Positive Detection of GPCR Antagonists Using a System for Inverted

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Positive detection of GPCR antagonists using a system for inverted expression of a fluorescent reporter gene Nobuo Fukuda, Misato Kaishima, Jun Ishii, and Shinya Honda ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 12 May 2017 Downloaded from http://pubs.acs.org on May 15, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Synthetic Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

Illustration for abstract 206x176mm (300 x 300 DPI)

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 31

1

Positive detection of GPCR antagonists using a system for

2

inverted expression of a fluorescent reporter gene

3 4

Nobuo Fukuda1*, Misato Kaishima2, Jun Ishii3 and Shinya Honda1

5 6

1

7

Technology (AIST), Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan

8

2

9

University, 1-1 Rokkodai, Nada, Kobe, Japan.

Biomedical Research Institute, National Institute of Advanced Industrial Science and

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe

10

3

11

Nada, Kobe, Japan.

Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai,

12 13

*Correspondence to: N. Fukuda

14

Biomedical Research Institute

15

National Institute of Advanced Industrial Science and Technology (AIST)

16

Higashi, Tsukuba, Ibaraki 305-8566, Japan

17

Fax: +81 29 861 6194; Tel: +81 29 849 1458

18

E-mail: [email protected]

19 20 21 22

Key words: antagonist, G-protein-coupled receptor, inverted expression system, PEST sequence, yeast

23 24 25

Running title: Positive detection of GPCR antagonists using a system for inverted expression of a fluorescent reporter gene

26


1

Page 3 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Abstract

2

3

The yeast Saccharomyces cerevisiae is a useful eukaryotic host organism for studying

4

GPCRs as monomolecular models. Fluorescent reporter gene assays for GPCRs provide a

5

convenient assay for measuring receptor activity using fluorometric instruments. Generally,

6

these assays detect receptor activation by agonistic ligands as the induction of fluorescent

7

reporter expression, whereas antagonistic activities are detected by competition with

8

agonistic ligands, resulting in decreases in fluorescence intensity.

9

In the current study, we established a system for inverted expression of a fluorescent

10

reporter by incorporating a PEST-tag and finding out a promoter inhibited by activation of

11

the GPCR signaling pathway from yeast endogenous promoters. Because agonists prevent

12

fluorescent reporter expression in this system, antagonists compete with agonists and yield

13

increased fluorescence intensity. We used the yeast endogenous pheromone receptor as a

14

model GPCR to demonstrate the feasibility of our system for positive detection targeted at

15

antagonists. Compared to results when only agonists were added to yeast cells, more than 10-

16

fold higher fluorescence intensity was observed when antagonists were added in combination

17

with agonists. The approach described here has the potential to markedly accelerate the

18

identification of GPCR antagonists by providing rapid and straightforward responses.

19


2


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Page 4 of 31

Introduction

2 3

G-protein-coupled receptors (GPCRs) are one of the largest families of membrane

4

proteins, and serve as key mediators of the effect of numerous neurotransmitters, hormones,

5

cytokines, and therapeutic drugs [1]. GPCR signaling is triggered by an agonist, and

6

conformational change of the receptor caused by agonist binding induces dissociation of

7

heterotrimeric G proteins into monomeric Gα and a Gβγ complex, which is accompanied by

8

the exchange of GDP for GTP on the Gα subunit. Both Gα-GTP and Gβγ complex can

9

modulate various cellular signaling pathways, such as regulation of adenylate cyclases,

10

phospholipases, the mitogen-activated protein kinase (MAPK) pathway, and target gene

11

expression [2]. GPCR signaling is negatively regulated by β-arrestins that also activate

12

different intracellular signaling pathways [3]. Based on insights into the functions of GPCRs,

13

numerous drug candidates have been designed and generated using approaches to modulating

14

the activity of these proteins [4]. In fact, over 30% of marketed medicines act on GPCRs,

15

which remain attractive targets for drug development [5].

16

The budding yeast Saccharomyces cerevisiae is an appealing host cell system for

17

studying GPCRs because the mechanisms of GPCR signaling are highly conserved among a

18

diverse range of eukaryotes [6]. Yeast possesses an uncompetitive and monopolistic GPCR

19

signaling pathway (the pheromone signaling pathway) [7]; hence the deletion and

20

replacement of the yeast endogenous GPCR with mammalian GPCRs permits reconstitution

21

of signaling pathways from higher organisms in the absence of signaling cross-talk [2].

22

Yeast-based analyses provide additional advantages, including rapid cell growth, simplicity,

23

low cost of cultivation [8-11], and amenability to robotic manipulation.

24 25

GPCR signaling in yeast has been detected and evaluated using a variety of reporter genes under control of pheromone-responsive promoters. Although the HIS3 and lacZ genes


3

Page 5 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

[12, 13] are conventional reporter genes that are used for growth screening and colorimetric

2

evaluation, respectively, recent research has successfully employed fluorescent reporter

3

genes as powerful and convenient tools for rapid quantification of the level of GPCR

4

signaling [14-16]. The coral-derived GFP, Umikinoko Green (ymUkG1), is one of the

5

brightest GFPs, having undergone codon-optimization for S. cerevisiae [16], permitting more

6

sensitive assays than ever before.

7

In yeast-based fluorescent reporter systems, agonistic ligands trigger GPCR signaling

8

accompanied by increased reporter expression. In contrast, antagonistic ligands induce

9

receptor inactivation by competing with agonistic ligands, resulting in a decrease in

10

fluorescence intensity by preventing reporter expression. In past reports, the GFP reporter

11

genes were introduced into yeast cells using episomal multi-copy plasmids to enhance the

12

signal-to-noise ratio in fluorescence intensity [17, 18], compared to chromosomally-inserted

13

reported genes. Unlike in the case of assay of agonists, however, it is extremely difficult to

14

distinguish yeast cells affected by antagonists from ones that have lost the episome-borne

15

GFP reporter genes; this distinction can become a critical issue, especially in single cell

16

isolation technologies [19] such as flow cytometry and cell picking systems.

