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(1)

Structure and Function

of the Receptor Activation Domain

of Parathyroid Hormone

Eun Jin Lee

Department of Medical Science

The Graduate School, Yonsei University

(2)

Structure and Function

of the Receptor Activation Domain

of Parathyroid Hormone

Directed by Professor Sung-Kil Lim

The Doctoral Dissertation submitted

to the Department of Medical Science,

the Graduate School of Yonsei University

in partial fulfillment of requirements for the degree

of Doctor of Philosophy

Eun Jin Lee

December 2003

(3)

This certifies that the Doctoral Dissertation for the

Doctoral Degree of Philosophy by Eun Jin Lee is

approved.

Thesis Supervisor: Sung-Kil Lim

Thesis Committee Member

Thesis Committee Member

Thesis Committee Member

Thesis Committee Member

Department of Medical Science

The Graduate School, Yonsei University

(4)

Acknowledgements

본 논문을 완성하기까지 세심한 지도와 격려를 아끼지 않으셨던 임승길 교수 님께 진심으로 감사드리며, 연구를 진행하는 과정에서 연구방향의 설정을 바로 잡아 주시고 많은 도움과 격려를 주신 이원태 교수님께 마음으로부터의 감사를 드립니다. 그리고 바쁘신 중에도 논문 내용을 세심하게 살펴주시고 각별한 조언 을 아끼지 않으신 김경환 교수님, 김경섭 교수님, 박정수 교수님께도 감사드립 니다. 또한 실험적인 조언과 함께 따뜻한 관심을 가져 주신 백자현 교수님께 머 리 숙여 감사드립니다. 실험실 생활을 즐겁고, 활기차게 할 수 있도록 도와준 이송철, 김용군, 황난주, 이유미, 김세화, 전수정, 정현주, 안연희 선생께 감사드리며, 내분비내과 및 임상 의학연구센터의 모든 동료들에게 깊은 감사를 전합니다. 오랜 기간 동안 편히 연구에만 전념 할 수 있도록 물심양면으로 도와주시고, 저의 부족한 면들을 따뜻한 믿음으로 보살펴 주신 아버지와 어머니, 그리고 언 제나 저의 건강을 걱정해 주시고 알뜰히 챙겨주신 아버님, 어머님께 감사드립니 다. 바로 옆에서 가장 실질적인 도움을 주시는 든든한 형부와 언니, 행복과 기 쁨을 느끼게 해 주는 조카들 혜성이, 용석이, 그리고 곧 세상에 나올 사랑이, 우 리 집의 기둥, 이수진과 임정수에게도 감사의 마음을 전합니다. 잦은 짜증과 트집에도 넉넉한 미소로 마음의 평온을 유지하게 해준 조성곤 님에게 저의 작은 결실을 드립니다. 2003년 12월 이 은 진

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Contents

Abstract ··· 1

I. INTRODUCTION ··· 4

II. MATERIALS AND METHODS ··· 8

1. Chemicals ··· 8

2. Peptide synthesis ··· 8

3. Subcloning of human PTH1 receptor cDNAs into mammalian expression vector ··· 9

4. Preparation of mammalian cell lines stably expressing the human PTH1 receptor ··· 9

5. cAMP generating assay ··· 10

6. Circular dichroism spectroscopy ··· 11

7. NMR Spectroscopy and modeling calculations ··· 11

III. RESULTS ··· 14

1. Synthesis of short-length amino terminal PTH analogues ··· 14

2. Preparation of LLC-PK1 cell lines stably expressing the hPTH1R ··· 14

3. Effects of the synthesized peptides on cAMP accumulation in LLC -PK1 cells stably expressing hPTH1R ··· 17

4. Effect of substituting the 10th and 11th residues ··· 17

5. Effect of substituting with the Glu10 and Ile11 residues ··· 21

6. Effect of substituting the 7th and 8th residues ··· 22

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rPTH(1-12)NH2··· 22

8. Circular dichroism (CD) ··· 24

9. Resonance assignment and solution structure ··· 27

IV. DISCUSSION ··· 31

V. CONCLUSION ··· 36

REFERENCES ··· 37

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LIST OF FIGURES

Figure 1. Selection of the LLC-PK1 cell line stably overexpressing hPTH1R by measuring the level

of PTH(1-34)-stimulated cAMP accumulation ··· 14

Figure 2. The effect of substituting the 11th residues of the

[Ala3,10,12(Leu7/Phe7)Arg11]rPTH(1-12)NH2

analogues upon cAMP accumulation in hPTH1R

expressing LLC-PK1 cells ··· 20

Figure 3. The effect of substituting the 10th residues of the

[Ala3,10,12(Leu7/Phe7)Arg11]rPTH(1-12)NH2 analogues upon cAMP accumulation in hPTH1R expressing

