THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 275, No. 20, Issue of May 19, pp. 15520 –15525, 2000
Printed in U.S.A.
120- and 160-kDa Receptors for Endogenous Mitogenic Peptide,
Phytosulfokine-␣, in Rice Plasma Membranes*
Received for publication, September 1, 1999, and in revised form, February 10, 2000
Yoshikatsu Matsubayashi‡ and Youji Sakagami
From the Laboratory of Bioactive Natural Products Chemistry, Graduate School of Bio-agricultural Sciences,
Nagoya University, Chikusa, Nagoya 464-8601, Japan
Plant cells in culture secrete a sulfated peptide named
phytosulfokine-␣ (PSK-␣), and this peptide induces the
cell division and/or cell differentiation by means of specific high and low affinity receptors. Putative receptor
proteins for this autocrine type growth factor were
identified by photoaffinity labeling of plasma membrane fractions derived from rice suspension cells. Incubation of membranes with a photoactivable 125I-labeled PSK-␣ analog, [N⑀-(4-azidosalicyl)Lys5]PSK-␣ (ASPSK-␣), followed by UV irradiation resulted in specific
labeling of 120- and 160-kDa bands in SDS-polyacrylamide gel electrophoresis. The labeling of both bands
was completely inhibited by unlabeled PSK-␣ and partially decreased by PSK-␣ analogs possessing moderate
binding activities. In contrast, PSK-␣ analogs that have
no biological activity showed no competition for 125I-ASPSK-␣ binding, confirming the specificity of binding
proteins. Analysis of the affinity of 125I incorporation
into the protein by ligand saturation experiments gave
apparent Kd values of 5.0 nM for the 120-kDa band and
5.4 nM for the 160-kDa band, suggesting that both proteins correspond to the high affinity binding site. Treatment of 125I-AS-PSK-␣ cross-linked proteins with peptide N-glycosidase F demonstrated that both proteins
contained approximately 10 kDa of N-linked oligosaccharides. Specific cross-linking of 125I-AS-PSK-␣ was
also observed by using plasma membranes derived from
carrot and tobacco cells, indicating the widespread occurrence of the binding proteins. Together, these data
suggest that the 120- and 160-kDa proteins are PSK-␣
receptors that mediate the biological activities of PSK-␣.
Plant cells in culture secrete a sulfated pentapeptide named
phytosulfokine-␣ (PSK-␣1; Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln)
in response to externally added auxin and cytokinin, and
PSK-␣ triggers cell proliferation at nanomolar concentrations
in collaboration with plant hormones (1, 2). Therefore, dispersed cells cannot proliferate under low cell density conditions
in which secreted PSK-␣ is diluted below the critical concentration with excess culture medium. This autocrine type peptide growth factor has been found in conditioned medium de* This work was supported by the Program for Promotion of Basic
Research Activities for Innovative Biosciences. The costs of publication
of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence and reprint requests should be addressed.
Tel.: 81-52-789-5552; Fax: ⫹81-52-789-4118; E-mail: matsu@agr.
nagoya-u.ac.jp.
1
The abbreviations used are: AS, azidosalicylic; PNGase F, peptide
N-glycosidase F; PSK, phytosulfokine; PSK-␣, phytosulfokine-␣; Fmoc,
N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; DMF, N,Ndimethylformamide.
rived from both monocot and dicot cell cultures (3, 4), implying
that PSK-␣ is the general factor involved in plant cell growth.
PSK-␣ also stimulates tracheary element differentiation of Zinnia mesophyll cells (3) and somatic embryogenesis in carrots
under defined conditions (5).
A cDNA encoding a PSK-␣ precursor has been isolated from
cDNA library constructed using poly(A)⫹ mRNA purified from
rice cells cultured for 10 days (6). The cDNA is 725 base pairs
in length, and the 89-amino acid product, preprophytosulfokine, has a 22-amino acid hydrophobic region that resembles a
cleavable leader peptide at its NH2 terminus. The PSK-␣ sequence occurs only once within the precursor, close to the
COOH terminus. The critical importance of preprophytosulfokine in cell growth was shown by transforming rice cells with
sense and antisense rice PSK gene that is regulated by the
constitutive rice actin promoter. The sense transgenic cells
divided about two times faster than the controls, whereas the
antisense transgenic cells had decreased mitogenic activity.