17

Here, we designed and constructed an inverted reporter expression system to allow

18

positive detection of GPCR antagonists. The general strategy is illustrated in Figure 1. In this

19

system, the ymUkG1 reporter gene is expressed in the absence of any ligands (Figure 1A),

20

and addition of agonists induces GPCR signaling, resulting in repression of the ymUkG1

21

reporter gene (in contrast to induction by agonist in the “standard” system) (Figure 1B).

22

Against such a background, antagonists inhibit signal transduction by binding to GPCRs in

23

competition with agonists, resulting in promotion (derepression) of ymUkG1 gene expression

24

(Figure 1C). Using the yeast endogenous pheromone receptor (Ste2) as a model GPCR, we

25

demonstrated the feasibility of our system for positive detection of GPCR antagonists.

26


4


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Page 6 of 31

Results and Discussion

2 3

Construction of the reporter expression system. As a first step, we searched for

4

endogenous promoters that were inhibited by activation of the GPCR signaling pathway in

5

yeast. Based on the ratio between transcription levels with and without α-factor (the

6

endogenous agonist of Ste2) [20], we narrowed down a list of yeast endogenous promoters.

7

From the 30 promoters with the highest values for this ratio, we selected three kinds of

8

characteristic promoters in accordance with the following viewpoints. Whereas the RNR1

9

promoter (PRNR1) yields the highest values for the above ratio, the PMA2 promoter (PPMA2)

10

and the HXT1 promoter (PHXT1) produce high and moderate levels of transcription

11

(respectively) in the absence of agonists. Separate constructs were designed by inserting the

12

ymUkG1 reporter gene downstream of each promoter. For comparison, we also generated a

13

construct using the strong constitutive promoter of the PGK1 gene (PPGK1).

14

To accelerate degradation of the ymUkG1 reporter protein within yeast cells

15

supplemented with agonists, a PEST-tag (corresponding to a 36-amino-acid segment

16

(residues 414 to 449) of Ste3 [21]), was fused to the C-terminus of the reporter as shown in

17

Figure 2A. The ymUkG1-PEST reporter gene was similarly inserted downstream of each

18

promoter, and fluorescence intensity of yeast cells was measured after overnight cultivation

19

without agonists. In comparison with PPGK1, PRNR1 allowed obviously low levels of reporter

20

gene expression in the absence of agonist, whereas PHXT1 produced moderate reporter gene

21

expression under these conditions. In contrast, the level of expression with PPMA2 was not

22

sufficient to permit use as a reporter construct; hence we eliminated PPMA2 from further

23

consideration in the current study. As shown in Figure 2A, attachment of the PEST-tag to the

24

ymUkG1 reporter caused a reduction of the fluorescence intensity. To investigate whether

25

the degradation of the ymUkG1-PEST reporter was accelerated within yeast cells,


5

Page 7 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

fluorescence intensity was measured over time after repressing transcription of the reporter

2

genes (see Supporting Information Figure S1A). While the half-life of the ymUkG1 reporter

3

was approximately 36 h, that of the ymUkG1-PEST reporter was approximately 12 h (see

4

Supporting Information Figure S1B), demonstrating that the Ste3-derived PEST-tag

5

contributes to degradation of the ymUkG1 reporter within yeast cells.

6

Subsequently, fluorescence intensity of yeast cells was measured and compared after

7

cultivation with agonist (Figure 2B). To grasp the degree of change in a comprehensible way,

8

relative fluorescence intensity (FI) was indicated by normalizing the FI values to those

9

obtained in the absence of agonist. Among the tested promoters, PHXT1 was most strongly

10

inhibited by the addition of the agonist. The relative FI of yeast cells expressing the

11

ymUkG1-PEST reporter gene under the control of PHXT1 decreased to 6.0% after 16 h of

12

cultivation with agonist at an initial concentration of 20 µM. Based on these results, we

13

adopted the PHXT1-ymUkG1-PEST-terminator construct as the appropriate expression cassette

14

for use as a reporter for the inverted expression system described in Figure 1.

15 16

Improvement of fluorescence intensity by exchanging the terminator. Although use of

17

PHXT1 and attachment of the PEST-tag successfully brought a significant change to the

18

relative FI value in response to the agonist, the absolute value of FI declined, even in the

19

absence of the agonist (Figure 2A). To compensate for the decline, we replaced the original

20

terminator within the expression cassette with one optimized according to yeast

21

“terminatome” data [22]. Terminator regions encode 3’ untranslated regions (3’-UTRs) of

22

mRNA, and influence the expression of upstream coding regions to varying degrees. In the

23

global ranking of yeast terminator region activity [22], the ADH1 terminator (TADH1) (which

24

we have used successfully in previous work) is not classified as one of the top 30 terminators,

25

despite the popularity of this fragment for use in constructs. We therefore instead focused on

26

the DIT1 terminator (TDIT1), which exhibits the strongest known termination activity among


6


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 31

1

yeast terminators, and replaced TADH1 with TDIT1 within the expression cassette (PHXT1-

2

ymUkG1-PEST-terminator). As a result, the absolute value of FI was doubled in the absence

3

of agonist (see Supporting Information Figure S2).