LLC-PK1 cells ··· 21 Figure 4. Comparison of the biological activity of

[Ala3,10,12(Leu7/Phe7) Arg11]rPTH(1-12)NH2 analogues obtained by substituting Ala10 and Arg11 with

Glu10 and Ile11, respectively ··· 22 Figure 5. cAMP formation of various PTH analogues in

LLC-PK1 cells stably transfected with hPTH1R ··· 23 Figure 6. Comparison of cAMP stimulating activity induced by

[Ala3,10,12(Leu7/Phe7)Arg11]rPTH(1-12)NH2,

[Ala3,10(Leu7/Phe7)Arg11] rPTH(1-11)NH2, and [Ser1Ala3,10 (Leu7/Phe7)Arg11]rPTH(1-11)NH2··· 24

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Figure 7. Circular dichroism spectra of [Ala3,10,12Leu7Arg11] rPTH(1-12)NH2 (A), [Ala3,10,12Phe7Arg11]rPTH(1-12) NH2 (B), [Ser1Ala3,10Leu7Arg11]rPTH(1-11)NH2 (C), [Ala3,10Phe7Arg11]rPTH(1-11)NH2

(D) in different solutions ··· 25

Figure 8. Circular dichroism spectra of Ala10→Glu10 and/or Arg11→Ile11

substituted analogues ··· 26

Figure 9. Summary of the NMR data of [Ala3,10,12Leu7Arg11]

rPTH(1-12)NH2 (A), [Ala3,10,12Phe7Arg11]rPTH(1-12)NH2 (B), [Ser1Ala3,10Leu7Arg11]rPTH(1-11)NH2 (C), and

[Ala3,10Phe7Arg11]rPTH(1-11)NH2 (D) ··· 27

Figure 10. NMR structure of the [Ala3,12Leu7Glu10Ile11]

rPTH(1-12)NH2 analogue ··· 28

Figure 11. NMR structures of the rPTH(1-12)NH2 and

rPTH(1-11)NH2 analogues ··· 29 Figure 12. Superimposed REM structures of rPTH(1-12)

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LIST OF TABLES

Table 1. Amino acid sequences of PTH analogues designed and

tested in this study ··· 16

Table 2. EC50 values (means SEM) of the rPTH(1-12)and rPTH

(1-11) analogues used in this study ··· 19 Table 3. Structural statistics for the 20 final simulated-annealing

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Abstract

Structure and Function

of the Receptor Activation Domain

of Parathyroid Hormone

Eun Jin Lee

Department of Medical Science

The Graduate School of Yonsei University

(Directed by Professor Sung-Kil Lim)

Osteoporosis is characterized by low bone mass, structural deterioration of bone and increased risk of fracture. The prevalence and cost of osteoporosis is increasing dramatically with increasing of aging population. All currently approved therapies for osteoporosis are anti-resorptive agents that act on osteoclasts to prevent further bone loss: estrogen, bisphosphonates, calcitonin, and selective estrogen receptor modulators. Anti-resorptive agents stabilize the bone remodeling by reducing the number and activity of osteoclast, and thereby reducing the risk of fracture without increments of true bone mass. In contrast, anabolic agents are not only to increase bone mass remarkably but also reduce fracture risk extraordinary. Therefore, it is necessary to develop the anabolic agents acting on bone.

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Parathyroid hormone (PTH) is vital for the regulation of calcium and phosphate homeostasis. PTH has both anabolic and catabolic effects on bone, depending on the route of administration. The anabolic effects of PTH on bone density have prompted considerable interest in the development of new PTH-1 receptor agonist analogues. Based on these understanding, PTH analogues have been regarded as one of most important candidates for treating osteoporosis.

In order to study the structure and function of short-length amino terminal PTH analogues, the cAMP generating activity of chemically synthesized PTH(1-11) and PTH(1-12) oligopeptides were analyzed in LLC-PK1 cell lines stably transfected with the wild-type human PTH-1 receptor, and

performed NMR studies. Replacement of the 10th residue with Ala, Asp, Gln,

or Glu, and the 11th residue with Arg, Met, Lys, or Ile in both

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH2 and [Ala3,10,12Phe7Arg11]rPTH(1-12) NH2

produced different magnitude of cAMP generating potency, dependent upon

the residue type. Especially, the substitution of Ala10 of [Ala3,10,12(Leu7

/Phe7)Arg11]rPTH(1-12)NH2 with Glu10 and/or the Arg11 with Ile11 markedly

decreased cAMP generating activity. The substitution of Leu7 with Phe in

most of the [Ala3,10,12Leu7Arg11]rPTH(1-12)NH2 analogues reduced the cAMP

formation. The substitution of Leu7 with Phe in [Ala3,10Leu7Arg11]

rPTH(1-11)NH2 did not. The substitution of Ala1 by Ser in [Ala3,10Leu7Arg11]

rPTH(1-11)NH2 caused a near abrogation of cAMP formation. NMR analysis

of both [(Ala1/Ser1)Ala3,10(Leu7/Phe7)Arg11]rPTH(1-11)NH2 and [Ala3,10,12(Leu7/

Phe7)Arg11]rPTH(1-12)NH2 revealed a similar α-helical backbone structure and

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dichroism and the NMR structural analysis of [Ala3,10,12(Leu7/Phe7)

