J Biol Chem, Vol. 274, Issue 44, 31713-31718, October 29, 1999
Determination of the Binding Site on the Extracellular Domain of
Guanylyl Cyclase C to Heat-stable Enterotoxin*
Makoto
Hasegawa
,
Yuji
Hidaka,
Yoshiko
Matsumoto,
Toshifumi
Sanni, and
Yasutsugu
Shimonishi§
From the Division of Protein Organic Chemistry, Institute for
Protein Research, Osaka University, Yamadaoka 3-2, Suita,
Osaka 565-0871, Japan
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ABSTRACT |
Guanylyl cyclase C, one of the family of
membrane-bound guanylyl cyclases, consists of an extracellular domain
and an intracellular domain, which are connected by a single
transmembrane polypeptide. The extracellular domain binds unique small
polypeptides with high specificity, which include the endogenous
peptide hormones, guanylin and uroguanylin, as well as an exogenous
enterotoxigenic peptide, heat-stable enterotoxin, secreted by
pathogenic Escherichia coli. Information on this specific
binding is propagated into the intracellular domain, followed by the
synthesis of cGMP, a second messenger that regulates a variety of
intracellular physiological processes. This study reports the design of
a photoaffinity labeled analog of heat-stable enterotoxin
(biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)),
which incorporates a Pap residue (p-azidophenylalanine) at
position 11 and a biotin moiety at the N terminus, and the use of this
analog to determine the ligand-binding region of the extracellular
domain of guanylyl cyclase C. The endoproteinase Lys-C digestion of the
extracellular domain, which was covalently labeled by this ligand, and
mass spectrometric analyses of the digest revealed that the ligand
specifically binds to the region (residue 387 to residue 393) of
guanylyl cyclase C. This region is localized close to the transmembrane
portion of guanylyl cyclase C on the external cellular surface. This
result was further confirmed by characterization of site-directed
mutants of guanylyl cyclase C in which each amino acid residue was
substituted by an Ala residue instead of residues normally located in
the region. This experiment provides the first direct demonstration of
the ligand-binding site of guanylyl cyclase C and will contribute
toward an understanding of the receptor recognition of a ligand and the
modeling of the interaction of the receptor and its ligand at the
molecular level.
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INTRODUCTION |
Guanylyl cyclase C
(GC-C),1 a member of the
growing family of membrane-bound guanylyl cyclases, catalyzes the
synthesis of cGMP as a second messenger in intestinal and kidney cortex
epithelial cells in response to stimulation by a ligand (1-3). It is
generally thought that this process is involved in the regulation of
intestinal and kidney fluids and electrolytes, via the endogenous
peptide hormone, guanylin or uroguanylin (4, 5). It is also noteworthy that GC-C greatly increases cGMP levels in intestinal epithelial cells,
on interaction with an exogenous peptide, heat-stable enterotoxin (STa), which is produced by enteric bacteria such as enterotoxigenic Escherichia coli. This results in an efflux of watery
electrolytes into the intestinal lumen, which leads to, in turn, acute
diarrhea in human and domestic animals (6).
GC-C is a single subunit protein molecule (1050 residues) with a unique
structure consisting of an N-terminal extracellular domain (ECD, ~407
residues), which is responsible for ligand binding, and a C-terminal
intracellular domain (~619 residues), for the catalysis of cGMP
synthesis, which are connected through a single transmembrane
polypeptide (~24 residues) (1, 7, 8). The C-terminal intracellular
domain is comprised of two functional regions, a kinase homology region
and a guanylyl cyclase catalytic region. The N-terminal domain is
structurally specific for a ligand such as guanylin and uroguanylin,
and the resulting information is then transferred to the cytoplasmic
intracellular domain via the transmembrane polypeptide, resulting in
the stimulation of the guanylyl cyclase catalytic region in the
intracellular domain. The molecular mechanism for recognizing the
structure of a ligand and leading to signal transduction by GC-C
currently is unknown, but one plausible scenario is that GC-C induces a
change in conformation or topology between each subunit when ligand
binding occurs, resulting in the removal of a negative regulatory
effect of the kinase homology region on the activation of the guanylyl
cyclase catalytic region and, thus, creating a favorable environment
for the synthesis of cGMP (9). GC-C exists as an oligomer in cultured
cells irrespective of the presence or absence of a ligand (9, 10). On
the contrary, the ECD molecule forms an oligomer in a
ligand-dependent manner (11). Thus, the relationship of the
oligomeric state of GC-C to the binding to a ligand remains unclear.
