Interaction between Peptide Pheromone or Its Truncated Derivatives and Pheromone Receptor of the Fission Yeast <i>Schizosaccharomyces pombe</i> Examined by a Force Spectroscopy Study and a GFP Reporter Assay

doi:10.4236/jsemat.2013.34A1005

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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.

Email: [email protected]

Received June 17th, 2013; revised July 25th, 2013; accepted August 3rd, 2013

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

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

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

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

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

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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