4

When the initial concentration of agonist was set at 20 µM, the relative FI value of

5

yeast cells possessing PHXT1-ymUkG1-PEST-TDIT1 fell to 5.6% of peak FI after 16 h of

6

cultivation, whereas green fluorescence fell to 22% of peak FI in cells possessing PPGK1-

7

ymUkG1-PEST-TDIT1 under the same conditions (Figure 3). These results suggested that we

8

would observe an 18-fold higher FI value by addition of antagonist (in competition with

9

agonist) in cells harboring the PHXT1-ymUkG1-PEST-TDIT1 expression cassette. Therefore, we

10

assumed that this expression cassette should be amenable to positive detection of GPCR

11

antagonists.

12 13

Redesign of operation according to promoter characteristics. Although the above-

14

described experiments demonstrated the feasibility of the inverted fluorescent reporter

15

expression system (Figure 1), this system required 16 h after agonist addition to undergo

16

clearance of fluorescent reporter molecules within yeast cells. Generally, researchers prefer

17

to use rapid and accurate responses for physiological analyses. In the previously reported

18

system (a negative detection system) [14], GPCR antagonism was detected only after 4-8 h

19

of cultivation. However, given the half-life of the ymUkG1-PEST reporter protein (12 h), it

20

was difficult to envision substantially shortening cultivation times for the system as

21

described in Figure 1.

22

To address the issue of responsivity, we redesigned the flow of operations according

23

to promoter characteristics. Because expression of the HXT1 gene is induced in the presence

24

of glucose [23], raffinose was selected as the alternative carbon source during pre-cultivation

25

without GPCR ligands (Figure 4A). According to this revised scheme, yeast cells are initially

26

grown in raffinose medium (a condition under which the ymUkG1-PEST reporter is not


7

Page 9 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

expressed; Figure 4B); the pre-cultivated cells then are transferred into glucose medium

2

containing antagonist and/or agonist. While agonist represses reporter gene expression even

3

in the presence of glucose (Figure 4C), antagonist blocks agonist binding to the receptor,

4

preventing repression and thereby permitting expression of the reporter and ensuing

5

fluorescence emission (Figure 4D). We examined the possibility of rapid detection of GPCR

6

antagonists in accordance with this revised scheme (Figure 4).

7 8

Detection of the synthetic antagonist against the model GPCR according to the

9

redesigned scheme. We initially investigated the relationship between the FI value and

10

glucose concentration (Figure 5A). Specifically, we examined ymUkG1-PEST reporter gene

11

expression as a function of glucose concentration by assessing fluorescence after cultivation

12

of yeast cells in the presence of total sugar (glucose and raffinose) at an initial concentration

13

of 2.0%. We observed only negligible levels of fluorescence from yeast cells grown in

14

raffinose medium (0.0% glucose in Figure 5A), an intensity that was equivalent to that of the

15

mock yeast (harboring a construct lacking a GFP gene; see Figure 2A).

16

To obtain rapid detection, we shortened cultivation from 16 h to 6 h, as shown in

17

Figure 5B. We utilized the chemically synthesized peptide, Fpep1 (WLQLKPGQP[Nle]Y)

18

[24] as an antagonist of the Ste2 receptor, and generated agonist concentration–response

19

curves both without and with 40 µM antagonist (Figure 5C). The curve was shifted to the

20

right through the effect of antagonist, and the IC50 value of agonist for reporter gene

21

expression was 34 µM in the presence of 40 µM antagonist, while that value was 4.0 µM in

22

the absence of antagonist. Additionally, we constructed agonist concentration–response

23

curves after 8 h of cultivation (see Supporting Information Figure S3). The IC50 values of the

24

agonist were 3.7 µM (in the absence of antagonist) and 46 µM (in the presence of 40 µM

25

antagonist). Although the difference between the two curves appeared to be enlarged by the


8


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

additional 2 h of cultivation, we concluded that 6 h of cultivation would be suitable for the

2

evaluation of GPCR antagonists with a special emphasis on responsivity.

3

Page 10 of 31

Subsequently, antagonist concentration–response curves were constructed in the

4

presence and absence of agonist at 20 µM (Figure 5D); these experiments yielded an EC50

5

value of 5.7 µM for the antagonist in this reporter gene assay. Compared to agonist, a 2.5-

6

fold higher concentration of antagonist was sufficient to induce 100% of green fluorescence

7

emission, presumably by occupying the majority of the receptor binding sites. However, the

8

background signal (the relative FI value without antagonist) rose to almost 20% in the

9

presence of 20 µM agonist (Figure 5C and D); contrast that value to the 5.6% obtained via

10

the previous method (see Figure 3). These results suggested that while exchange of carbon

11

source in pre-cultivation (from glucose to raffinose) permitted rapid detection of the

12

antagonist by decreasing baseline reporter gene expression, higher concentrations of ligands

13

would be required in cultivation to provide a sufficient signal-to-noise ratio.

14 15

Introduction of short incubation with ligands for reducing background signal. Despite

16

our expectation, a small but notable amount of fluorescence was detected within yeast cells

17

even during cultivation in the presence of 20 µM agonist (Figure 5D). We hypothesized that

18

activation of PHXT1 by glucose was initiated before the promoter underwent repression caused

19

by agonist via the GPCR signaling pathway. To permit earlier inactivation of PHXT1 by

20

agonist, we introduced a short additional step into our scheme, whereby yeast cells were

21

incubated with ligands for 15 minutes before cultivation in glucose medium (Figure 6A).