Evidence for the existence of high affinity binding sites for
PSK-␣ in rice plasma membrane was provided initially by us
using [35S]PSK-␣ (7) and later using [3H]PSK-␣ (8). The observed binding was saturable, reversible, and localized on the
outer surface of the plasma membrane of rice cells. Because the
specific binding was significantly altered by the external pH
and ionic strength, the ligand-receptor binding may be mainly
controlled by ionic interactions. Ligand saturation analysis
using [3H]PSK-␣ revealed the existence of both high and low
affinity binding sites with Kd values of 1.4 and 27 nM, respectively. Specific binding activities for [3H]PSK-␣ have been detected in plasma membrane fractions derived from cell lines of
many plant species containing carrot, maize, asparagus, and
tomato, indicating the widespread occurrence of [3H]PSK-␣binding sites (8). At present, however, the molecular structures
of PSK-␣-binding sites remain rather unclear. To understand
the molecular mechanism of how plant cells perceive and transduce the PSK-␣ signal, it is critically important to identify and
characterize the receptor molecules that initially perceive the
PSK-␣ signal.
Derivatization of small peptide hormones with photoactivable groups has been utilized for characterization and purification of hormone receptors (9). In these cases, a key factor in
the application of such functional groups may be how to modify
peptides without loss of binding activity and biological activity.
Structure-activity studies for PSK-␣ have shown that the active core of PSK-␣ is an NH2-terminal tripeptide containing
two sulfated groups (10). Thus, we focused on the COOHterminal region of PSK-␣ for the functional derivatization of
PSK-␣.
In this study, we prepared an 125I-labeled photoactivable
PSK-␣ analog containing 4-azidosalicylic acid, [N⑀-(4azidosalicyl)Lys5]PSK-␣, which possesses comparable binding
activity with that of PSK-␣ in order to specifically label mem-
15520
This paper is available on line at http://www.jbc.org
Receptors for Endogenous Mitogenic Peptide in Plant
brane proteins. After photoaffinity cross-linking, proteins of
120 and 160 kDa in rice plasma membranes were labeled with
this ligand. These proteins fulfill the criteria expected for the
high affinity PSK-␣ receptor(s) in terms of their affinity and
specificity.
EXPERIMENTAL PROCEDURES
Materials—[125I]NaI solution (carrier-free) was obtained from ICN
Biomedicals (Costa Mesa, CA). N-Hydroxysuccinimidyl-4-azidosalicylic
acid was obtained from Pierce. A reverse-phase HPLC column, Develosil ODS-5 and ODS-10, was purchased from Nomura Chemicals (Seto,
Japan). Molecular weight standards were from Amersham Pharmacia
Biotech. Fmoc amino acids were from Peptide Institute (Osaka, Japan).
Preloaded HMP resin was from Applied Biosystems (Chiba, Japan).
DMF-SO3 was from Sigma. Peptide N-glycosidase F was from Takara
(Tokyo, Japan). Nonidet P-40 was from Nacalai Tesque (Kyoto, Japan).
All the other inorganic and organic chemicals were obtained from Wako
Pure Chemicals (Osaka, Japan).
Preparation of Plasma Membranes—Cell lines of rice Oryza sativa L.
(Oc) and carrot Daucus carota L. (NC) were subcultured every 2 weeks
in Murashige and Skoog medium supplemented with 1.0 mg/liter of
2,4-dichlorophenoxyacetic acid and 30 g/liter of sucrose. A cell line of
tobacco Nicotiana tabacum L. (BY-2) was maintained with subculturing
every week in Murashige and Skoog medium supplemented with 0.2
mg/liter of 2,4-dichlorophenoxyacetic acid and 30 g/liter sucrose. Cultures were incubated at 25 °C in the dark with rotary shaking at 120
rpm as described previously (7). Cells (150 g fresh weight) were homogenized in a blender, and a microsomal fraction was obtained by ultracentrifugation as described previously (8). Plasma membranes were
further enriched by the two-phase partitioning protocol (11). Pellets of
plasma membrane fraction were suspended in suspension buffer containing 10 mM Tris-HCl, pH 7.0, and 250 mM sucrose using a PotterElvehjem type homogenizer and stored at ⫺80 °C until use. Protein
concentration was measured as described by Bradford (12) with bovine
serum albumin as a reference.