Studies of the site-directed mutagenesis of porcine GC-C revealed two
regions in the ECD that are sensitive to point mutations (12); one is a
highly conserved region from residue 91 to residue 155 (numbers denote
the positions of amino acid residues relative to the N terminus of
porcine GC-C) in the amino acid sequences of GC-Cs determined thus far,
and the other is in the sequence from residue 274 to residue 409 which
is close to the transmembrane portion. A mutation in the region
from residue 347 to residue 401 in the latter, which is positioned
near the transmembrane portion, causes a significant reduction in both
ligand binding activity and guanylyl cyclase catalytic activity. These
results suggest that the region on the ECD for interacting with a
ligand is focused on or located within these regions, the mutation of which strongly affects the biological properties of GC-C.
We describe herein the identification of the specific region on the ECD
of porcine GC-C bound to a ligand. For defining the region on the ECD
that confronts a ligand, we designed and synthesized a photoaffinity
labeling analog of STa
(biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17))
(Fig. 1) with a Pap residue
(p-azido-L-phenylalanine) at position 11 and a
biotin moiety at the N terminus. This analog of STa would be expected
to undergo UV-induced binding to the ECD, and a peptide fragment bound
to this analog could then be salvaged from the enzymatic digest of the
photoaffinity labeled ECD by using an avidin-immobilized matrix.
MALDI-TOF mass spectrometry was used to map a peptide on ECD for
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17),
which has been specifically limited to the amino acid sequence from
residue 387 to residue 393, close to the transmembrane portion. This
finding was confirmed by site-directed mutagenesis because substitution
by an Ala residue for Thr-389, Phe-390, or Trp-392 in the sequence,
respectively, greatly affects binding ability to the ligand, as well as
cyclase catalytic activity of GC-C. These results provide novel
insights into the elucidation of the recognition and activation
mechanism of GC-C by a ligand.
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EXPERIMENTAL PROCEDURES |
Materials--
The reagents used for peptide synthesis were
purchased from the Peptide Institute Inc. (Minoh, Japan) and Nacalai
Tesque, Inc. (Kyoto, Japan). T4 DNA ligase and restriction enzymes were from Takara Shuzo Co. (Kyoto, Japan) and New England Biolabs, Inc.
(Beverly, MA), respectively. Na125I (carrier free) was
purchased from NEN Life Science Products and used for the iodination of
STp (4-17). Other reagents and solvents were purchased from Sigma and
Katayama Chemicals Inc. (Osaka, Japan), all of which were reagent grade.
Synthesis of
Biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)--
Peptides
were synthesized using standard solid-phase procedures using
tert-butyloxycarbonyl chemistry (13). STp(4-17) and ANB-STp(4-17) were synthesized as described previously (14). Details
of the procedures for synthesis of
[Gly4,Pap11]STp(4-17) in which the amino
acid residues at positions 4 and 11 of STp(4-17) were replaced by Gly
and Pap, respectively, that is synthesis of intermediates, protection
of the functional side chains of amino acid residues, removal of
peptides from the resin, conversion from linear forms of peptides to
their oxidized forms, conversion of
p-amino-L-phenylalanine to Pap, and purification of peptides on reversed-phase HPLC were as described previously (15).
In the final step of the synthesis, the biotin group was attached to
the N terminus of [Gly4,Pap11]STp(4-17) by
the following procedure:
[Gly4,Pap11]STp(4-17) (100 nmol) was
mixed with biotinyl-(AC5)2-OSu (Dojin Chemical Inc., Kumamoto, Japan) and triethylamine (50 eq each of the
amount of the peptide) in N,N-dimethylformamide
(200 µl). After standing for 2 h at room temperature, the
reaction product (biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17))
was isolated by reversed-phase HPLC. The binding activity of
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
to GC-C was determined by a competitive inhibition assay involving the
binding of [125I-Tyr4]STp(4-17), as
described in a previous paper (11).