22

This pre-incubation with 10 µM agonist successfully reduced the background signal detected

23

after the subsequent 6 h of cultivation in the presence of glucose (see Supporting Information

24

Figure S4A). Setting antagonist concentration at 100 µM, the relative FI value of yeast cells

25

supplemented with 10 µM agonist remained at approximately 80%, whereas that value

26

approached 100% in the presence of 5 µM agonist. The subsequent 6 h of cultivation in the


9

Page 11 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

presence of glucose was sufficient for the synthesis of the fluorescent reporter (see

2

Supporting Information Figure S4B).

3

Finally, we performed rapid detection of antagonist under the conditions described in

4

Figure 6B. The relative FI values were 8.2% (in the presence of 10 µM agonist) and 100%

5

(in the presence of 10 µM agonist and 300 µM antagonist), representing a 12-fold increase in

6

response. These results demonstrated the feasibility of our system for rapid and

7

straightforward detection of GPCR antagonists. Because the background level in

8

fluorescence intensity was extremely low (Figure 6B and Supporting Information Figure

9

S4A), detection of activity would be possible with much lower ratio of antagonist to agonist,

10

especially in identification of antagonistic compounds. Furthermore, we carried out

11

fluorescence microscope observation of ligand-treated yeast cells to understand phenotypic

12

heterogeneity in populations. Specifically, in addition to changes in fluorescence emissions

13

caused by the episomal reporter gene expression cassettes, yeast cells treated with agonist are

14

known to undergo morphological changes in response to changes in gene expression induced

15

by GPCR signaling [25]. Notably, we did not observe detectable green fluorescent signals

16

from yeast cells grown in the presence of agonist (Figure 6C), despite the observation (to

17

various degrees) of morphological changes (“shmoo” formation) in response to agonist. On

18

the other hand, approximately half of the cells exhibited obvious fluorescent signals when

19

grown in the presence of both antagonist and agonist, while all of these cells had normal

20

morphologies (Figure 6D). The copy number of plasmids (multi-copy) is presumed to be a

21

major factor in the production of heterogeneity in the fluorescence of yeast cells treated with

22

the combination of agonist and antagonist. However, it is obvious that yeast cells emitting

23

green fluorescence had responded to antagonistic ligands, which would exclude false results

24

in identification of antagonistic compounds.

25


10


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 31

1

Concluding remarks. In the current work, we established an inverted fluorescent reporter

2

gene expression system in which expression is induced in the presence of GPCR antagonists;

3

this system is expected to allow positive detection of antagonists. Our system consists of a

4

single expression cassette (encoding a fluorescent reporter protein) carried on a multi-copy

5

episome; hence, introduction of the system into other host strains should be easy, even if the

6

selection marker and/or color of the fluorescent reporter would need to be changed for use in

7

specific backgrounds.

8 9

Characterization of human GPCRs in a yeast platform has provided deep insights into GPCR signaling and facilitated identification of the ligands of orphan GPCRs. Use of our

10

system in combination with the already established platform is expected to facilitate further

11

progress, especially in identification of GPCR antagonists that might serve as potential

12

therapeutic compounds.

13 14


11

Page 13 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Methods

2 3

Strains and media. Detailed information about S. cerevisiae laboratory yeast strain HR41-K

4

[26] is shown in Table 1. Yeast cells were grown in YPD medium (1% yeast extract, 2%

5

peptone, and 2% glucose), SD medium (0.67% yeast nitrogen base without amino acids

6

[Becton Dickinson and Company, Franklin Lakes, NJ], and 2% glucose), SGR medium

7

(0.67% yeast nitrogen base without amino acids, 2% galactose, and 2% raffinose), or SDR

8

medium (0.67% yeast nitrogen base without amino acids, 2% total sugar consisting of

9

glucose and raffinose). Amino acids and nucleotides (20 mg/L lysine, 30 mg/L leucine, and

10

20 mg/L uracil) were supplemented into SD, SGR, and SDR media to compensate for

11

relevant auxotrophies. A final concentration of 2% agar was added to the liquid media when

12

preparing solid media.

13 14

Construction of plasmids. The sequences of the oligonucleotides used in this study are

15

listed in Table 2. The plasmids used in this study (Table 1) were constructed as follows.

16

Using the pGK416-yUmikinoko-Green [16] plasmid as a template, the ymUkG1 gene was

17

amplified with oligonucleotide pair o1 and o2, and the resulting fragment was digested with

18

NotI and BamHI. The digested DNA fragment was inserted into NotI-, BamHI-digested pHY-

19

PGA plasmid [8], permitting replacement of the EGFP gene with the ymUkG1 gene; the

20

resulting plasmid was designated pHYP-yUG. PGAL1 was prepared by digesting the pUY-

21

GGA plasmid [26] with SacII and NotI. PRNR1, PPMA2, and PHXT1 were amplified from strain

22

HR41-K genomic DNA using oligonucleotide pairs o3 and o4, o5 and o6, and o7 and o8

23

(respectively); the resulting fragments were digested with SacII and NotI. Each promoter

24

fragment was inserted into SacII-, NotI-digested pHYP-yUG plasmid, permitting


12


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 31

1

replacement of PPGK1 with the respective promoters; the resulting plasmids were designated

2

pHYG-yUG, pHYR-yUG, pHYPM-yUG, and pHYHX-yUG, respectively.