Preparation of 125I-Labeled Photoactivable Analog of PSK-␣—The
partially protected peptide resin, Fmoc-Tyr-Ile-Tyr-Thr(t-butyl)-Lys(tbutoxycarbonyl)-linker resin, was synthesized by Fastmoc chemistry
with a peptide synthesizer (Applied Biosystems model 433A). This
peptide resin (0.05 mmol) was suspended in DMF/pyridine mixture (4:1,
1.5 ml) and sulfated by the addition of 230 mg of DMF/SO3. After 16 h,
peptides were cleaved and deprotected as described previously (10). The
peptides were purified by HPLC on a Develosil ODS-10 column (20 ⫻
250 mm) by an isocratic elution of 30% acetonitrile containing 0.1%
ammonium acetate at a flow rate of 10 ml/min. After lyophilization, a
precursor peptide, Fmoc-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Lys was obtained. AS-PSK-␣, a PSK-␣ derivative conjugated with 4-azidosalicylic
acid (AS), was prepared by coupling N-hydroxysuccinimidyl-4-azidosalicylic acid and the synthesized precursor peptide. N-Hydroxysuccinimidyl-4-azidosalicylic acid (3.3 mg), Fmoc-Tyr(SO3H)-Ile-Tyr(SO3H)Thr-Lys (3.2 mg), and NaHCO3 (1.0 mg) were dissolved in 1.0 ml of 50%
acetone and stirred at room temperature for 3 h. The reaction mixture
was then evaporated to dryness, dissolved in 1.0 ml of DMF/piperidine
mixture (1:1), and further stirred for 1.0 h. Deprotected peptides were
purified by HPLC on a Develosil ODS-5 column (10 ⫻ 250 mm) by an
isocratic elution of 20% acetonitrile containing 0.1% ammonium acetate
at a flow rate of 4.0 ml/min. After lyophilization, Tyr(SO3H)-IleTyr(SO3H)-Thr-Lys(N⑀-(4-azidosalicyl)) (AS-PSK-␣) was obtained. ASPSK-␣ was radioiodinated using chloramine T as the oxidizing agent.
Six microliters of Na125I (20 MBq), 0.6 ␮l of unlabeled 4 mM NaI as a
carrier, 50 ␮l of 0.4 mM AS-PSK-␣, and 10 ␮l of 0.5 M phosphate buffer,
pH 7.5, were mixed in an Eppendorf tube. Reaction was started by
adding 2.5 ␮l of 4 mM chloramine T to this solution, and the mixture was
allowed to stand for 30 min at room temperature. Iodinated peptides
were purified by HPLC on a Develosil ODS-5 column (10 ⫻ 250 mm) by
an isocratic elution of 25% acetonitrile containing 0.1% ammonium
acetate at a flow rate of 2.0 ml/min. Under this condition, two monoiodinated (ortho or para position of hydroxyl group) peptides were obtained at a retention time of 6.8 and 7.7 min, respectively. From the
results of binding assay, the monoiodinated AS-PSK-␣ eluted at 7.7 min
showed higher affinity for PSK-␣-binding sites. Thus we used this
labeled ligand designated 125I-AS-PSK-␣ (230 Ci/mmol) in the following
experiments. Mitogenic activities of the synthesized peptides were detected by bioassay using asparagus mesophyll cells as described previously (1).
Ligand Binding Assay—Procedures for preparation of [3H]PSK-␣
and ligand binding assay were previously described (8). In short, ali-
15521
quots of plasma membrane fraction (125 ␮g of protein) were incubated
for 30 min at 4 °C in a total volume of 250 ␮l of binding buffer containing 10 nM [3H]PSK-␣ and various concentrations of ligands as a competitor. Incubations were terminated by layering the reaction mixture
onto 900 ␮l of buffer containing 0.5 M sucrose and centrifuged for 5 min
at 100,000 ⫻ g at 4 °C. After discarding the supernatant, the radioactivity contained in the pellet was determined with a liquid scintillation
counter.
