Journal of Surface Engineered Materials and Advanced Technology, 2013, 3, 36-42
http://dx.doi.org/10.4236/jsemat.2013.34A1005 Published Online October 2013 (http://www.scirp.org/journal/jsemat)
Interaction between Peptide Pheromone or Its Truncated
Derivatives and Pheromone Receptor of the Fission Yeast
Schizosaccharomyces pombe Examined by a Force
Spectroscopy Study and a GFP Reporter Assay
Sho Hidaka1, Osamu Nikaido1, Shoichi Kiyosaki1, Atsushi Ikai2, Toshiya Osada1
1Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan;
2Innovation Laboratory, Tokyo Institute of Technology, Yokohama, Japan.
Received June 17th, 2013; revised July 25th, 2013; accepted August 3rd, 2013
Copyright © 2013 Sho Hidaka et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
In our previous study, the specific interaction between P-factor, a peptide pheromone and its receptor, Mam2, on the
cell surface of the fission yeast Schizosaccharomyces pombe was investigated by two methods, an atomic force micro-
scope (AFM) and a GFP reporter assay. The removal of Leu at C-terminal of P-factor resulted in an inactivation of
P-factor function to bind Mam2 and induce the signal transduction pathway. Here, we used truncated P-factor deriva-
tives lacking N-terminal of P-factor (P12 ~ P22: 12 ~ 22 amino acid residues from C-terminal) as ligands for Mam2.
From the dose-dependent analysis of the GFP reporter assay ranging from 1 nM to 100 µM of the peptide concentration,
the peptides can be classified into three groups based on EC50 and maximal GFP production level, group1 (P-factor),
group2 (P17 ~ P22), and group3 (P12 ~ P16). At 0.1 µM, only P-factor induced the signal transduction pathway. At 1
µM, peptides from group2 partially induced the pathway and peptides from group3 induced the pathway a little. At 10
µM, all peptides induced the pathway mostly depending on the length of peptides. We also performed AFM experi-
ments using P-factor and peptides from group3 to investigate the interaction between the peptides and Mam2 for com-
parison between the two methods.
Keywords: GPCR; Mam2; Pheromone; GFP; AFM; Yeast
1. Introduction
G-protein-coupled receptors (GPCRs) are integral mem-
brane proteins characterized by seven transmembrane
helices and constitute a large family of transmembrane
proteins. They play an important role in transducing cell
signals by binding to extracellular substances, which
invoke alterations of cell physiology. Peculiarly, GPCRs
receive considerable attention in drug research, as ap-
proximately 70% of medication under development and
more than half of drugs currently on the market targeting
these proteins [1-3].
The fission yeast Schizosaccharomyces pombe (S.
Pombe) is a popular model organism as a host system for
analyzing heterogenous GPCR due to ease in handling of
transgenesis and culture [4-7]. S. pombe multiplies in the
haploid state. This organism has a two-haploid mating
type, h+ (P) and h− (M) [8]. These cells initiate sexual
development when they are starved for nutrients. Under
nutrition depletion, the cells cease to be divided with the
cAMP cascade and conjugate with cells of the opposite
mating type to form diploid zygotes. Diffusible phero-
mones are involved in the conjugating process. h+ cells
secrete P-factor, which bind to the P-factor receptor
Mam2 on the surface of h− cells, whereas h− cells se-
crete M-factor, which bind to the M-factor receptor
Map3 on the surface of h+ cells. The binding of the pher-
omones to their receptors activates the pheromone re-
sponse pathway [9-13]. The active receptors induce Gα
subunit Gpa1 to facilitate a GDP-to-GTP exchange factor
and the dissociation of Gα from the G protein complex.
The Gα subunit with GTP then interacts with downstream
effectors [14-17]. The Gα subunit with GTP then interacts
with downstream effectors to engage signaling cascades
including the MAP kinase pathway consisting of Byr2,

Interaction between Peptide Pheromone or Its Truncated Derivatives and Pheromone Receptor of the Fission Yeast
Schizosaccharomyces pombe Examined by a Force Spectroscopy Study and a GFP Reporter Assay
37
Byr1, Spk1 etc. [18] Since its invention by Binnig et al.
[19], the atomic force microscope (AFM) has become a
powerful tool to study biological samples for measuring
interaction between biomolecules. The AFM tip makes
contact with the cell surface, allowing binding between
ligand and receptor. The tip retraction then induces
stretching of the complex molecules followed by forced
dissociation of the complex. This technique has already
permitted us to quantify unbinding forces of numerous
ligand-receptor pairs, either on an artificial surface or on
the surface of living cells [20-27].
In our previous paper, we revealed that P-factor lack-
ing C-terminal Leu had no ability to bind Mam2 or in-
duce the signal transduction pathway using AFM and the
reporter assay.