Photoaffinity Labeling of ECD6H with
Biotinyl-(AC5)2-[Gly4, Pap11]STp(4-17)--
The
expression and purification of ECD6H (ECD with a hexahistidine sequence
attached to the C terminus) from Sf21 insect cells were carried
out according to methods described in an earlier report (11). That is
the recombinant baculovirus carrying the cDNA of ECD6H was infected
into Sf21 insect cells (5 × 107, 80%
confluent state) in a 175-cm2 flask for 1 h. The
infected cells were cultured for an additional 2 days in spinner flasks
(5 × 105 cells/ml). The culture supernatant (1200 ml)
was treated batchwise with 10-ml bed volumes of concanavalin
A-immobilized agarose. The concanavalin A-agarose was filtered and
washed with buffer A (50 mM Tris (pH 7.5), 100 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2) (50 ml). The adsorbed protein was
eluted with buffer A (50 ml) containing 500 mM
-methyl-D-mannoside. The non-adsorbed fraction was
treated repeatedly by the same procedure, and the eluted fractions were
pooled. The eluate was concentrated to 20 ml using an Ultrafiltration Cell (Amicon Inc., MA). The solution contained about 800 pmol of the
purified ECD6H. The resulting purified ECD6H was mixed with
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
(100 nmol) and stored at 37 °C in the dark for 1 h. The
solution was then exposed to UV irradiation (302 nm, model UVM-57, UVP
Inc., CA) for 30 min on ice. After the photoaffinity labeling, the
labeled protein was separated from excess ligand and extraneous
materials by using Ni2+-chelating affinity chromatography
according to the procedure described in Ref. 11. Tris was added into
the reaction mixture as a scavenger to inhibit nonspecific
cross-linking reaction as well as serve as a buffer for the reaction
solution (16, 17). These purification procedures yielded about 260 pmol
of the labeled ECD6H, based on the starting crude ECD6H (800 pmol), as
judged from the ligand-binding capacity of the ECD6H treated without photoaffinity labeling (control experiments).
Digestion of Photoaffinity Labeled ECD6H--
The solution that
contained the photoaffinity labeled ECD6H, described above, was
directly diluted with 1 ml of buffer (100 mM Tris (pH 9.0)
and 8 M urea) and incubated at 4 °C for 2 h. The
solution was then diluted with 100 mM Tris buffer (pH 9.0) (5 ml) containing 0.01% SDS and concentrated to 0.5 ml on a
Centricon-10 (Amicon Inc.). Peptide N-glycosidase F (0.4 units) was added to the solution for removal of carbohydrate chains at
the N-linked glycosylation sites and allowed to incubate at
37 °C for 2 h. The resulting solution was then incubated with
endoproteinase Lys-C (3 µg) at 37 °C for 16 h.
Isolation of a Photoaffinity Labeled Peptide--
The above
digested ECD6H solution was mixed with avidin-immobilized agarose
(50-µl bed volume) and incubated at room temperature for 30 min. The
avidin-immobilized agarose was collected on an Ultrafree-MC 0.1 µm
filter unit (Millipore Inc.). The agarose was washed twice with 100 mM Tris (pH 9.0) (300 µl) containing 100 mM
NaCl and then with sufficient 0.05% trifluoroacetic acid to remove
salts. The peptides were removed from the agarose by heating in a
mixture of 0.05% trifluoroacetic acid and 50% acetonitrile at
100 °C for 15 min. The supernatant, which contained the peptides, was concentrated in vacuo and subjected to mass measurements
with an Voyager Elite-XL MALDI-TOF mass spectrometer (Japan PerSpective Biosystems, Inc., Tokyo, Japan) using -cyano-4-hydroxycinnamic acid
as a matrix.