Photoaffinity Labeling—Aliquots of plasma membrane fraction (125
␮g of protein) was incubated for 30 min at 4 °C in a total volume of 250
␮l of binding buffer (10 mM citric acid-KOH, pH 4.0, 100 mM sucrose)
containing 10 nM 125I-AS-PSK-␣. Incubations were terminated by layering the reaction mixture onto 900 ␮l of wash buffer (10 mM citric acid
KOH, pH 4.0, 0.5 M sucrose) and centrifuged for 5 min at 100,000 ⫻ g
for 5 min at 4 °C. After discarding the supernatant, the pellet was
irradiated on ice for 10 min with an UV lamp (model ENF-260C/J (365
nm), Spectronics Co. Ltd, NY) at a distance of 1 cm. The cross-linked
membrane proteins were solubilized in 62.5 ␮l of SDS-PAGE sample
buffer and heated at 95 °C for 5 min. Twenty microliters of each sample
(40 ␮g of protein) was loaded onto a 7.5% gel and separated according
to the method of Laemmli (13). The dried gels were exposed to the
bio-imaging plate for 2 h at room temperature, and the plates were
analyzed using an imaging plate reader and bio-imaging analyzer (BAS
2000, Fujifilm, Tokyo, Japan). Protein bands in SDS-PAGE gels under
the reducing condition were visualized by the fluorescent dye Nile Red
(14).
Endoglycosidase Treatments of Labeled Proteins—125I-AS-PSK-␣-labeled membranes were suspended in 62.5 ␮l of 0.5 M Tris-HCl buffer,
pH 8.6, containing 0.5% SDS and incubated at 95 °C for 3 min. Aliquots
of this solution (20 ␮l; corresponds to 40 ␮g of protein) were incubated
with Nonidet P-40 (1.0% final concentration) and peptide N-glycosidase
F (PNGase F) (2 milliunits) at 37 °C for 24 h at a total volume of 25 ␮l.
After deglycosylation, samples were mixed with SDS-PAGE buffer and
subjected to SDS-PAGE.
RESULTS
Preparation and Characterization of 125I-AS-PSK-␣—Preparation of AS-PSK-␣ is summarized in Fig. 1. Structure-activity
studies of PSK-␣ showed that receptor binding affinity of
PSK-␣ was not significantly affected by a substitution of Gln by
Lys in which ⑀ amino group was coupled with some reporter
molecules (15). Thus we conjugated photoactivable AS acid
with the similar procedure. The NH2-terminal protected peptide was prepared by a peptide synthesizer without final deprotection of Fmoc group, and 4-azidosalicylic acid was coupled
with ⑀ amino group of COOH-terminal Lys. After deprotection
of Fmoc group and HPLC purification, AS-PSK-␣ was obtained
with an overall yield of ⬃20%.
Iodination of AS-PSK-␣ was conducted at the ligand/iodine
ratio of 7:1 to obtain monoiodinated peptides, because the cold
run experiments showed that the binding activity of diiodinated AS-PSK-␣ was lower than that of monoiodinated ASPSK-␣ (data not shown). Tyrosine phenyl rings were not iodinated under this condition owing to the presence of sulfate
ester. The competition by PSK-␣ and two isomers of monoiodinated AS-PSK-␣ was assessed in experiments in which increasing amounts of unlabeled PSK-␣ and PSK-␣ derivatives competed with [3H]PSK-␣ for receptor binding (Fig. 2). Unlabeled
PSK-␣, monoiodinated AS-PSK-␣ eluted at 6.8 min, and monoiodinated AS-PSK-␣ eluted at 7.7 min, all competed for the
binding sites with half-maximal inhibitory concentrations
(IC50) of ⬃30, ⬃300, and ⬃100 nM, respectively. Although the
monoiodinated positions on phenyl ring of 4-azidosalicylic acid
could not be chemically determined owing to the limited
amounts of products, the peptide eluted at 7.7 min showed
higher affinity for PSK-␣-binding sites than that eluted at 6.8
min.
The affinity of the monoiodinated AS-PSK-␣ eluted at 7.7
min was calculated by nonlinear regression analysis software
(GraphPad PRISM娂) using the equation for competitive binding to two classes of receptor. By fitting competition data from
Fig. 2, the Ki1 and Ki2 values for the peptide to rice microsomes
15522
Receptors for Endogenous Mitogenic Peptide in Plant
FIG. 1. Preparation of the photoactivable PSK-␣ analog, ASPSK-␣. The partially protected peptide resin, Fmoc-Tyr-Ile-Tyr-Thr(tbutyl)-Lys(t-butoxycarbonyl)-linker resin, was sulfated by dimethyformamide/SO3. Sulfated peptide was cleaved from resin by trifluoroacetic
acid (TFA) treatment and coupled to N-hydroxysuccinimidyl-4-azidosalicylic acid by the formation of an amide bond between ⑀ amino group
of Lys and carboxyl group of 4-azidosalicylic acid. After deprotecting
Fmoc group by piperidine, photoactivable PSK-␣ analog, AS-PSK-␣,
was obtained with an overall yield of ⬃20%. Asterisks show the iodinated positions on the phenyl ring.
were determined to be 5.1 and ⬃500 nM, respectively.