Arg11]rPTH(1-12)NH2 revealed stable α-helical structures, while the Glu10

and/or Ile11 substituted analogues showed unstable α-helical structures. The

overall results suggest that short oligopeptides, based on the first 11 or 12 amino acids of PTH, tend to form α-helical structure, and that the different potency of these analogues is probably associated with residue specificity

rather than secondary structure. In addition, 10th and 11th residues are

important for stabilizing its helical conformation and destabilization of α -helical structure, induced by substituting the residues, remarkably affect its biological potency.

Key Words: Parathyroid hormone receptor; cAMP-generating activity; NMR; α-helix

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Structure and Function

of the Receptor Activation Domain

of Parathyroid Hormone

Eun Jin Lee

Department of Medical Science

The Graduate School of Yonsei University

(Directed by Professor Sung-Kil Lim)

I. INTRODUCTION

Parathyroid hormone (PTH) is a major regulator of calcium and phosphate homeostasis. It is known that the delivery route of administration significantly

determines its effect on bone1. The continuous infusion of PTH causes bone

loss, but intermittent injections of low doses repress osteoblast apoptosis and stimulate the differentiation of bone lining cells and mesenchymal cells to

osteoblasts, resulting in bone mass increase2,3. The anabolic effect of PTH on

bone has prompted considerable interest in the development of novel derivatives of PTH and PTH-related peptide (PTHrP). Successful PTH analogue development is viewed with considerable importance for the

(14)

treatment of osteoporosis.

PTH is composed of 84 amino acids, but its full biological effects are limited to its N-terminal region, PTH(1-34). However, this 34-amino acid- peptide has limited effectiveness when administered by parenterally, nasal

sprays and pulmonary inhalation4. To resolve this problem, wider and deeper

studies have been conducted on the structures and functions of PTH and

PTH/PTHrP receptor using site-directed mutagenesis5, photoaffinity labeling6,

NMR7, and crystallographic analysis8. However, many questions remain

regarding the nature of the ligand-receptor interaction, particularly the interaction between short length PTH analogues and the PTH/PTHrP receptor.

Recently, Jin et al. reported that hPTH(1-34) crystallizes as a slightly

bent, long helical dimer9. Whereas recent NMR analysis of hPTH(1-34)

revealed that it forms an N-terminal helix and a C-terminal helix connected

by a highly flexible region in physiologic solution10. According to the report

of Jin et al., the N-terminal region of PTH(1-34) binds to a pocket consisting of the extra cellular portion of TM3, TM4, and TM6 and the

second and third extra cellular loops of the PTH/PTHrp receptor11. The

middle region of PTH(1-34) is sandwiched between the first extra cellular loop and the N-terminal extra cellular region of the receptor adjacent to TM1, and the C-terminal region of hPTH(1-34) interacts extensively with the putative binding domain of the PTH/PTHrp receptor. After comparing their crystallographic data with published NMR data upon PTH(1-34), Jin et al. proposed that only one orientation of hPTH(1-34) satisfied all the known ligand receptor interactions. Furthermore, it was recently reported that Tip39

(15)

is an endogenous ligand of the PTH2 receptor12,13, and that the NMR structure of Tip39 helps to explain the manner in which PTH(1-34) binds to

the PTH/PTHrp receptor14.

PTH and PTHrP interact with the PTH/PTHrP receptor (or PTH1R),

which is a member of the B family of G-protein coupled receptors15. Residues

25-34 of PTH(1-34) are important for binding to the PTH/PTHrP receptor, whereas the first 13 amino acids of the N-terminus are essential for receptor

binding and activation16. In terms of the short length PTH analogues,

Gardella et al. itemized the residues of chemically synthesized PTH(1-14)

and PTH(1-13) analogues that could be substituted by other amino acids17.

On the other hand, Monticelli et al. performed a computer simulation study

using a PTH1R/[Arg11]PTH(1-11) tethered system and proposed a

mechanism involving the binding of the PTH N-terminus residue with

receptors. These workers also found that the 1st, 2nd, 4th, 5th, 7th, and 8th

residues of PTH are very important components of receptor

binding/activation18. However, too many degrees of freedom remained within

the PTH N-terminus are unknown19,20, and in particular, its structure and the

elements facilitating its receptor binding remained to be defined.