Site-directed Mutagenesis of Porcine GC-C--
The preparation
of the constructs of the recombinant GC-C and its mutant proteins was
carried out according to procedures reported previously (12), with
minor modifications. These recombinant proteins were transiently
expressed in 293T human embryonic kidney cells, which were grown in
Dulbecco's modified Eagle's medium (Sigma) supplemented with 10%
fetal bovine serum, using the Superfect transfection reagent according
to the manufacturer's specifications (Qiagen). Briefly, the
1.0-kilobase pair cDNA fragment between the KpnI and
HindIII sites was isolated from pCG-STaR. The cDNA fragment was subcloned into pBluescript and used as a template for the
PCR reaction as follows. The first PCR fragment was prepared by using a
universal primer (M13-20) and sense primers for the mutation
(CCGGTGGATAAGG*C*CCCCACATTCATC for S387A; GTGGATAAGAGCG*CCACATTCATCTGG for P388A; GATAAGAGCCCCG*CC*TTCATCTGGAAG for T389A;
AAGAGCCCCACAG*C*CATCTGGAAGAAC for F390A; AGCCCCACATTCG*C*CTGGAAGAACCAC
for I391A; CCCACATTCGCCG*C*GAAGAACCACAAA for W392A: asterisks
indicate position of the mutated nucleotides), and the second PCR
fragment was prepared by another universal primer (M13 reverse) and the
antisense primers. The third PCR fragment was constructed with the
universal primers (M13-20 and M13 reverse) using the first and second
PCR fragments as templates. The third PCR fragment was digested with
KpnI and HindIII in order to subclone to the
XbaI/HindIII sites of pBluescript SK(
) together with the cDNA fragment between the XbaI and
KpnI sites. These fragments were individually inserted into
the pCG vector. The vectors were ligated with a 1.7-kilobase pair
cDNA fragment that encodes for the C-terminal portion of porcine
GC-C with HindIII sites at both ends. The DNA sequences of
the vectors constructed in this study were confirmed by an Applied
Biosystems 373A DNA Sequencing System using an ABI PRISM Dye terminator
cycle sequencing kit (Perkin-Elmer).
Assay of cGMP Production of the Mutant Proteins of
GC-C--
cGMP production of the mutant proteins of GC-C, expressed on
293T cells, was assayed as described previously (8). Specifically, the
293 cell membranes, which expressed the mutant proteins, were mixed
with STp(4-17) and incubated at 37 °C for 10 min. The reaction was
terminated by the addition of 10% trichloroacetic acid, and the cells
were then rapidly frozen at 80 °C and thawed at room temperature.
The cell debris was removed by centrifugation at 15,000 rpm
(18,500 × g) for 15 min. The resulting supernatant was
extracted three times with water-saturated ether. The cGMP concentration was determined by a radioimmunoassay kit according to the
manufacturer's specifications (YAMASA, Chiba, Japan). Other related
assays have been described in an earlier paper (18).
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RESULTS |
Synthesis of
Biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)--
A
novel STa analog
(biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17),
Fig. 1) was synthesized as a probe for
mining a peptide fragment that encompasses the ligand-binding site on
the ECD of GC-C. The synthetic ligand contained two functional residues
as follows: one was a photosensitive amino acid (Pap) with azido group,
which is easily converted to nitrene by radiation with UV light (300 nm) and covalently anchored to electron-rich groups such as N-H, O-H,
etc. on the receptor molecule (17); and the other was a biotinyl
moiety, which noncovalently binds to avidin with an extremely high
affinity (Kd, 10
15 M) and
is widely used as a carrier in purification of proteins using
avidin-based affinity chromatography. This synthetic compound was
confirmed not only to have the same conformation as that of STp(4-17)
by comparison of their CD spectra but also to be activated by UV
radiation, showing it to be useful as a ligand for ECD. In addition,
this ligand was confirmed to be efficiently adsorbed on
avidin-immobilized agarose and easily removed from the avidin-agarose by heating at 100 °C for 10 min (data not shown).
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Fig. 1.
Amino acid sequences of STp produced by a
porcine strain of enterotoxigenic E. coli (19)
(A) and
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
(B). Six Cys residues are intramolecularly linked
by disulfide linkages between Cys5 and Cys10,
Cys6 and Cys14, and Cys9 and
Cys17.
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Binding Affinity of
Biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
to ECD6H--
To confirm whether
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
can sufficiently bind to ECD6H, we examined the binding activity of biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
to ECD6H using a competitive ligand binding assay in the presence of a
constant amount of [125I-Tyr4]STp(4-17).
Fig. 2 shows the competitive binding
curves in the binding equilibrium between ECD6H and
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
or STp(4-17) with IC50 values of 8 × 107 and 2 × 108 M,
respectively, indicating that the binding potency of
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
to ECD6H is 40-fold lower than that of STp(4-17). Previous experiments
demonstrated that [Pap11]STp(4-17), in which a Pap
residue was introduced at position 11 in STp(4-17), retained a
high efficiency of photoaffinity labeling to GC-C, although it
diminished the binding potency to GC-C to a level about 30-fold lower
than that of STp(4-17) (15). Therefore, we concluded that
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
has the same level of the binding potency as
[Pap11]STp(4-17) and is a good probe for the efficient
photoaffinity labeling of ECD6H. On the basis of the finding shown in
Fig. 2, we determined that the optimum concentration of the ligand for a photoaffinity labeling experiment is 5 × 106
M.