The high affinity of the monoiodinated peptide eluted at 7.7
min for PSK-␣ receptor was also confirmed by the bioassay
using asparagus mesophyll cells (Fig. 3). Half-maximal induction of cell division occurred ⬃3 nM, which is virtually equal
concentration for half-maximal activity of unmodified PSK-␣.
Based on these results, we prepared the monoiodinated peptide
eluted at 7.7 min using Na125I and used as a photoaffinity
ligand in the following experiments. The specific radioactivity
of this radioiodinated peptide designated as 125I-AS-PSK-␣ was
230 Ci/mmol.
Identification of High Affinity Binding Proteins by Photoaffinity Labeling—Photoaffinity labeling was performed after incubation of plasma membrane fraction with 10 nM 125I-ASPSK-␣ for 30 min on ice. Cross-linked membrane proteins were
solubilized by SDS sample buffer and analyzed by SDS-PAGE
under the reducing and the nonreducing conditions. Autoradiographic analysis of the SDS-PAGE gels showed that 120- and
160-kDa proteins were labeled under the reducing condition
(Fig. 4). The incorporation of 125I-AS-PSK-␣ into these bands
was completely inhibited with a 320-fold excess (3.2 ␮M) of
unlabeled PSK-␣, indicating the specific binding of the ligands
to the proteins. Under the nonreducing condition, relative mobility of 120-kDa protein was not altered at all, but 160-kDa
protein showed a relatively smear band. The 160-kDa protein
may contain intramolecular disulfide bonds that define the
tertiary structure of the binding protein.
To verify the specificity of the interaction of 125I-AS-PSK-␣
FIG. 2. HPLC profile of iodinated AS-PSK-␣ and their binding
activity to rice plasma membranes. A, AS-PSK-␣ was iodinated
using chloramine T as the oxidizing agent. Iodinated peptides were
purified by HPLC on a reverse-phase column by an isocratic elution of
25% acetonitrile containing 0.1% ammonium acetate. Under this condition, two monoiodinated (ortho or para position of hydroxyl group)
peptides were obtained at a retention time of 6.8 and 7.7 min, respectively. B, binding affinities of iodinated peptides were determined by
the competitive ligand binding assay. Aliquots of plasma membrane
fraction were incubated for 30 min at 4 °C in the binding buffer containing 10 nM [3H]PSK-␣ and various concentrations of the peptide
eluted at 6.8 min (square), the peptide eluted at 7.7 min (triangle), or
unlabeled PSK-␣ (circle) as a competitor.
FIG. 3. Mitogenic activity of iodinated AS-PSK-␣. The mitogenic
activities of monoiodinated AS-PSK-␣ eluted at 7.7 min (circle) and
unlabeled PSK-␣ (square) were measured in triplicate by the bioassay
system using asparagus mesophyll.
with its binding sites, rice plasma membranes were incubated
with the radioligand in the presence of increasing concentrations of unlabeled PSK-␣. Photoaffinity cross-linking revealed
a dose-dependent reduction in the label intensity of the 120and 160-kDa proteins (Fig. 5). By quantifying the radioactivities of each band using an image analyzer, the ligand concentrations required to half-maximally displace 125I-AS-PSK-␣
from the binding sites were shown to be ⬃30 nM for 120-kDa
Receptors for Endogenous Mitogenic Peptide in Plant
15523
FIG. 6. Binding specificity of PSK-␣-binding proteins. Rice
plasma membranes were incubated with 10 nM 125I-AS-PSK-␣ in the
presence of 320-fold excesses (3.2 ␮M) of various PSK-␣ analogs as
competitors. After UV irradiation, cross-linked membrane proteins
were solubilized by SDS sample buffer and analyzed by SDS-PAGE and
bio-imaging analyzer. Lane 1, no competitor; lane 2, Tyr(SO3H)-IleTyr(SO3H)-Thr-Gln (PSK-␣); lane 3, Tyr-Ile-Tyr(SO3H)-Thr-Gln; lane 4,
Tyr(SO3H)-Ile-Tyr-Thr-Gln; lane 5, Tyr-Ile-Tyr-Thr-Gln.