Shimizu et al. showed that PTH(1-14)NH2 stimulated cAMP formation

weakly, to ca. 1/1000 of that of hPTH(1-34), in LLC-PK cells stably

expressing a high level of the hPTH/PTHrp receptor21. Furthermore, they

substituted PTH residues and analyzed the functional tolerability of each residue substitution. Finally, they proposed that the short amino-terminal peptides of PTH could possibly be optimized to significantly increase signaling potency by modifying the interactions involving receptor regions containing the

(16)

transmembrane domains and the extra cellular loops22-24. However, although a part has just been explored and is being tested by some research group recently, such shorter and more potent PTH analogues have not been fully developed to date. Furthermore, few studies have been conducted upon the structures and functions of derivatives of PTH(1-11), PTH(1-12) or

PTH(1-13)21,25-26.

PTH(1-14) is regarded as the basic entity required for receptor activation, but the functionality of PTH(1-14), required for PTH/PTHrp receptor

activation, is retained in the first 9 amino acids24. Accordingly, little work

has been reported upon the relationships between the structures and

functions of amino acids 9 to 14 N-terminal PTH analogues21,25-26. Moreover, in

previous studies, the substitution of the 1st∼6th amino acids residues was

found to cause loss of the receptor activating function27,28. To better

understand the ligand and receptor interactions of the short N-terminal PTH fragment activating PTH/PTHrP receptor, and to develop a potent analogue, it is systemically synthesized more than forty-three short-length amino

terminal PTH analogues substituted at the 7th, 8th, 10th, and 11th amino-acids

whilst preserving the 2nd∼6th amino acids residues. The biological activity of

these analogues were examined using a cAMP-generating assay, in LLC-PK1 cell lines stably transfected with the wild-type human PTH1 receptor, and their conformational characteristics were determined by NMR to study the relationships between the biological activity of the short-length PTH analogues and the solubilized NMR structures.

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II. MATERIALS AND METHODS

1. Chemicals

All media and sera for cell cultivation were purchased from Gibco-BRL. hPTH(1-34) and the other chemicals mentioned were purchased from Sigma, unless specified otherwise.

2. Peptide synthesis

The truncated rPTH(1-12) analogues were synthesized by modifying their

7th, 10th, and 11th residues serially. The substitution of these residues is

known to be more tolerated than the substitution of the first 6 residues of

the N-terminus, as described by Shimizu et al21. In addition, rPTH(1-11)

analogues were also synthesized and made substitutions at the 1st, 7th or 8th

residues (Table 1). All synthesized peptides had an amide group at the C-terminus. The peptides used in this study were synthesized at the Korea Basic Science Institute (Seoul, Korea) using the solid phase approach and purified by HPLC. Peptide sequences were assembled with a Milligen 9050 (Fmoc Chemistry). MS was used to confirm the molecular masses of the synthesized peptides. For deprotection, a reagent mixture (88% trifluoroacetic

acid, 5% phenol, 2% triisopropylsilane, 5% H2O; 2h) was used. The raw

peptides formed were purified by HPLC (Delta PAK 15 C18 300 3.9×150mm column, with a 240 nm detector).

(18)

3. Subcloning of human PTH receptor cDNAs into mammalian expression vector

Human PTH1R cDNA, kindly provided by the Mogam Biotech. Institute in korea, was cloned into the expression vector pcDNA3 neo. By using a PCR cloning technique, they were able to obtain a 1.8 kb hPTH1R cDNA, which included the total open reading frame of hPTH1R, from the human kidney library.

4. Preparation of mammalian cell lines stably expressing the human PTH1 receptor (hPTH1R)

For receptor expression, LLC-PK1 porcine kidney cells were grown in

Medium-199 with 3% fetal bovine serum (FBS) in 95% O2/5% CO2 and stably

transfected with hPTH1R-pcDNA3 neo using the calcium phosphate method.

Briefly, 5∼7×105 cells (approximately 80% confluent cultures) were plated per

10cm culture dish the day before transfection. Cells were fed with fresh complete culture medium containing 20mM Hepes and incubated in 95%

O2/5% CO2. After 3×4h, the medium was discarded and 5 ml of calcium

phosphate-DNA precipitate containing, 25 μg DNA, 124 mM CaCl2, 140mM

NaCl, 25 mM Hepes and 1.41 mM Na2HPO4 (pH 7.12) was added. The cells

were then incubated for 4h in 95% O2/5% CO2, washed with NaCl/Pi (137mM

NaCl, 2.68 mM KCL, 4.3 mM N2HPO4, 1.47 mM KH2PO4, pH 7.12), shocked

with glycerol buffer (15% glycerol, 140mM NaCl, 25mM Hepes and 1.41mM

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36-48h in complete Medium-199. Cells were then cultured in complete Medium-199 containing 0.5 mg/ml G418 (Geneticin, Life Technologies) until G418-resistant colonies appeared, and these were then picked out and subcultured for at least 10∼14 days. Human PTH1R-expressing cells were identified by screening more than 30 colonies and confirmed using the hPTH(1-34) induced cAMP accumulation assay, as described below. The highest reactivity LLC-PK1 porcine kidney cell, stably transfected with human PTH1R cDNA, was selected for further study. The number of hPTH1 receptors was determined to be 800,000 receptors/cell, by competitive radioligand binding

assay using 125I-[Nle8,18Tyr34]bPTH(1-34) and Scatchard plotting, as

previously described29.