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Fig. 2.
Binding of
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
(open circles) and STp(4-17) (closed
circles) to ECD6H. Curves show inhibition of
binding of [125I-Tyr4]STp(4-17) to ECD6H by
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
or STp(4-17). Each data point represents the mean values of triplicate
data sets.
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Photoaffinity Labeling of ECD6H with
Biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
and Isolation of a Labeled Peptide Fragment--
UV radiation of the
purified ECD6H in the dark, in the presence of
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17),
resulted in extensive and irreversible binding of the ligand to ECD6H.
Approximately 50% of the ECD6H was occupied with the ligand, and the
remaining 50% was free, in comparison with the ECD6H treated under the
same conditions except for the absence of
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
(data not shown). This suggested that half of the ECD6H was involved in
cross-linking with
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17).
The photoaffinity labeled ECD6H was treated with peptide
N-glycosidase F for removal of carbohydrate moieties and
then digested with endoproteinase Lys-C. The digest was passed through
a column of the avidin-immobilized matrix to adsorb the photoaffinity
labeled peptide fragments, which were then removed from the
avidin-immobilized matrix by heating. The eluate from the avidin matrix
was analyzed by direct MALDI-TOF mass spectrometry. Fig.
3 shows a typical mass spectrum of the
eluate from the avidin-immobilized matrix. The mass values of two major
signals, observed at m/z = 878.6 and 2716.4, were in
complete agreement with the calculated mass values for the peptide
fragment (residue 387 to residue 393) of ECD (calculated mass value for
SPTFIWK, 878.5) and the same peptide fragment bound to
biotinyl-(AC5)2-[Gly4, Pap11]STp(4-17)
(calculated mass value for SPTFIWK + biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17),
2716.1), respectively. Moreover, the mass values of the other
two signals observed at m/z = 2316.1 and 2430.4 were quite close to the values of PTFI (residue 388 to residue 391) and FIWK
(residue 390 to residue 393), bound to
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
(calculated mass values, 2314.9 and 2430.9, respectively). These
fragments could arise via the unexpected cleavage of the labeled
peptide, detected at m/z = 2716.4. The mass values of the signals observed in Fig. 3 conform only to peptides that encompass residues 387-393, bound to
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
but did not fit those of any other sequences (Fig. 4). These results are also supported by
the observation that the signals (Fig. 3) were not detected in the
digest of ECD6H that was treated in the absence of
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
by the same procedure as that described above (control experiment) (not
shown).
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Fig. 3.
MALDI-TOF mass spectrum of the digest of
ECD6H labeled by
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
and treated with endoproteinase Lys-C.
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Fig. 4.
Schematic representation of the ECD of
porcine GC-C, displaying the subdomains based on its secondary
structure predicted according to the method of Chou and Fasman
(26). Vertical bars indicate the location of Cys
residues, and the region bound to
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17)
is shown by the amino acid sequence, which is compared with those of
other species. Asterisk shows identity of the amino acid
sequence of the extracellular domain.
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Site-directed Mutational Analysis of the Ligand-binding
Site--
To investigate the issue of whether the ligand-binding
region is truly localized within the amino acid sequence (residue 387 to residue 393) in ECD, it was mutagenized using site-directed mutagenesis to give six mutant proteins (Fig.
5), in which each of the amino acid
residues in the sequence (residue 387 to residue 392) were individually
substituted by an Ala residue by employing a recombinant GC-C
expression system using 293T human embryonic kidney cells. The
expression of the mutant proteins was confirmed by Western blotting and
staining with an anti-GC-C antibody raised against a synthetic peptide
covering the C-terminal region (residue 1036 to residue 1050) of
porcine GC-C (20), as shown in Fig. 5A. The recombinant GC-C
gave two protein bands on SDS-PAGE (lane 1 in Fig.
5A), in which the heterogeneity of molecular weight of GC-C
was likely caused by differences in the extent of N-linked glycosylation, as has been reported previously (20). The mutant proteins exhibited two protein bands on SDS-PAGE, which were stained by
an antibody detection reagent, similar to the wild type of GC-C
(lanes 2-7), suggesting that all the mutant proteins were expressed in the same quantity as that of the wild type GC-C.
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Fig. 5.
Analysis of site-directed mutant proteins in
the region (residues 387-392) of GC-C expressed in 293T cells.