In this study, we investigated the interaction between
the series of N-terminal truncated P-factors and Mam2
with the same two methods [28]. Our study showed that
peptides were able to be divided into three groups based
on EC50 and maximal GFP production level with the
reporter assay. Although some peptides from group3
were able to induce the signal transduction pathway only
at high concentration, the distribution pattern of force
curve histogram of group3 peptides from the AFM study
was very similar to that of P-factor. For the evaluation of
the interaction between receptors and ligands, we found
that the result of the AFM experiment was not always in
agreement with that of the GFP assay.
2. Materials and Methods
2.1. Peptides
Peptides used in this study are listed in Table 1. The
customized peptides were obtained from Operon Co.
Ltd. (Tokyo, Japan). Each peptide was prepared as a
stock solution of 1 mM in Milli-Q water and stored at
−80˚C.
2.2. GFP Reporter Assay for Mam2 Signaling
Reporter strains which were previously designed in our
laboratory were grown in YES10 media at 32˚C for 24 -
36 h and were inoculated into 5 mL of the fresh YES10
media [28]. Then the cells were grown at 30˚C for 18 h
and harvested. After having been washed twice with ster-
ile water, cells were transferred to YCB media at OD600
of 1.0 for nitrogen starvation. The cells were incubated at
30˚C for 2 h and used for AFM study and Mam2 signal-
ing assay. For the signaling assay, 1 mL aliquots of cells
were transferred to 24-well microplate containing 1 μL of
peptide solution (final concentration of 0.001, 0.01, 0.1,
1, 3.1, 10, 31, 100 μM each). After incubation at 30˚C for
20 h, the cells were washed three times with PBS and
resuspended in the same volume of PBS. Fluorescence
Table 1. List of peptides used in this study.
Peptides Sequences
P-factor TYADFLRAYQSWNTFVNPDRPNL
P22 YADFLRAYQSWNTFVNPDRPNL
P21 ADFLRAYQSWNTFVNPDRPNL
P20 DFLRAYQSWNTFVNPDRPNL
P19 FLRAYQSWNTFVNPDRPNL
P18 LRAYQSWNTFVNPDRPNL
P17 RAYQSWNTFVNPDRPNL
P16 AYQSWNTFVNPDRPNL
P15 YQSWNTFVNPDRPNL
P14 QSWNTFVNPDRPNL
P13 SWNTFVNPDRPNL
P12 WNTFVNPDRPNL
Cys-P-factor CTYADFLRAYQSWNTFVNPDRPNL
Cys-P15 CYQSWNTFVNPDRPNL
Cys-P14 CQSWNTFVNPDRPNL
Cys-P13 CSWNTFVNPDRPNL
Cys-P12 CWNTFVNPDRPNL
intensity of GFP was measured by a fluorescence spec-
trophotometer (Hitachi F-3010, Japan).
The cells expressing GFP were excited at 491 nm, and
fluorescence emission was detected at 515 nm.
2.3. AFM Measurement
AFM tip preparation was done in the same manner as
described previously [28]. The addition of cysteine at
N-terminal of P-factor and P12 ~ P15 was carried out for
the AFM tip preparation. Force measurements were car-
ried out at room temperature with an NVB-100 AFM
(Olympus, Inc., Tokyo, Japan), which was set on an in-
verted optical microscope (IX70, Olympus, Inc., Tokyo,
Japan) [29,30]. The modified AFM tips were placed on
the nitrogen starved cell surface, and force curve meas-
urements were executed on different positions with a
scan speed of around 1.74 μm/s and using a relative trig-
ger of 20 - 40 nm on the cantilever deflection. The force
curves from about 1024 positions (32 × 32) were re-
corded in each experimental condition to make a histo-
gram of the rupture force in force curves. In the inhibi-
tion experiments, the force measurements were per-
formed in an experimental buffer with free P-factor (final
concentration of 1 μM). To calibrate the response of the
cantilever deflection signal as a function of piezoelectrics,

Interaction between Peptide Pheromone or Its Truncated Derivatives and Pheromone Receptor of the Fission Yeast
Schizosaccharomyces pombe Examined by a Force Spectroscopy Study and a GFP Reporter Assay
38
standard force curve measurements were carried out on
the bottom of the dish, and the spring constant of the
cantilever was calibrated by the thermal vibration me-
thod.