3

Using the pHYP-yUG plasmid as a template, the ymUkG1 gene was amplified with

4

oligonucleotide pair o1 and o9. A DNA fragment encoding the PEST-tag and derived from

5

STE3 was amplified from strain HR41-K genomic DNA using oligonucleotide pair o10 and

6

o11. The two amplified DNA fragments (corresponding to ymUkG1 and PEST) were

7

combined using the In-Fusion HD Cloning kit (Takara Bio, Inc., Shiga, Japan). Using the

8

combined DNA fragment as a template, ymUkG1-PEST was amplified with oligonucleotide

9

pair o1 and o11, and then digested with NotI and BamHI. The digested DNA fragments then

10

were inserted into NotI-, BamHI-digested pHYP-yUG, pHYG-yUG, pHYR-yUG, pHYPM-

11

yUG and pHYHX-yUG, permitting replacement of the original ymUkG1 gene with ymUkG1-

12

PEST; the resulting plasmids were designated pHYP-yU36, pHYG-yU36, pHYR-yU36,

13

pHYPM-yU36, and pHYHX-yU36, respectively.

14

TDIT1 was amplified from strain HR41-K genomic DNA using oligonucleotide pair

15

o12 and o13; the resulting fragment was then digested with BamHI and XhoI. The digested

16

DNA fragment was inserted into BamHI-, XhoI-digested plasmids pHYP-yU36 and pHYHX-

17

yU36, permitting replacement of TADH1 with TDIT1; the resulting plasmids were designated

18

pHYP-yU36D and pHYHX-yU36D, respectively.

19 20

Each of the resulting plasmid constructs was introduced into yeast cells using the lithium acetate method [27].

21 22

GPCR ligands. The agonist for yeast Ste2 receptor, α-factor, was obtained from Zymo

23

Research Corp. (Irvine, CA, Unite States). The antagonist against Ste2, Fpep1, was obtained

24

from Biologica Co. (Nagoya, Aichi, Japan).

25


13

Page 15 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Fluorescent Reporter Assay. The ymUkG1 or ymUkG1-PEST gene was used as a

2

fluorescent reporter. The yeast cells grown in the respective medium with or without ligands

3

were harvested and washed with distilled water. The cells then were resuspended in 100 µL

4

of distilled water to an optical density of 1.0 at 600 nm (OD600 = 1.0). Green fluorescence

5

intensities were measured using an Infinite 200 fluorescence microplate reader (Tecan Japan

6

Co., Ltd., Kawasaki, Japan). For detection of the signal, the excitation wavelength was set at

7

485 nm with a bandwidth of 20 nm, and the emission wavelength was set at 535 nm with a

8

bandwidth of 25 nm. The gain was set at 60.

9 10

Microscopic observation. Each yeast strain was cultured in SD medium with antagonist

11

and/or agonist at 30 °C. The cells then were harvested, washed with 100 µL distilled water,

12

and resuspended in 100 µL distilled water to an OD600 of 1.0. Resuspended cells were

13

observed using a fluorescent microscope (BZ-X700, Keyence Co., Ltd., Tokyo, Japan). Each

14

image was photographed with the same exposure time using a 40x objective lens. Green

15

fluorescence images were acquired with a 470/40 nm band-pass filter for excitation and a

16

525/50 nm band-pass filter for emission. Fluorescent images were overlaid on the

17

corresponding bright-field images.

18 19 20

Author Contributions

21

N.F. designed the study and conducted the experiments. N.F., M.K., and J.I. analyzed

22

the data. N.F., J.I., and S.H. cowrote the manuscript. All authors read and approved the final

23

manuscript.

24 25

Acknowledgments


14


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Page 16 of 31

Plasmid pYO323 was provided by the National Bio-Resource Project (NBRP) of the

2

MEXT, Japan. This work was supported in part by a grant from JSPS KAKENHI (Grant

3

Number 16K14497).

4 5

Abbreviations

6

FI, fluorescence intensity; GFP, green fluorescent protein; GPCR, G-protein-coupled

7

receptor; MAPK, mitogen-activated protein kinase; ymUkG1, Umikinoko Green subjected to

8

codon optimization for S. cerevisiae

9 10 11 12

Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors.

13 14 15

Conflict of interest All authors declare that they have no competing interests.

16 17


15

Page 17 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

References

2

[1] Teitler, M., and Herrick-Davis, K. (2014) Determining the oligomer number of native

3

GPCR using florescence correlation spectroscopy and drug-induced inactivation-

4

reactivation. Curr Pharm Biotechnol. 15, 927-937.

5 6

[2] Liu, R., Wong, W., IJzerman, A.-P. (2016) Human G protein-coupled receptor studies in Saccharomyces cerevisiae. Biochem Pharmacol. 114, 103-115.

7

[3] Liu, C.-H., Gong, Z., Liang, Z.-L., Liu, Z.-X., Yang, F., Sun, Y.-J., Ma, M.-L., Wang, Y.-

8

J., Ji, C.-R, Wang, Y.-H., Wang, M.-J., Cui, F.-A., Lin, A., Zheng, W.-S., He, D.-F., Qu,

9

C.-X., Xiao, P., Liu, C.-Y., Thomsen, A.-R., Joseph Cahill, T. 3rd., Kahsai, A.-W., Yi, F.,

10

Xiao, K.-H., Xue, T., Zhou, Z., Yu, X., Sun, J.-P. (2017) Arrestin-biased AT1R agonism

11

induces acute catecholamine secretion through TRPC3 coupling. Nat Commun. 8, 14335.

12 13 14 15 16

[4] Eglen, R.-M., and Reisine, T. (2009) New Insights into GPCR Function: Implications for HTS. Methods Mol Biol. 552, 1-13. [5] Hopkins, A.-L., and Groom, C.-R. (2002) The druggable genome, Nat. Rev. Drug Discov. 1, 727–730. [6] Fukuda, N., Ishii, J., Kaishima, M., Kondo, A. (2011) Amplification of agonist

17

stimulation of human G-protein-coupled receptor signaling in yeast. Anal Biochem. 417,

18

182-187.