A, SDS-PAGE and Western blot analysis of the mutant proteins
of GC-C contained in the membrane fractions of the 293T cells. Proteins
were visualized by an anti-GC-C antibody raised against a synthetic
peptide (residue 1036 to residue 1050 of GC-C). Lane 1, wild
type; lane 2, S387A; lane 3, P388A; lane
4, T389A; lane 5, F390A; lane 6, I391A;
lane 7, W392A. B, SDS-PAGE of the mutant proteins
photoaffinity radiolabeled with
ANB-[125I-Tyr4]STp(4-17) in the absence or
presence of 105 M STp(4-17)
(lanes of odd or even numbers,
respectively). Lanes 1 and 2, wild type;
lanes 3 and 4, S387A; lanes 5 and
6, P388A; lanes 7 and 8, T389A;
lanes 9 and 10, F390A; lanes 11 and
12, I391A; lanes 13 and 14, W392A.
C, cGMP production of 293T cells expressing the mutant
proteins of GC-C in the presence of 105 M
STp(4-17). Lane 1, wild type; lane 2, S387A;
lane 3, P388A; lane 4, T389A; lane 5,
F390A; lane 6, I391A; lane 7, W392A.
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The mutant proteins were photoaffinity labeled with
ANB-[125I-Tyr4]STp(4-17), as shown in Fig.
5B. Among the six mutant proteins, one (S387A) showed a
binding potency identical to that of the wild type GC-C. On the
contrary, two mutant proteins (P388A and I391A) showed a reduced
binding ability, and the remaining three mutant proteins (T389A, F390A,
and W392A) were nearly completely devoid of binding ability to
ANB-[125I-Tyr4]STp(4-17). These results
suggest that substitution by an Ala residue of the amino acid residues
at Thr-389, Phe-390, and Trp-392 resulted in a strong deficiency in
ligand binding ability.
The response of the guanylyl cyclase activation of the mutant proteins
for exposure to STp(4-17) was compared with that observed for the wild
type GC-C by assaying the cGMP concentration in the 293T cells which
express either these mutant proteins or the wild type GC-C (Fig.
5C). The formation of cGMP for each of the mutant proteins
was correlated to the binding ability to STa, even though all the
mutant proteins, when stimulated by STa, exhibited cGMP formation to
some degree. The mutant protein (S387A) gave rise to cGMP at a level
similar to that of the wild type GC-C. On the contrary, the mutant
proteins (P388A and I391A) showed 6.0 and 24% cGMP formation, and the
other three mutant proteins (T389A, F390A, and W392A) showed only
0.5-1%, compared with the wild type GC-C, respectively. This again
indicates that the mutant proteins (T389A, F390A, and W392A) have
practically no ability to bind to STa (1 × 105
M). Collectively, these experiments demonstrate that the
substitution by an Ala residue for amino acid residues Thr-389,
Phe-390, and Trp-392 in GC-C abolishes their ability to bind to a
ligand and, hence, the cyclase catalytic activity of GC-C.
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DISCUSSION |
The membrane-bound guanylyl cyclases represent a single
transmembrane type receptor with distinct ligand specificities and are
found in both mammals and lower eukaryotes (2, 3). The extracellular
domains of these receptor proteins show sequence diversity, compatible
with their ability to recognize and discriminate a diversity of ligand
structures. On the contrary, the intracellular domains, in particular
the guanylyl cyclase catalytic regions, have high sequence homology
among the receptors, consistent with their unique function in the
synthesis of cGMP. This suggests that the receptors have a similar
structural topology and regulate diverse physiological processes
through ligand-specific synthesis of intracellular cGMP as an
intracellular message. The identification of the binding sites of
ligands to the receptors on the external surfaces of membranes will
lead to a better understanding of the molecular basis of ligand binding
and receptor activation.
GC-C serves as a receptor protein for the polypeptide ligands: guanylin
(4) and uroguanylin (5) for endogenous ligands as well as heat-stable
enterotoxin (STa) (21) for an exogenous ligand produced by enteric
bacteria, such as enterotoxigenic E. coli. Our previous
experiments (15, 22, 23) demonstrated that
[Pap11]STp(4-17) substituted by a Pap residue in place
of Asn11, located near the key amino acid residue
Ala13 in the context of binding to GC-C, is an efficient
probe for photoaffinity cross-linking of STa to GC-C. In addition, we
demonstrated that the ECD of GC-C, which is expressed in a system
consisting of insect cells and a recombinant baculovirus, retains a
ligand-binding ability that is similar to that of GC-C and could be
prepared as a soluble, homogeneous protein (11). These data allowed us to examine the interaction of ECD with a ligand at the molecular level.