FIG. 4. Detection of PSK-␣-binding proteins by photoaffinity
labeling. Rice plasma membrane was incubated with 10 nM 125I-ASPSK-␣ and irradiated with UV light after the removal of excess unbound ligand. Cross-linked membrane proteins were solubilized by SDS
sample buffer and analyzed by SDS-PAGE and autoradiography under
the reducing or the nonreducing conditions in the presence or absence
of excess unlabeled PSK-␣. Total protein bands in SDS-PAGE gels were
visualized by the fluorescent dye Nile Red. Numbers at left indicate
molecular mass markers given in kilodaltons.
FIG. 5. Competitive displacement of 125I-AS-PSK-␣ incorporation by unlabeled PSK-␣. Rice plasma membranes were incubated
with 10 nM 125I-AS-PSK-␣ in the presence of increasing concentrations
of unlabeled PSK-␣. After UV irradiation, cross-linked membrane proteins were solubilized by SDS sample buffer and analyzed by SDSPAGE and autoradiography.
bands. Binding specificity of the 120- and 160-kDa protein to
the structurally related PSK-␣ analogs was further analyzed
by competitive displacement of 125I-AS-PSK-␣ with the unlabeled analogs. A 320-fold molar excess of the analogs was
added concomitantly with the radioligand to the assay mixture,
and the samples were proceeded as described above. Results
from a representative experiment are shown in Fig. 6. PSK-␣
analogs with moderate mitogenic activity such as Tyr-IleTyr(SO3H)-Thr-Gln and Tyr(SO3H)-Ile-Tyr-Thr-Gln showed
some competition for cross-linking of 125I-AS-PSK-␣ to rice
plasma membrane. In contrast, PSK-␣ analogs that have no
biological activity such as Tyr-Ile-Tyr-Thr-Gln showed no competition for 125I-AS-PSK-␣ binding, confirming the specificity of
binding proteins.
The binding affinities of 125I-AS-PSK-␣ to the 120- and 160kDa band were estimated from the concentration dependence
of the ligand incorporation into the protein band (Fig. 7). The
specific binding between the ligand concentration range from
0.32 to 10 nM determined by quantifying band densities using a
bio-imaging analyzer showed the saturable mode of incorporation of radioactivity to the 120- and 160-kDa bands (Fig. 7A).
The Scatchard analysis of the binding data showed linear
profile and yielded the following parameters: Kd 120,000 ⫽ 5.0
FIG. 7. Binding constants of PSK-␣-binding proteins. A, rice
plasma membranes were incubated with various concentrations of 125IAS-PSK-␣. After UV irradiation, cross-linked membrane proteins were
solubilized in SDS sample buffer and analyzed by SDS-PAGE and
bio-imaging analyzer. B, Scatchard analysis of the binding data showing linear profiles with the following parameters: Kd 120,000 ⫽ 5.0 nM,
Bmax 120,000 ⫽ 81 fmol/mg protein (square), and Kd 160,000 ⫽ 5.2 nM,
Bmax 160,000 ⫽ 34 fmol/mg protein (circle).
nM, Bmax 120,000 ⫽ 216 fmol/mg protein, and Kd 160,000 ⫽ 5.4 nM,
Bmax 160,000 ⫽ 88 fmol/mg protein (Fig. 7B). Although the binding constants obtained by Scatchard analysis of the irreversibly
cross-linked ligands may not reflect the value determined under equilibrium binding situations, these Kd values are in good
agreement with the Ki1 value observed in competitive binding
assay (5.1 nM; Fig. 2), indicating that these proteins correspond
to high affinity PSK-␣-binding site. We also tried to characterize the binding affinities of 125I-AS-PSK-␣ to these proteins in
higher ligand concentrations, but the labeled proteins could not
be quantified well owing to high background (data not shown).