19

[7] Stewart, G.-D., Valant, C., Dowell, S.-J., Mijaljica, D., Devenish, R.-J., Scammells, P.-J.,

20

Sexton, P.-M., Christopoulos, A. (2009) Determination of adenosine A1 receptor agonist

21

and antagonist pharmacology using Saccharomyces cerevisiae: implications for ligand

22

screening and functional selectivity. J Pharmacol Exp Ther. 331, 277-286.

23 24

[8] Fukuda, N., Doi, M., Honda, S. (2013) Yeast one-hybrid Gγ recruitment system for identification of protein lipidation motifs. PLoS One. 8, e70100.


16


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

[9] Kaishima, M., Fukuda, N., Ishii, J., Kondo, A. (2014) Desired alteration of protein

2

affinities: competitive selection of protein variants using yeast signal transduction

3

machinery. PLoS One. 9, e108229.

Page 18 of 31

4

[10] Fukuda N, Honda S. (2015) Rapid evaluation of tyrosine kinase activity of membrane-

5

integrated human epidermal growth factor receptor using the yeast Gγ recruitment

6

system. ACS Synth Biol. 4, 421-429.

7

[11] Fukuda, N., and Honda, S. (2015) Functional analyses and affinity-alteration of

8

receptors and enzymes based on membrane recruitment of yeast guanine nucleotide-

9

binding protein gamma subunit. Receptors Clin Investig. 2, e373.

10

[12] Manfredi, J.-P., Klein, C., Herrero, J.-J., Byrd, D.-R., Trueheart, J., Wiesler, W.-T.,

11

Fowlkes, D.-M., Broach, J.-R. (1996) Yeast alpha mating factor structure-activity

12

relationship derived from genetically selected peptide agonists and antagonists of Ste2p.

13

Mol Cell Biol. 16, 4700-4709.

14

[13] Pausch, M.-H., Lai, M., Tseng, E., Paulsen, J., Bates, B., Kwak, S. (2004) Functional

15

expression of human and mouse P2Y12 receptors in Saccharomyces cerevisiae.

16

Biochem Biophys Res Commun. 324, 171-177.

17

[14] Nakamura, Y., Ishii, J., Kondo, A. (2015) Applications of yeast-based signaling sensor

18

for characterization of antagonist and analysis of site-directed mutants of the human

19

serotonin 1A receptor. Biotechnol Bioeng. 112, 1906-1915.

20

[15] Kaishima, M., Ishii, J., Fukuda, N., Kondo, A. (2015) Gγ recruitment systems

21

specifically select PPI and affinity-enhanced candidate proteins that interact with

22

membrane protein targets. Sci Rep. 5, 16723.

23

[16] Kaishima, M., Ishii, J., Matsuno, T., Fukuda, N., Kondo, A. (2016) Expression of varied

24

GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected

25

expression and fluorescence intensity. Sci Rep. 6, 35932.


17

Page 19 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

[17] Iguchi, Y., Ishii, J., Nakayama, H., Ishikura, A., Izawa, K., Tanaka, T., Ogino, C.,

2

Kondo, A. (2010) Control of signalling properties of human somatostatin receptor

3

subtype-5 by additional signal sequences on its amino-terminus in yeast. J Biochem. 147,

4

875-884.

5

[18] Ishii, J., Yoshimoto, N., Tatematsu, K., Kuroda, S., Ogino, C., Fukuda, H., Kondo, A.

6

(2012) Cell wall trapping of autocrine peptides for human G-protein-coupled receptors

7

on the yeast cell surface. PLoS One. 7, e37136.

8 9

[19] Gross, A., Schoendube, J., Zimmermann, S., Steeb, M., Zengerle, R., Koltay, P. (2015) Technologies for Single-Cell Isolation. Int J Mol Sci. 16, 16897-16919.

10

[20] MacKay, V.-L., Li, X., Flory, M.-R., Turcott, E., Law, G.-L., Serikawa, K.-A., Xu, X.-

11

L., Lee, H., Goodlett, D.-R., Aebersold, R., Zhao, L.-P., Morris, D.-R. (2004) Gene

12

expression analyzed by high-resolution state array analysis and quantitative proteomics:

13

response of yeast to mating pheromone. Mol Cell Proteomics. 3, 478-489.

14

[21] Roth, A.-F., Sullivan, D.-M., Davis, N.-G. (1998) A large PEST-like sequence directs

15

the ubiquitination, endocytosis, and vacuolar degradation of the yeast a-factor receptor. J

16

Cell Biol. 142, 949-961.

17

[22] Yamanishi, M., Ito, Y., Kintaka, R., Imamura, C., Katahira, S., Ikeuchi, A., Moriya, H.,

18

Matsuyama, T. (2013) A genome-wide activity assessment of terminator regions in

19

Saccharomyces cerevisiae provides a “terminatome” toolbox. ACS Synth Biol. 2, 337-

20

347.

21

[23] Lewis, D.-A., and Bisson, L.-F. (1991) The HXT1 gene product of Saccharomyces

22

cerevisiae is a new member of the family of hexose transporters. Mol Cell Biol. 11,

23

3804-3813.

24

[24] Eriotou-Bargiota, E., Xue, C.-B., Naider, F., Becker, J.-M. (1992) Antagonistic and

25

synergistic peptide analogues of the tridecapeptide mating pheromone of Saccharomyces

26

cerevisiae. Biochemistry. 31, 551-557.


18


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Page 20 of 31

[25] Banderas, A., Koltai, M., Anders, A., Sourjik, V. (2016) Sensory input attenuation allows predictive sexual response in yeast. Nat Commun. 7, 12590.