3. Results and Discussion
The interaction between P-factor and Mam2 was inves-
tigated in an S. pombe strain containing sxa2 >
GFPpMAM3G/pAL7 reporter constructs. The binding of
P-factor to Mam2 on the cell surface activates the intra-
cellular signaling pathway that leads to the expression of
GFP. The expression of GFP in response to P-factor is
monitored by a fluorescence spectrophotometer. Fluo-
rescence intensities for P-factor and truncated peptides
(from P12 to P22) were measured at 0.001, 0.01, 0.1, 1,
3.1, 10, 31, and 100 µM as shown in Figure 1. At 0.001
and 0.01 µM, no peptides induced the signal transduction
pathway. At 0.1 µM, only P-factor induced the pathway
and the production level of GFP was about 60% com-
pared with maximal production level (Emax). The pro-
duction level of GFP almost reached the plateau at 1 µM
of P-factor. At the same concentration, peptides from
group2 partially induced the pathway and peptides from
group3 induced the pathway a little. At 3.1 and 10 µM,
all peptides induced the production of GFP mostly de-
pending on the length of peptides. At 31 µM and more,
most of the peptides reached the plateau. EC50s of each
peptide according to fitting a sigmoid function were cal-
culated to be 066 µM for P-factor, 0.42 - 0.69 µM for
peptides from group2, and 1.2 - 9.4 µM for peptides from
group3 (Table 2). A force-volume mode of AFM was
Table 2. List of the maximum GFP production levels, EC50
and the isoelectric points of each peptide.
Emax EC50 (µM) pI
P-factor 67.34 0.066 5.63
P22 56.10 0.606 5.96
P21 63.06 0.421 6.00
P20 58.56 0.524 5.96
P19 58.15 0.485 8.75
P18 57.15 0.693 8.75
P17 61.63 1.20 8.75
P16 56.26 3.98 5.88
P15 55.05 3.85 5.84
P14 52.32 4.79 5.84
P13 50.41 6.07 5.55
P12 41.28 9.35 5.84
(a)
(b)
Figure 1. GFP production levels of the reporter strain (sxa2 >
GFPpMAM3G/pAL7) were exposed to P-factor and trun-
cated peptides (from P12 to P22). The concentration of pep-
tide was varied over the range of 0.001-100 µM, in stimula-
tion of induction for 24 h. Dose-responses of P-factor and
truncated peptides for the reporter strain were shown in (a).
Each peptide can be classified into three groups that consist
of group1 (red line), group2 (blue lines), and group3 (green
lines), based on the affinity with Mam2. GFP production
levels of the reporter strain exposed to 10 μM peptides were
shown in (b).
carried out to examine specific interactions between pep-
tides and the pheromone receptor. Using the AFM tip
cross-linked with Cys-P-factor, Cys-P15, Cys-P14, Cys-
P13, or Cys-P12 peptides via a heterobifunctional PEG
linker, 1024 AFM force curves from each peptide were
then obtained over different spots on mam2+ strain cells
expressing pheromone receptors. Although most of the
retraction curves showed no interaction, some retraction
curves presented a downward deflection abruptly ending
with a force jump. The distribution of unbinding force
greater than 50 pN is shown in Figure 2. Force curves
were obtained in the presence of or absence from 1 µM
free P-factor to evaluate the specificity of the unbinding
force. Ranging from 90 to 160 pN, 100 interaction peaks

Interaction between Peptide Pheromone or Its Truncated Derivatives and Pheromone Receptor of the Fission Yeast
Schizosaccharomyces pombe Examined by a Force Spectroscopy Study and a GFP Reporter Assay
39
Figure 2. Force histogram of unbinding events obtained after analysis of 1024 force curves using the AFM tip cross-linked
with Cys-P-factor (a), or Cys-P15 (b) or Cys-P14 (c), or Cys-P13 (d), or Cys-P12 (e) with (red columns) or without (blue col-
umns) free 1 µM P-factor. In the presence of or absence from 1 µM free P-factor, the unbinding probability decreased from
9.8% to 4.9% for Cys-P-factor (a), from 10% to 5.2% for Cys-P15 (b), from 9.7% to 4.7% for Cys-P14 (c) from 5.2% to 3.2%
for Cys-P13 (d) in the range of 90 to 160 pN. For Cys-P12, the unbinding probabilities were almost the same in the presence
of or absence from free P-factor (e).

Interaction between Peptide Pheromone or Its Truncated Derivatives and Pheromone Receptor of the Fission Yeast
Schizosaccharomyces pombe Examined by a Force Spectroscopy Study and a GFP Reporter Assay
40
were detected without free P-factor while 50 unbinding
events were detected with free P-factor. The number of
events clearly decreased and the unbinding probability
fell from 9.8% to 4.9% for Cys-P-factor (Figure 2(a)).