In this work, we focused on the determination of the specific region of
the ECD that is involved in binding to STa. For this purpose, we used
the following four-step strategy: covalent binding of an STp analog
with a photoaffinity functional group to ECD, digestion of the ECD
which is covalently coupled with a photoaffinity labeled STp analog,
isolation of the photoaffinity labeled fragment from the digest by
affinity chromatography, and identification of the labeled fragment by
mass spectrometry. An STp analog
(biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17))
not only covalently linked to the ECD but permitted the isolation of a
peptide fragment from the digest of the photoaffinity labeled ECD by
affinity chromatography, a technique that takes advantage of the
non-covalent binding between biotin and avidin. In general, the binding
of a photoaffinity labeled ligand to its receptor protein appears to
proceed with considerable low efficiency, as has been shown in other
cases (24). Therefore, an expedient technique is needed, in order to
detect the photoaffinity labeled receptor protein or its fragment from
a photoaffinity labeled reaction mixture. In the present case, the
photoaffinity labeled peptide fragment was successfully recovered by
the attachment of a biotin moiety at the N terminus of the ligand from
the digest of the photoaffinity labeled ECD6H but in only a tiny
amount. Mass spectrometry identified the peptide fragment, which
encompasses the amino acid sequence from residues 387 to 393 (SPTFIWK)
(Fig. 4), positioned near the transmembrane portion of GC-C, in the digest of the ECD6H photoaffinity labeled with
biotinyl-(AC5)2-[Gly4,Pap11]STp(4-17).
Moreover, the peptide fragments that are bound to the photoaffinity
ligand with the sequence from residue 388 to residue 391 (PTFI) and
that from residue 390 to residue 393 (FIWK) were observed by mass
spectrometry (Fig. 3). These findings strongly suggest that the ligand
binds to the amino acids, Phe or Ile, at positions 390 and 391, respectively, which are common in the three peptide fragments observed
by mass spectrometry. The result was again supported by site-directed
mutagenesis of the amino acid residues in the binding region, which
show that the substitution of an Ala residue at positions Pro-388,
Thr-389, Phe-390, or Trp-392 caused the complete loss of binding
ability to the ligand (Fig. 5).
In our earlier work (12), the site-directed mutational analysis of the
extracellular domain of porcine GC-C revealed that amino acid
replacement (residues 347-348, 363-365, 373-375, and 399-401) in a
region (residue 347-401), which is close to the transmembrane region
and surrounds the peptide fragment found in this study, causes a strong
reduction in both binding activity to STa and guanylyl cyclase
catalytic activity. In addition, the mutation of the Asn-379 residue to
Ala, which is positioned at an N-glycosylation consensus
site, lost the ability to contain an oligosaccharide at this residue
and resulted in a strong reduction in binding ability of GC-C to STa
(18). These previous results provide support for the conclusion that
the region of GC-C involved in the binding to a ligand is located from
Ser-387 to Lys-393, very near the transmembrane domain. It is also
interesting to note that the amino acid sequence of the ligand-binding
region that was found in porcine GC-C in this experiment is highly
homologous to those not only of mammalian species but also of an
amphibian (Xenopus laevis, African clawed frog) and a fish
(Oryzias latipes, Japanese medaka), although this homology
is not found in the amino acid sequences of the entire extracellular
domains (1, 7, 8,
25)2-5
(Fig. 4).
The prediction of the secondary structure of ECD, as examined according
to the method of Chou and Fasman (26), suggests that ECD is composed of
two subdomains as follows: an /
-rich region (residue 1 to residue
331) and a -rich region (residue 332 to residue 401), as described
in Fig. 4. The / region consists of alternating -helixes and
-strands and contains eight Cys residues, probably linked by
intramolecular disulfide bridges, suggesting that this subdomain, which
consists of 75% of the amino acid residues of the extracellular
domain, functions as a determinant of the basic architecture of the
entire molecule. In contrast, the -rich region has a low propensity
for -helix in contrast to high propensities for a -strand and
turn and is comprised of hydrophilic amino acid residues, including a
Cys residue which is not found in other species and perhaps is not
involved in a disulfide linkage, suggesting that this subdomain forms a
flexible conformation. The x-ray crystallography of the complex of
human growth hormone and its receptor protein suggests that the
ligand-binding region on the receptor protein is largely comprised of
turn structures (27). The same may be inferred in the case of GC-C,
because the sequence from Ser-387 to Pro-388 present in the
ligand-binding region is assumed to form a turn structure, and in
particular, Pro-388 could represent an element of a turn structure.