3

[26] Fukuda, N., Matsukura, S., Honda, S. (2013) Artificial conversion of the mating-type of

4

Saccharomyces cerevisiae without autopolyploidization. ACS Synth Biol. 2, 697-704.

5 6

[27] Gietz, D., St. Jean, A., Woods, R.-A., Schiestl, R.-H. (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20, 1425.

7


19

Page 21 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22


Table 1. Yeast strain and plasmids used in this study. Name Yeast strain

Description

Reference source

HR41-K

MATa his3∆1 ura3∆0 leu2∆0 lys2∆0

25

Plasmids pYO323 2µ ori and HIS3 marker (mock) pHYP-yUG 2µ ori, HIS3 marker and PPGK1-ymUkG1-TADH1 pHYG-yUG, 2µ ori, HIS3 marker and PGAL1-ymUkG1-TADH1 pHYR-yUG 2µ ori, HIS3 marker and PRNR1-ymUkG1-TADH1 pHYPM-yUG 2µ ori, HIS3 marker and PPMA2-ymUkG1-TADH1 pHYHX-yUG 2µ ori, HIS3 marker and PHXT1-ymUkG1-TADH1 pHYP-yU36 2µ ori, HIS3 marker and PPGK1-ymUkG1-PEST-TADH1 pHYG-yU36 2µ ori, HIS3 marker and PGAL1-ymUkG1-PEST-TADH1 pHYR-yU36 2µ ori, HIS3 marker and PRNR1-ymUkG1-PEST-TADH1 pHYPM-yU36 2µ ori, HIS3 marker and PPMA2-ymUkG1-PEST-TADH1 pHYHX-yU36 2µ ori, HIS3 marker and PHXT1-ymUkG1-PEST-TADH1 pHYP-yU36D 2µ ori, HIS3 marker and PPGK1-ymUkG1-PEST-TDIT1 pHYHX-yU36D 2µ ori, HIS3 marker and PHXT1-ymUkG1-PEST-TDIT1 a) Resource was provided by the National Bio-Resource Project (NBRP) of the MEXT, Japan


NBRPa Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study Present study

20


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Page 22 of 31

Table 2. Sequences of oligonucleotides used to construct plasmids.

3

Number

Sequence

4

1

5’-TACTTTAAAgcggccgcATGGTCAGTGTCATCAAA-3’

5

2

5’-CCCCAGTTTGggatccTTACTTAGAAGCTTGAGA-3’

6

3

5’-AATTGGAGCTCCAccgcggGGGTTATGGAGAGTATGCTG-3’

7

4

5’-CACTGACCATgcggccgcGATGTTAATATATCAACAAA-3’

8

5

5’-AATTGGAGCTCCAccgcggACTTTGTTGTTAGCAGACA-3’

9

6

5’-CACTGACCATgcggccgcAACGCAAAACAGTTTCCTT-3’

10

7

5’-AATTGGAGCTCCAccgcggGCCACAATGAAACTTCAAT-3’

11

8

5’-CACTGACCATgcggccgcGATTTTACGTATATCAACT-3’

12

9

5’-CTTAGAAGCTTGAGATGGCA-3’

13

10

5’-TCTCAAGCTTCTAAGGACGACGAAATATCACTTGG-3’

14

11

5’-CCCCAGTTTGggatccTTAAGAGTAGCAAAGACTTTCC-3’

15

12

5’-TGTACAAGTAAggatccTAAAGTAAGAGCGCTACA-3’

16

13

5’-CGGGCCCCCCctcgagGATTCACATTTCAACCAC-3’

17 18


21

Page 23 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure legends

3

Figure 1. Schematic outline of the concept of the inverted fluorescent reporter gene

4

expression system for positive detection of GPCR antagonists. (A) Fluorescent reporter

5

genes (ymUkG1 with/without the sequence encoding the PEST-tag) are carried on multi-copy

6

episomal plasmids and introduced into yeast cells. These cells express the reporter genes in the

7

absence of GPCR ligands. (B) Exposure of yeast to agonist triggers GPCR signaling, which

8

prevents expression of the fluorescent reporter gene. After the passage of a time interval

9

sufficient for decay of existing GFP, these cells should no longer emit green fluorescence. (C)

10

Addition of antagonist blocks binding of agonist to the receptor, thereby inhibiting GPCR

11

signaling and allowing expression of the fluorescent reporter gene.

12 13

Figure 2. Evaluation of the expression levels of the fluorescent reporter genes. (A) Two

14

kinds of reporter genes, ymUkG1 and ymUkG1-PEST, were prepared and expressed under the

15

control of PPGK1, PPMA2, PRNR1, or PHXT1. The expression levels of the reporter genes were

16

quantified and compared among promoters. (B) Inhibition assay with 20 µM agonist (α-factor)

17

was carried out after 16 h of cultivation. Relative fluorescence intensity (FI) value was

18

calculated by setting each value without agonist at 100%. Values are presented as means ±

19

standard deviations from three independent experiments.

20 21

Figure 3. Inhibition assay with 20 µM agonist for ymUkG1-PEST reporter gene expression.

22

Two kinds of expression cassettes were prepared and the relative FI values were compared

23

between PPGK1 and PHXT1. Cultivation and calculation of the relative FI values were as in Fig. 2B.

24

Values are presented as means ± standard deviations from three independent experiments.


22


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 31

1 2

Figure 4. Schematic outline of the redesigned scheme for positive detection of GPCR

3

antagonists. (A) The expression cassette, PHXT1-ymUkG1-PEST-TDIT1, is carried on a multi-copy

4

episomal plasmid and introduced into yeast cells. These cells do not express the reporter gene in

5

the absence of glucose and GPCR ligands. (B) Yeast cells grown in the presence of glucose

6

express the reporter gene in the absence of GPCR ligands. (C) Yeast cells stimulated with

7

agonist trigger GPCR signaling, preventing expression of the reporter gene and precluding

8

emission of green fluorescence, even in the presence of glucose. (D) Addition of antagonist

9

blocks binding of agonist to the receptor, thereby inhibiting GPCR signaling and allowing

10

expression of the fluorescent reporter gene.