The difference of the unbinding probability with or
without free P-factor is expected to come from specific
interaction. Next, we carried out force curve measure-
ments to examine the interaction force between the AFM
tip modified with Cys-P15, Cys-P14, Cys-P13, or Cys-
P12 and the cell surface. In the presence of or absence
from 1 µM free P-factor, the unbinding probability fell to
5.2% from 10.0% for Cys-P15 (Figure 2(b)), and to
4.7% from 9.4% for Cys-P14 (Figure 2(c)). The change
of the unbinding probability for Cys-P15 and Cys-P14 is
very similar to that for Cys-P-factor. When the AFM tip
was modified with Cys-P13, the specific interaction was
observed with the decreased number of events, and the
unbinding probabilities were from 5.2% to 3.2% (Figure
2(d)). When the AFM tip was modified with Cys-P12,
the unbinding probabilities were almost the same with
(3.1%) or without (3.8%) free P-factor (Figure 2(e)).
As described in our previous report, the removal of
Leu at C-terminal of P-factor resulted in a complete loss
of P-factor function. This result suggested that C-termi-
nal Leu of P-factor was important for the unbinding force
between peptide and Mam2 examined by AFM and in-
duction of the signal transduction pathway examined by
the GFP reporter assay [28]. In this report, the amino
acid residue at N-terminal of P-factor is removed little by
little, and the resulting peptides were examined by the
GFP reporter assay. At 3.1 and 10 µM, all peptides in-
duced the production of GFP depending on the length of
peptides, indicating that the N-terminal region of P-factor
was expected to be important for an initial interaction
between peptides and Mam2. The initial interaction be-
tween the N-terminal region of P-factor and Mam2 might
be followed by the tight binding between the C-terminal
region of P-factor and Mam2. P17, P18, and P19 induced
the production of GFP slightly higher than expected.
This might be due to a higher isoelectric point (pI) of
three peptides (Table 2). As P15 and P14 were able to
induce the signal transduction pathway only at high con-
centration, we expected that the specific interaction
would not be observed by AFM measurement. But the
distribution patterns of force curve histogram of P15 and
P14 are very similar to that of P-factor. In the AFM ex-
periment, peptides were forced to interact with Mam2
with the applied force by AFM cantilever, which might
compensate the initial interaction between peptide and
Mam2. The length of P12 and P13 might be not enough
for the tight binding to induce specific interaction ob-
served by AFM measurement. The initial interaction be-
tween the N-terminal region of P-factor and Mam2 might
be weak and not detected by AFM. The tight binding be-
tween the C-terminal region of P-factor and Mam2 might
occur after the initial interaction or the applied force by
AFM cantilever and then be detected by AFM experi-
ment.
Figure 3 shows a possible model of an interaction
between the peptide and Mam2 on the cell surface. At the
first step, the N-terminal region of P-factor binds Mam2
weakly. At the next step, the C-terminal region of P-
factor manages to fit a Mam2 binding pocket (Figures
3(a)-(c)) followed by the induction of the signal trans-
duction pathway and the strong interaction between pep-
tide and Mam2. Peptide losing the N-terminal region
cannot bind to the binding pocket at lower concentration
due to a lacking of the initial interaction between the
N-terminal region of P-factor and Mam2 (Figure 3(d)).
For AFM study, peptide with cantilever is forced to make
contact with Mam2. Therefore, the C-terminal region of
P-factor binds to the binding pocket of Mam2 without the
initial interaction between the N-terminal region of
P-factor and Mam2 (Figures 3(e) and (f)). This explains
why we were not able to detect the difference in the
number of events between P14 or P15 and P-factor by
AFM study, although P14 and P15 from group3 have
quite different EC50 from P-factor with the GFP reporter
assay. P13 might be small and P12 might be too small to
occupy the binding pocket of Mam2.
4. Acknowledgements
We would like to thank Dr. H. Tohda (Asahi Glass Co.,
Figure 3. The possible motif of binding of P-factor to Mam2.
First, the N-terminal region of P-factor binds Mam2 weakly
(a). Then, the C-terminal region fits a binding pocket (b)
and (c). The N-terminal region truncated P-factor fails to
bind to the binding pocket since the first binding cannot be
performed (d). Irrespective of the existence of the N-ter-
minal region, The C-terminal region is forcibly combined
with the pocket by pushing of the cantilever in AFM study
(e) and (f).

Interaction between Peptide Pheromone or Its Truncated Derivatives and Pheromone Receptor of the Fission Yeast
Schizosaccharomyces pombe Examined by a Force Spectroscopy Study and a GFP Reporter Assay
41
LTD, Kanagawa, Japan) for his technical assistance and
for kindly providing plasmids (pAL and pSU1) and yeast
strain ARC010 (h− leu1-32 ura4-D18), from which all
strains in this study derived. This work was supported by
a Grant-in-Aid for Challenging Exploratory Research
(25650032) and a Grant-in-Aid for Creative Scientific
Research (19GS0418) to A.I.
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