5. cAMP generating assay

LLC-PK1 cells expressing the receptors were grown to confluence in 48-well plates. Cell culture medium was changed to complete Medium-199 containing 3% FBS 3∼4hrs before the cells were treated with peptides. For the assays, the medium was first removed and the cells were washed with

0.25 ml of cAMP-generating medium containing 3% FBS, 2 mM

3-isobutyl-1-methyl-xanthine, 0.1% BSA, 20mM Hepes, and 0.002% ascorbic acid in complete Medium-199. cAMP-generating media (0.15 ml) containing various concentrations of peptides were added and the cells were incubated for 30mins at 37℃. The media were then completely discarded, and the cells frozen at -70℃ for 30min and thawed at room temperature for 15∼20mins; this freeze-thawing process was repeated twice. The cells were then

(20)

detached from the plates with 0.5 ml of 50mM HCl solution per well, transferred to a 1.5ml Eppendorf tube, and centrifuged at 1900×g for 10min. The supernatants were then diluted 50-fold with radio-immunoassay (RIA)

buffer and the cAMP concentration was measured using a cAMP 125I RIA Kit

(bti, USA), according to the manufacturer's instructions. The mean values of the data were fitted to a sigmoid curve with a variable slope factor using the nonlinear squares regression technique in the GRAPHPAD PRISM. EC50 (nM) values are represented by means SE. All of the cAMP assays were performed in triplicate wells and repeated twice. (p<0.05 was considered significant).

6. Circular Dichroism Spectroscopy

Circular Dichroism spectra of the peptide samples in H2O with various

concentrations of trifluoroethanol (TFE) were recorded at room temperature using a Jasco J-715 spectropolarimeter. The concentrations of the peptides used ranged from 79M to 81M in 50mM phosphate buffer, at pH 7.0, and the CD spectra were recorded from 190 to 250nm at a scanning rate of

50nmmin-1 with a time constant of 0.5s. CD data were obtained from an

average of 7 scans with a resolution of 0.2nm and bandwidth of 2.0nm. A standard noise reduction was applied to the final spectrum. Ellipticity is

reported as the mean residue molar ellipticity, [θ]r.

(21)

NMR spectroscopy was carried out in both 70% H2O(D2O)/30% 2,2,2-

trifluoro-(d3)-ethanol (TFE) and 90% H2O/10% D2O at pH 7.0. The peptide

concentrations were:-2.2 mM for [Ala3,10,12(Leu7/Phe7)Arg11]rPTH(1-12)NH2,

and 3.2 mM for [Ser1Ala3,10Leu7Arg11]rPTH(1-11)NH2 and [Ala3,10Phe7Arg11]

rPTH(1-11)NH2, in 50mM of sodium phosphate buffer. NMR spectra were

recorded at 298K on a Bruker DRX-500 spectrometer equipped with a triple resonance probe, and triple axis gradient coils. The spectra were also recorded over the temperature range 10-25℃, to calculate the temperature

coefficients. Pulsed-field gradient (PFG) techniques were used in all H2O

experiments to suppress solvent signals. Two-dimensional total correlation

spectroscopy (TOCSY)30, with an MLEV-17 mixing pulse of 69.7ms, and

two-dimensional nuclear Overhauser effect spectroscopy (NOESY)31, with

mixing times of 100∼600 ms, was also performed. Two-dimensional

double-quantum-filtered (DQF) COSY32 spectra were collected in H2O to

obtain the vicinal coupling constants. A series of one-dimensional NMR measurements were obtained to identify the slowly exchanging amide hydrogen

resonances in freshly prepared D2O solutions after the H2O sample had been

lyophilized. All NMR experiments were performed in the phase-sensitive

mode using the time proportional phase incrementation (TPPI) method33 with

2048 data points in the t2, and 256 points in the t1 domains. NMR data were

processed using XWIN-NMR (Bruker Instruments) software, and the processed data further analyzed using Sparky 3.60 software, which was

developed at UCSF on a Silicon Graphics Indigo2 workstation. Starting

structures were generated using distance geometry (DG) by employing a refinement protocol using distance restraints assigned as strong, medium, or

(22)

weak, on the basis of cross-peak volumes in the NOESY spectra. All distances had a lower limit of 1.8, with upper limits of 2.7, 3.3, or 5.0 for strong, medium or weak intensities, respectively. The dihedral angle

constraints were also deduced from the3 JHNα coupling constants from the 2D

DQF-COSY spectra in H2O. Structure calculations were performed using

hybrid distance geometry and the dynamically simulated-annealing

protocol34-37 using the X-PLOR 3.81 (Biosym/Molecular Simulations, Inc.)