11 12

Figure 5. Positive detection of antagonist activity after short cultivation. (A) Quantification

13

of the reporter expression levels in yeast cells grown in SDR medium (2% total sugar consisting

14

of glucose and raffinose). Relative FI values were calculated by setting the value of yeast cells

15

grown in medium without raffinose (2% glucose) at 100%. (B) Flow diagram of detection of

16

antagonist activity according to the redesigned scheme described in Figure 4. (C) Dose–response

17

curves for agonist (α-factor). Relative FI value was calculated by setting each value without

18

agonist at 100%. (D) Dose–response curves for antagonist (Fpep1) in the presence of 20 µM

19

agonist. Relative FI value was calculated as in C. Values are presented as means ± standard

20

deviations from three independent experiments.

21 22

Figure 6. Comparison of green fluorescent signals between yeast cells pre-incubated with

23

antagonist and/or agonist before short cultivation. (A) Flow diagram for detection of the

24

antagonist. Yeast cells were treated with ligands and then transferred into medium containing


23

Page 25 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

glucose and ligands. (B) Comparison of reporter gene expression levels. Relative FI value was

2

calculated as in Fig. 5C. (C) Fluorescence microscope image of yeast cells treated with 10 µM

3

agonist. (D) Fluorescence microscope image of yeast cells treated with 10 µM agonist and 300

4

µM antagonist. Scale bar: 10 µm.

5


24


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Schematic outline of the concept of the inverted fluorescent reporter gene expression system for positive detection of GPCR antagonists. (A) Fluorescent reporter genes (ymUkG1 with/without the sequence encoding the PEST-tag) are carried on multi-copy episomal plasmids and introduced into yeast cells. These cells express the reporter genes in the absence of GPCR ligands. (B) Exposure of yeast to agonist triggers GPCR signaling, which prevents expression of the fluorescent reporter gene. After the passage of a time interval sufficient for decay of existing GFP, these cells should no longer emit green fluorescence. (C) Addition of antagonist blocks binding of agonist to the receptor, thereby inhibiting GPCR signaling and allowing expression of the fluorescent reporter gene. 191x185mm (300 x 300 DPI)


Page 26 of 31

Page 27 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Evaluation of the expression levels of the fluorescent reporter genes. (A) Two kinds of reporter genes,ymUkG1 and ymUkG1-PEST, were prepared and expressed under the control of PPGK1, PPMA2, PRNR1, or PHXT1. The expression levels of the reporter genes were quantified and compared among promoters. (B) Inhibition assay with 20 µM agonist (α-factor) was carried out after 16 h of cultivation. Relative fluorescence intensity (FI) value was calculated by setting each value without agonist at 100%. Values are presented as means ± standard deviations from three independent experiments. 204x131mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Inhibition assay with 20 µM agonist for ymUkG1-PEST reporter gene expression. Two kinds of expression cassettes were prepared and the relative FI values were compared between PPGK1 and PHXT1. Cultivation and calculation of the relative FI values were as in Fig. 2B. Values are presented as means ± standard deviations from three independent experiments. 135x152mm (300 x 300 DPI)


Page 28 of 31

Page 29 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Schematic outline of the redesigned scheme for positive detection of GPCR antagonists. (A) The expression cassette, PHXT1-ymUkG1-PEST-TDIT1, is carried on a multi-copy episomal plasmid and introduced into yeast cells. These cells do not express the reporter gene in the absence of glucose and GPCR ligands. (B) Yeast cells grown in the presence of glucose express the reporter gene in the absence of GPCR ligands. (C) Yeast cells stimulated with agonist trigger GPCR signaling, preventing expression of the reporter gene and precluding emission of green fluorescence, even in the presence of glucose. (D) Addition of antagonist blocks binding of agonist to the receptor, thereby inhibiting GPCR signaling and allowing expression of the fluorescent reporter gene. 208x180mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Positive detection of antagonist activity after short cultivation. (A) Quantification of the reporter expression levels in yeast cells grown in SDR medium (2% total sugar consisting of glucose and raffinose). Relative FI values were calculated by setting the value of yeast cells grown in medium without raffinose (2% glucose) at 100%. (B) Flow diagram of detection of antagonist activity according to the redesigned scheme described in Figure 4. (C) Dose–response curves for agonist (α-factor). Relative FI value was calculated by setting each value without agonist at 100%. (D) Dose–response curves for antagonist (Fpep1) in the presence of 20 µM agonist. Relative FI value was calculated as in C. Values are presented as means ± standard deviations from three independent experiments. 201x182mm (300 x 300 DPI)


Page 30 of 31

Page 31 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Comparison of green fluorescent signals between yeast cells pre-incubated with antagonist and/or agonist before short cultivation. (A) Flow diagram for detection of the antagonist. Yeast cells were treated with ligands and then transferred into medium containing glucose and ligands. (B) Comparison of reporter gene expression levels. Relative FI value was calculated as in Fig. 5C. (C) Fluorescence microscope image of yeast cells treated with 10 µM agonist. (D) Fluorescence microscope image of yeast cells treated with 10 µM agonist and 300 µM antagonist. Scale bar: 10 µm. 199x172mm (300 x 300 DPI)


Positive Detection of GPCR Antagonists Using a System for Inverted

Recommend Documents