15524
Receptors for Endogenous Mitogenic Peptide in Plant
FIG. 8. Peptide N-glycosidase F treatment of photoaffinity labeled membranes. 125I-AS-PSK-␣ labeled membranes were suspended Tris-HCl buffer containing 0.5% SDS and incubated at 95 °C for
3 min. Aliquots of this solution were incubated with Nonidet P-40 (1.0%
final concentration) and peptide PNGase F (2 milliunits) at 37 °C for
24 h. After deglycosylation, samples were mixed with SDS-PAGE buffer
and subjected to SDS-PAGE.
Peptide N-Glycosidase F Treatment of Cross-linked Membrane Proteins—Peptide N-glycosidase F (PNGase F) treatment of the photoaffinity labeled membranes demonstrated the
presence of N-linked carbohydrate side chains on the receptor
protein (Fig. 8). PNGase F is known to cleave asparaginebound N-glycans to give proteins free of N-linked carbohydrate
chains. After 24 h of treatment in the presence of SDS, the
labeled 120- and 160-kDa proteins were reduced to apparent
molecular masses of 110 and 150 kDa, respectively, suggesting
the presence of approximately 10 kDa of N-linked carbohydrate
chains in both proteins.
Widespread Occurrence of the PSK-␣-binding Proteins—Recent studies revealed that a significant amount of PSK-␣ is
produced by several plant cell lines including dicots as well as
monocots and stimulates cell division and differentiation at a
nanomolar level.2 Therefore, we investigated the distribution of
PSK-␣-binding proteins in plasma membrane fractions derived
from carrot and tobacco cells. In both species, specific 125I-ASPSK-␣ cross-linked proteins were detected by incubating
plasma membrane fractions in the buffer containing 10 nM of
the ligand followed by UV irradiation (Fig. 9). In the membrane
fraction derived from D. carota L. (NC) cell line, 130- and
170-kDa proteins were specifically labeled at a relatively high
efficiency. Tobacco (BY-2) membrane also possessed specific
binding proteins with apparent molecular masses of 110 and
150 kDa. The differences in labeling efficiency of these proteins
between rice, carrot, and tobacco membranes are probably due
to the differences in the population of the binding sites (8).
DISCUSSION
As part of a research program to characterize the PSK receptor protein, we prepared a fully active photoactivable probe
that could be used in receptor visualization experiments
through UV irradiation, SDS-PAGE, and autoradiography. In
designing this analog, the ⑀ amino group of [Lys5]PSK-␣ appeared particularly appropriate as the site of modification for
the following reasons. First, structure-activity studies demonstrated that the COOH-terminal dipeptide of PSK-␣ does not
play a critical role in the expression of its biological activity.
Second, the allylazido moiety would not be expected to interfere
with receptor binding due to its remote location relative to the
message portion of PSK-␣.
The present study demonstrated cross-linking of 125I-ASPSK-␣ to rice plasma membrane proteins of 120 and 160 kDa.
These proteins have all the properties expected of components
of the PSK-␣ receptor that mediates proliferation and differentiation of plant cells. (a) The photoaffinity labeling of these two
species occurs at biologically relevant concentrations of the
2
Y. Matsubayashi and Y. Sakagami, unpublished data.
FIG. 9. Widespread occurrence of the PSK-␣-binding proteins.
Plasma membrane fractions derived from rice, carrot, and tobacco cells
were incubated with 10 nM 125I-AS-PSK-␣ and irradiated with UV light
after the removal of excess unbound ligand. Cross-linked membrane
proteins were solubilized by SDS sample buffer and analyzed by SDSPAGE and autoradiography under the reducing conditions in the presence or absence of excess unlabeled PSK-␣.
ligand, in the range 0.1–10 nM; (b) the labeling of 120- and
160-kDa proteins is inhibited by native PSK-␣ in a dose-dependent manner; (c) the cross-linking is not affected by PSK-␣
analogs that have no biological activities; (d) the binding constants of 120- and 160-kDa proteins for 125I-AS-PSK-␣ are at
the nanomolar level, in agreement with the binding constant of
[3H]PSK-␣ for the high affinity site in the rice plasma membrane fraction (1.4 nM) and also the ED50 of PSK-␣ (3.8 nM)
determined by bioassay using asparagus mesophyll cells (1).
