably
retained in either the insoluble or soluble form by a possible
inter-fragment interaction. Joseph et al. (17) previously
reported that prolonged trypsin digestions of rat cerebellar microsomes
caused release of a 68-kDa fragment of IP3R with
concomitant appearance of higher IP3 binding activity in
the soluble fraction, whereas with mild digestion the 68-kDa fragment
was retained in the membrane-bound insoluble form with lower activity.
In the present study, however, we have shown that the affinity of the
insoluble fraction from the mildly trypsinized samples is slightly
higher than that of un-trypsinized samples. We consider that the 68-kDa
fragment is likely to correspond to the 64-kDa fragment II identified
in the present study and that the binding activity observed by Joseph et al. (17) could be attributed to a native-like interaction between fragments I and II which are simultaneously released, although
Joseph et al. (17) did not note fragment I in both the
soluble and insoluble fractions. This discrepancy between our results
and the previous study (17) may be due to differences in preparation of
membrane fractions, trypsin digestions, and/or binding assays
(filtration versus PEG precipitation).
Neither the SI plus (mIP3R1SI+) nor minus
(mIP3R1SI
) subtype showed any significant alteration in
IP3 binding activity,2 whereas deletions of any
other region within the IP3-binding core tested so far
completely abolished the activity (12-14), suggesting that the SI
region is the only redundant part of the binding core. Arg-343 and
Arg-345, which lie between fragments I and II and are close to the
alternative splicing SI region (residues 318-332), are very
susceptible to limited trypsinolysis. Within the
IP3-binding core of all members of the IP3R
family, the most divergent sequences are found around the SI region.
Thus, we suggest that in the vicinity of the SI region, Arg-343 and
Arg-345 may form a flexible loop-like structure (residues
~318 to ~345) that interconnects two non-covalently but tightly
associated well folded structural components; "binding domains" I (residues 1 or N terminus to ~317) and
II (residues ~346 to ~923) corresponding to the
trypsin-resistant fragments I and II, respectively (Fig.
7).

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Fig. 7.
Schematic model of the domain structure of
the IP3-binding site. Trypsin-resistant fragments I
and II of the mIP3R1 are designated as the binding
domains I (residues 1 or N terminus to ~317) and II
(residues ~346 to ~923). The N terminus of domain I in the native
mIP3R1 could not been determined by amino acid sequencing
(18). Around the region including the SI segment (residues 318-332),
Arg-343 and Arg-345 probably form a flexible loop between
two domains, exposed to the outside, thereby being hypersensitive to
limited trypsinolysis. The domain II consists of at least two
subdomains of the N-terminal 29 kDa (probably residues ~346 to
~604) and the C-terminal 38 kDa (probably residues ~605 to ~923),
which are cleaved off with a more extensive trypsinolysis (18). The
IP3-binding core has been experimentally determined to
reside within residues 226-578, and therefore there are at least two
putative core domains, I (residues at least 226 to ~317) and II (residues ~346 to ~604). The inner
surface of the core domains may be lined with 10 basic amino acid
residues for which Gln substitutions caused significant reduction of
the binding activity (14). Of them, Arg-265, Lys-508, and Arg-511 are
critical residues for the specific IP3 binding, because
their single amino acid substitutions to either Gln or Ala caused a
loss of the activity (14). The functional roles for the ligand binding
of the binding domain I, core domains I and II, and the region between
residues 1 and ~225 are indicated in italics as described
under "Discussion."
|
|
Residues 224-579 of the mIP3R1 expressed in E. coli which almost correspond to the IP3-binding core
(residues 226-578) show specific and high affinity IP3
binding activity, indicating that the binding core itself forms a
functional structure (14). We recently showed that more extensive
trypsin digestion further split fragment II into two subfragments, an
N-terminal 29-kDa and a C-terminal 38-kDa fragment, and that the
carboxyl side of Arg-603 and/or Lys-604 would be the sites for this
extensive trypsinization, which lie in the vicinity of the C-terminal
boundary of the IP3-binding core (18). Therefore, we
suggest that the IP3-binding core consists of two
"core domains" I (at least residues ~226 to ~317)
and II (residues ~346 to ~604) in the binding domains I
and II, respectively (Fig. 7).
The present study showed that the GST-fused N-terminal binding domain I
(GN) had no significant binding activity, whereas the GST-fused
C-terminal core domain II (GC) exhibited low affinity. Strikingly,
simple mixing of these regions led to increased binding activity. The
Kd value of this reconstituted high affinity IP3-binding site was almost comparable to that of the
intact mIP3R1. These results suggested that the two
separate GST fusion proteins mutually recognized and cooperatively
formed a virtually native high affinity IP3-binding pocket.