(23)

III. RESULTS

1. Synthesis of short-length amino terminal PTH analogues

The truncated rPTH(1-12) analogues were synthesized by modifying their 7th, 10th, and 11th residues serially. The substitution of these residues is known to be more tolerated than the substitution of the first 6 residues of the N-terminus,

as described by Shimizu et al21. In addition, rPTH(1-11) analogues were also

synthesized and made substitutions at the 1st, 7th or 8th residues (Table 1).

2. Preparation of LLC-PK1 cell lines stably expressing the hPTH1R

With stably transfected by the calcium phosphate method, the G418-resist

Figure 1. Selection of the LLC-PK1 cell lines stably over- expressing hPTH1R by measuring the level of PTH(1-34)-stimulated cAMP accumulation.

0 50 100 150 200 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Clone number cA M P le v el (f ol d s)

(24)

-ant cell lines were selected by of PTH(1-34)-induced cyclic AMP accumulation. As shown in Figure 1, compared to the vehicle-treated group, the

4th cell colony in hPTH1R exhibited the most significant increasement of

(25)

All peptides have an amide group on the C-terminus. The main substituted amino-acid residues are shown in bold italics.

Table 1. Amino-acid sequence of synthetic PTH analogues used in this study P e p t i d e A m i n o a c i d s e q u e n c e s [ L e u7] r P T H ( 1 - 1 2 ) a n a l o g u e s 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 [ A la3 ,1 0 ,1 2 L e u7 A r g1 1 ] rP T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s A l a A r g A la [ A la3 ,1 0 ,1 2 L e u7 L y s1 1 ] rP T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s A l a L y s A la [ A la3 ,1 0 ,1 2 L e u7 I l e1 1 ] rP T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s A l a I l e A la [ A la3 ,1 0 ,1 2 L e u7 M e t1 1 ] rP T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s A l a M e t A la [ A la3 ,1 2 L e u7 A s p1 0 A r g1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s A s p A r g A la [ A la3 ,1 2 L e u7A s p1 0L y s1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s A s p L y s A la [ A la3 ,1 2 L e u7A s p1 0I le1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s A s p I l e A la [ A la3 ,1 2 L e u7 A s p1 0 M e t1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s A s p M e t A la [ A la3 ,1 2 L e u7 G l n1 0 A r g1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s G l n A r g A la [ A la3 ,1 2 L e u7 G l n1 0 L y s1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s G l n L y s A la [ A la3 ,1 2 L e u7 G l n1 0 I le1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s G l n I l e A la [ A la3 ,1 2 L e u7 G l n1 0 M e t1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s G l n M e t A la [ A la3 ,1 2 L e u7 G l u1 0 A r g1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s G l u A r g A la [ A la3 ,1 2 L e u7 G l u1 0 L y s1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s G l u L y s A la [ A la3 ,1 2 L e u7 G l u1 0 I le1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s G l u I l e A la [ A la3 ,1 2 L e u7 G l u1 0 M e t1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s G l u M e t A la [ P h e7] r P T H ( 1 - 1 2 ) a n a l o g u e s [ A la3 ,1 0 ,1 2 P h e7 A r g1 1 ] rP T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s A l a A r g A la [ A la3 ,1 0 ,1 2 P h e7 L y s1 1 ] rP T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s A l a L y s A la [ A la3 ,1 0 ,1 2 P h e7 I l e1 1 ] rP T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s A l a I l e A la [ A la3 ,1 0 ,1 2 P h e7 M e t1 1 ] rP T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s A l a M e t A la [ A la3 ,1 2 P h e7 A s p1 0 A r g1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s A s p A r g A la [ A la3 ,1 2 P h e7A s p1 0L y s1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s A s p L y s A la [ A la3 ,1 2 P h e7A s p1 0I l e1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s A s p I l e A la [ A la3 ,1 2 P h e7 A s p1 0 M e t1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s A s p M e t A la [ A la3 ,1 2 P h e7 G l n1 0 A r g1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s G l n A r g A la [ A la3 ,1 2 P h e7 G l n1 0 L y s1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s G l n L y s A la [ A la3 ,1 2 P h e7 G l n1 0 I l e1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s G l n I l e A la [ A la3 ,1 2 P h e7 G l n1 0 M e t1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s G l n M e t A la [ A la3 ,1 2 P h e7 G l u1 0 A r g1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s G l u A r g A la [ A la3 ,1 2 P h e7 G l u1 0 L y s1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s G l u L y s A la [ A la3 ,1 2 P h e7 G l u1 0 I l e1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s G l u I l e A la [ A la3 ,1 2 P h e7 G l u1 0 M e t1 1 ] r P T H ( 1 - 1 2 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s G l u M e t A la r P T H ( 1 - 1 1 ) a n a l o g u e s [ A la3 ,1 0 L e u7 A r g1 1 ] rP T H ( 1 - 1 1 ) N H2 A la V a l A la G lu Ile G ln L e u M e t H i s A l a A r g [ S e r1 A la3 ,1 0 L e u7 A r g1 1 ] r P T H ( 1 - 1 1 ) N H2 S e r V a l A la G lu Ile G ln L e u M e t H i s A l a A r g [ A la3 ,1 0 d P h e7 A r g1 1 ] rP T H ( 1 - 1 1 ) N H2 A la V a l A la G lu Ile G ln d P h e M e t H i s A l a A r g [ A la3 ,1 0V a l7A r g1 1 ] rP T H ( 1 - 1 1 ) N H2 A la V a l A la G lu Ile G ln V a l M e t H i s A l a A r g [ A la3 ,1 0I l e7A r g1 1 ] r P T H ( 1 - 1 1 ) N H2 A la V a l A la G lu Ile G ln I l e M e t H i s A l a A r g [ A la3 ,1 0 P h e7 A r g1 1 ] r P T H ( 1 - 1 1 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s A l a A r g [ S e r1 A la3 ,1 0 P h e7 A r g1 1 ] r P T H ( 1 - 1 1 ) N H2 S e r V a l A la G lu Ile G ln P h e M e t H i s A l a A r g [ A la3 P h e7 A s p1 0 A r g1 1 ] r P T H ( 1 - 1 1 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s A s p A r g [ A la3 P h e7 G l n1 0 A r g1 1 ] r P T H ( 1 - 1 1 ) N H2 A la V a l A la G lu Ile G ln P h e M e t H i s G l n A r g [ A la3 ,1 0 P h e7 L e u8 A r g1 1 ] r P T H ( 1 - 1 1 ) N H2 A la V a l A la G lu Ile G ln P h e L e u H i s A l a A r g [ A la3 ,1 0 P h e7 N l e8 A r g1 1 ] r P T H ( 1 - 1 1 ) N H2 A la V a l A la G lu Ile G ln P h e N l e H i s A l a A r g