Although we detected both high and low affinity binding
sites in the rice plasma membrane fraction by the ligand binding assay using [3H]PSK-␣ (8), the photoaffinity labeling described here only revealed the presence of two binding proteins
with high affinity binding constants. The absence of a low
affinity protein detected by photoaffinity labeling might be
explained by the following. Low affinity binding site could be
produced by ligand-induced changes in receptor affinities and,
therefore, not present at low ligand concentrations. A negatively cooperative model for hormone-receptor interaction has
been reported for several mammal growth factor receptors (16,
17). We therefore tried to determine the binding constants of
125
I-AS-PSK-␣ for the two proteins at high ligand concentrations, but unfortunately the high background precluded meaningful results. Alternatively, these may be two distinct PSK-␣binding sites that have different binding affinities in the rice
plasma membrane fraction, but the low affinity site could not
be labeled due to its conformational characteristics.
The 160-kDa protein is not a multicomponent complex resulting from covalent cross-linking of the 120-kDa protein with
other membrane proteins, since 125I-AS-PSK-␣ possesses only
one photoactivable site which usually reacts with only one
target molecule. This conclusion is further supported by the
fact that the relative amount of labeling of the 120- and 160kDa proteins did not change over a wide range of 125I-ASPSK-␣ concentrations. The finding of two different PSK-␣binding proteins with different molecular weights can be
interpreted in several ways. (a) Only the 160-kDa protein is the
biologically relevant receptor, whereas the lower molecular
mass 120-kDa protein is a proteolytic breakdown product. (b)
The 120-kDa protein is a translation product of a truncated
form of the mRNA encoding the 160-kDa protein. (c) The high
affinity PSK-␣ receptor is associated with both of these two
proteins without covalent links such as disulfide bonds. (d)
Iodine radicals generated by UV irradiation may react at a site
somewhat distant from the site of nitrene insertion (18). (e) The
two high affinity binding sites are structurally unrelated and
are involved in different biological functions of PSK-␣.
In the case of mammal growth factor receptor, limited and
Receptors for Endogenous Mitogenic Peptide in Plant
specific proteolytic processes are known to transform the native
insulin receptor (19) and the epidermal growth factor (20) to
lower molecular weight forms. Moreover, it has been reported
that in addition to the mature 175-kDa epidermal growth factor receptor, A431 cells also possess a 95-kDa form originating
from such a truncated mRNA (21). In this context, interpretation a or b shown above may be a better explanation for the
presence of two different PSK-␣-binding proteins with different
molecular weights.
Several well characterized mammal growth factor receptors,
including epidermal growth factor receptor (22), platelet-derived growth factor receptor (23), and insulin receptor (24),
contain N-linked carbohydrate side chains. It has been demonstrated that core oligosaccharide addition is essential for the
acquisition of epidermal growth factor binding activity (25). In
addition, the oligosaccharide moieties of the insulin receptor
precursor are crucial for proper processing, intracellular translocation, and formation of functionally competent insulin receptors (26). Although the function of the carbohydrate moiety
in PSK-␣ receptors is still unclear, the presence of glycosylated
side chain allows us to predict that immobilized lectins will be
a useful tool in the purification of the PSK-␣ receptors.
Occurrence of specifically 125I-AS-PSK-␣ cross-linked proteins with similar size across distantly related plant species,
rice, carrot, and tobacco, is in good agreement with the widespread occurrence of PSK-␣. Although the presence of a number of receptor-like membrane proteins involved in plant
growth and development has been predicted based on sequence
similarities (27–30) and biochemical characterization (31), little is known about the receptor ligand(s) that ultimately activates the receptor function through the receptor-ligand interaction. Recent evidence implies that plants, like animals, may
actually make wide use of peptide signaling (1, 32, 33), so that
plant cell-to-cell communication is mediated by peptide-receptor interactions. Although further analysis of the PSK-␣-binding proteins is needed to determine whether a relationship
exists between the two proteins detected here, our work provides a basis for the purification and sequence analysis of
PSK-␣ receptor(s) that perceive the extracellular peptide signal
and transduce the intracellular secondary messengers activating sets of genes involved in plant cell proliferation and
differentiation.
15525
Acknowledgments—We thank Dr. K. Syono (Japan Women’s University) for providing rice (Oc) suspension cell, Dr. H. Kamada (University
of Tsukuba) for carrot (NC), and Dr. K. Nakamura (Nagoya University)
for tobacco (BY-2).
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