However, we could only reconstitute about 2% of the total input of the
GC by just mixing the GN and the GC. This low rate of functional
complementation may be due to physical interference by the N-terminal
GST moiety attached to both fusion proteins, and/or due to low
efficiency in direct interactions between GN and GC or in proper
folding of expressed GN and GC, under the present conditions.
The present study provides evidence for the modular construction of the
IP3-binding region with discrete functional domains. We
suggest that the low affinity core domain II is a prototype of the IP3-binding structure (Fig. 7). The binding domain
I, although it has no binding capacity by itself, could contribute to
form a high affinity site cooperatively with the core domain II. Thus, the domain I seems to act as a "modulator" to potentiate the
affinity of the IP3 binding prototype, core domain II. The
functional property of this N-terminal binding domain I, however, is a
little complicated as described previously (14); a short deletion of
the N-terminal 31 amino acids from the N-terminal 734 amino acid region
(T734) resulted in a significant reduction in the binding activity,
although the resultant mutant included the entire
IP3-binding core sequence. Such contradictory mutational
effects were also found in serial N-terminal deletions up to residue
215. However, the authentic binding activity was markedly recovered,
when deleted up to the first N-terminal 220, 223, or 225 amino acids,
thereby indicating that the N-terminal boundary of
IP3-binding core is at most residue 226. Notably, the
mutant lacking the first N-terminal 223 amino acids showed more than
10-fold higher affinity for IP3 than that of the parental
T734. Furthermore, a substitution of GST (26 kDa) for the first 223 amino acids of the N-terminal 604 amino acids (T604) significantly
increased the affinity, as compared with the parental
T604.2 These lines of peculiar evidence led us to
hypothesize that the N-terminal first ~225 amino acids are not
directly responsible for the binding but are somewhat related to its
suppression. Then, as shown in Fig. 7 we hypothesize that there are at
least three functional modules, two modulators in the domain I
(N-terminal suppressor and C-terminal enhancer
(core domain I)) and one proto-IP3-binding site
in the core domain II. Co-operative regulation among these modules for ligand binding may influence the channel gating.
Mignery et al. (12) reported that the N-terminal cytoplasmic
region of rat IP3R1 expressed in COS cells displayed an
altered mobility (apparent decrease in mass of >50 kDa) on gel
chromatography in the presence of IP3 and suggested that
any conformational change induced upon binding to IP3 might
be involved in coupling the ligand binding to the channel gating. We
suppose that relative movement of the core domains I and II
non-covalently associated with each other would occur upon ligand
binding as described below. We previously demonstrated the significance
of the 10 basic amino acid residues (Arg or Lys) in specific
IP3 binding, all of which are well conserved within the
IP3R family (14). Of these, Arg-265, Lys-508, and Arg-511
are critical. We thus suggested that these 10 basic residues,
especially Arg-265, Lys-508, and Arg-511, contribute to form a
positively charged pocket for binding to the negative charges on the
three phosphate groups of IP3. Four of them (Arg-241, Lys-249, Arg-265, and Arg-269) are positioned in the core domain I and
the other six (Arg-504, Arg-506, Lys-508, Arg-511, Arg-568, and
Lys-569) in the core domain II (Fig. 7). Thus, the
IP3-binding pocket constituting two core domains may be
relatively expanded due to repulsion among the positive charges on the
inner surface in the nonligand-bound state (open) and become narrow due
to neutralization of the positive charges by interactions with the
negative charges on IP3 in the ligand-bound state (closed).
Finally, studies on the higher order structures of the
IP3-binding site and the possible relative movement upon
IP3 binding will shed light on the molecular basis of the
gating of the mIP3R1 channel as well as the ligand binding.
 |
ACKNOWLEDGEMENT |
We thank K. Yoshikawa for preparation of manuscript.
 |
FOOTNOTES |
*
This work was supported by grants from the Ministry of
Education, Science, Sports and Culture of Japan and from the Ministry of Health and Welfare of Japan.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Howard Hughes Medical Institute, Department of
Physiology, University of California San Francisco, Box 0725, San
Francisco, CA 94143-0725.
¶
To whom correspondence should be addressed: Dept. of Molecular
Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: 81-3-5449-5320; Fax: 81-3-5449-5420; E-mail: tfuruich{at}ims.u-tokyo.ac.jp.
2
F. Yoshikawa and T. Furuichi, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
IP3, inositol 1,4,5-trisphosphate;
IP3R, inositol
1,4,5-trisphosphate receptor;
IP3R1, type 1 inositol
1,4,5-trisphosphate;
mIP3R1, mouse type 1 inositol
1,4,5-trisphosphate;
PEG, polyethylene glycol;
GST, glutathione
S-transferase;
PMSF, phenylmethylsulfonyl fluoride;
PAGE, polyacrylamide gel electrophoresis..
 |
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