(26)

3. Effects of the synthesized peptides on cAMP accumulation in LLC- PK1 cells stably expressing hPTH1R

LLC-PK1 cells stably transfected with human PTH1R cDNA were stimulated

with the synthesized PTH analogues substituting at 7th, 8th, 10th, and 11th

positions. Parental LLC-PK1 cells, which do not express hPTH1 receptors, were unresponsive to hPTH(1-34) or any of the PTH analogues used in this study. However human PTH1R-expressing cells were responsive to hPTH (1-34) (EC50=50.9±30.13pM) and the synthesized PTH analogues, and they showed a basal activity that was similar to that of untransfected LLC-PK1 cells, without hPTH(1-34) treatment. A typical dose-dependent induction of cAMP generating activity was observed upon increasing the concentrations of the PTH analogues, and the EC50 values calculated from the dose-response curves proved reproducible over several experiments; results are summarized in Table 2.

4. Effect of substituting the 10th and 11th residues

Most of the [Ala3,10,12Leu7Arg11]rPTH(1-12)NH2 analogues, in which Leu7 was

substituted by Phe7, showed lower cAMP formation, except [Ala3,10,12

Leu7Lys11]rPTH(1-12)NH2, [Ala3,12Leu7Asp10Lys11]rPTH(1-12)NH2, and [Ala3,12

Leu7Gln10Lys11]rPTH(1-12)NH2 (Table 2). All of the oligopeptides in which Arg11

of [Ala3,10,12(Leu7/Phe7)Arg11]rPTH(1-12)NH2 were substituted by Met11 or

Lys11 displayed lower cAMP accumulation. In particular, the substitution of

Arg11 by Ile11 led to remarkable decrease in cAMP formation, even at high

(27)

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH2 analogues substituted at the 11th residue was Arg11≈ Met11> Lys11> Ile11 (Figure 2A), and that of the [Ala3,10,12Phe7Arg11]

(28)

All peptides have an amide group at the C-terminus. The main substituted amino-acid residues are shown in bold italics. The amino acid sequence of

rPTH(1-12) is AVSEIQLMHNLG. EC50 values were obtained by computer analysis of

the respective dose-response curves on hPTH1R stably transfected LLC-PK1 cell lines.

"-", the mature cAMP generation failed to reach the maximum levels achieved by other ligands. Data are represented as means±SE of three independent experiments.