Indeed Pro-388 was identified as a significant amino acid residue, as evidenced by site-directed mutational analysis of GC-C (Fig. 5). Furthermore, the data herein are in agreement with the conclusions that
the region around this site points to a high content of hydrophilic amino acid residues, suggesting that this region is located on the
surface of the molecule, thus facilitating interaction with a ligand.
In addition, we recently found that the -rich region has a binding
ability to STa, when the interaction of a recombinant protein,
comprising the -rich region, with STp(4-17) was
examined.6 This experiment
also provides evidence that the -rich region near the transmembrane
domain plays a role in ligand binding.
In this study, we identified a region of GC-C that is involved in
ligand binding and that is critical for signaling via the ligand to the
intracellular domain of GC-C, which is located near the transmembrane
portion on the external surface of the cellular membrane. On the basis
of the present finding, together with previously reported data, we
hypothesize that the binding of a ligand to the extracellular domain
induces a clustering (or oligomerization) of the extracellular domain
and, in turn, modulates the topological relationship between each of
the ligand-binding regions of ECD, as has been recently proposed in the
case of the epidermal growth factor binding protein (28, 29). This
process may facilitate a change of the conformation of the
intracellular cytoplasmic domain, resulting in the activation of the
catalytic domain. It would then be interesting to see whether a
principle for the transmission of a ligand signal from an extracellular
domain into an intracellular domain exists in GCs, perhaps in a manner
analogous to GC-C. The extracellular domain of GC-C activates the
intracellular domain in the same manner as in other GCs, when
generating cGMP, a common cytoplasmic second messenger, because the
intracellular domain of GC-C is highly homologous to other GCs in terms
of primary structure, although the extracellular domain of GC-C has a
quite different amino acid sequence from those of other GCs (2). In any
event, the determination of the binding site of GC-C for interacting
with its ligand will provide new insights into our current
understanding of the recognition and activation mechanism of GC-C by a
ligand. A three-dimensional structural analysis of the extracellular
domain and its complex with a ligand will be required, in order to
answer the issue of how the extracellular domain propagates a signal to
the intracellular cyclase catalytic domain on binding with a ligand.
| |
ACKNOWLEDGEMENTS |
The use of the facility at the Radioisotope
Research Center of Osaka University is acknowledged. We thank Hiroko
Sakamoto for preparing the manuscript.
| |
FOOTNOTES |
*
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.
Supported by a Research Fellowship of the Japan Society of the
Promotion of Science for Young Scientists.
§
To whom correspondence should be addressed: Division of Protein
Organic Chemistry, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan. Tel.: +81-6-6879-8601; Fax: +81-6-6879-8603; E-mail: simonisi@protein.osaka-u.ac.jp.
2
R. T. MacFarland,
DDBJ/GenBankTM/EBI accession number D49837.
3
M. Kruhoeffer, Y. Cetin, U. Kaempf, and W.-G.
Forssmann, DDBJ/GenBankTM/EBI accession number
P70106.
4
R. M. Goraczniak, T. Duda, and R. K. Sharma, DDBJ/GenBankTM/EBI accession number AF081464.
5
T. Mantoku and N. Suzuki,
DDBJ/GenBankTM/EBI accession number AB007192.
6
Y. Hidaka, M. R. Giraud, M. Haseguawa, and Y. Shimonishi, unpublished data.
| |
ABBREVIATIONS |
The abbreviations used are:
GC, guanylyl
cyclase;
STa, heat-stable enterotoxin produced by enterotoxigenic
E. coli;
ECD, the extracellular domain of GC-C;
STp(4-17), porcine STa with the amino acid sequence from position 4 to 17;
Pap, p-azido-L-phenylalanine;
MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight;
ANB, 5-azido-2-nitrobenzoyl;
HPLC, high performance liquid chromatography;
AC5, -NH(CH2)5CO-
(
-aminocaproyl);
ECD6H, the ECD with the hexa-histidine tag at the C
terminus;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel
electrophoresis.
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Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.
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ABSTRACT
INTRODUCTION