Table 2. EC50 values (means ± SEM) of the rPTH(1-12) and rPTH(1-11) analogues used

in this study

Peptide EC50(nM) Peptide EC50(nM)

Leu7 oligopeptides Phe7 oligopeptides

rPTH(1-12) analogues

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH

2 1404 ± 82.278 [Ala3,10,12Phe7Arg11]rPTH(1-12)NH2 7845 ± 507.64

[Ala3,10,12Leu7Lys11]rPTH(1-12)NH

2 22330 ± 493.55 [Ala3,10,12Phe7Lys11]rPTH(1-12)NH2 14560 ± 892.79

[Ala3,10,12Leu7Ile11]rPTH(1-12)NH

2 171600 ± 5683.34 [Ala3,10,12Phe7Ile11]rPTH(1-12)NH2 546300 ± 38888.9

[Ala3,10,12Leu7Met11]rPTH(1-12)NH

2 3358 ± 146.263 [Ala3,10,12Phe7Met11]rPTH(1-12)NH2 108200 ± 5016.56

[Ala3,12Leu7Asp10Arg11]rPTH(1-12)NH

2 1969 ± 122.455 [Ala3,12Phe7Asp10Arg11]rPTH(1-12)NH2 6159 ± 483.69

[Ala3,12Leu7Asp10Lys11]rPTH(1-12)NH

2 28890 ± 514.39 [Ala3,12Phe7Asp10Lys11]rPTH(1-12)NH2 13240 ± 884.63

[Ala3,12Leu7Asp10Ile11]rPTH(1-12)NH

2 _ [Ala3,12Phe7Asp10Ile11]rPTH(1-12)NH2 _

[Ala3,12Leu7Asp10Met11]rPTH(1-12)NH

2 3759 ± 559.62 [Ala3,12Phe7Asp10Met11]rPTH(1-12)NH2 48850 ± 7626.96

[Ala3,12Leu7

Gln10Arg11]rPTH(1-12)NH2 2301 ± 240.562 [Ala3,12Phe7Gln10Arg11]rPTH(1-12)NH2 4257 ± 445.11

[Ala3,12Leu7Gln10Lys11]rPTH(1-12)NH

2 27531 ± 5033.62 [Ala3,12Phe7Gln10Lys11]rPTH(1-12)NH2 9463 ± 946.52

[Ala3,12Leu7Gln10Ile11]rPTH(1-12)NH

2 _ [Ala3,12Phe7Gln10Ile11]rPTH(1-12)NH2 _

[Ala3,12Leu7Gln10Met11]rPTH(1-12)NH

2 3163 ± 698.62 [Ala3,12Phe7Gln10Met11]rPTH(1-12)NH2 19490 ± 3207.54

[Ala3,12Leu7Glu10Arg11]rPTH(1-12)NH

2 10890 ± 2339.33 [Ala3,12Phe7Glu10Arg11]rPTH(1-12)NH2 22030 ± 4037.15

[Ala3,12Leu7Glu10Lys11]rPTH(1-12)NH

2 _ [Ala3,12Phe7Glu10Lys11]rPTH(1-12)NH2 _

[Ala3,12Leu7Glu10Ile11]rPTH(1-12)NH

2 _ [Ala3,12Phe7Glu10Ile11]rPTH(1-12)NH2 _

[Ala3,12Leu7Glu10Met11]rPTH(1-12)NH

2 _ [Ala3,12Phe7Glu10Met11]rPTH(1-12)NH2 _ rPTH(1-11) analogues

[Ala3,10Leu7Arg11]rPTH(1-11)NH

2 1529 ± 81.695 [Ala3,10Phe7Arg11]rPTH(1-11)NH2 2132 ± 126.24

[Ser1Ala3,10Leu7Arg11]rPTH(1-11)NH

2 _ [Ser1Ala3,10Phe7Arg11]rPTH(1-11)NH2 4230 ± 136.26

[Ala3,10dPhe7Arg11]rPTH(1-11)NH

2 _ [Ala3Phe7Asp10Arg11]rPTH(1-11)NH2 _

[Ala3,10

Val7Arg11]rPTH(1-11)NH2 _ [Ala3Phe7Gln10Arg11]rPTH(1-11)NH2 _

[Ala3,10Ile7Arg11]rPTH(1-11)NH

2 _ [Ala3,10Phe7Leu8Arg11]rPTH(1-11)NH2 _

[Ala3,10Phe7Nle8Arg11]rPTH(1-11)NH

(29)

-9 -8 -7 -6 -5 -4 0 100 200 300 400

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH

2

[Ala3,10,12Leu7Lys11]rPTH(1-12)NH

2

[Ala3,10,12Leu7Met11]rPTH(1-12)NH

2

[Ala3,10,12Leu7Ile11]rPTH(1-12)NH

2 peptide(log[M]) cA M P Le ve l ( Fol d of ba sa l)

A

-9 -8 -7 -6 -5 -4 0 100 200 300 400

[Ala3,10,12Phe7Arg11]rPTH(1-12)NH

2

[Ala3,10,12Phe7Lys11]rPTH(1-12)NH

2

[Ala3,10,12Phe7Met11]rPTH(1-12)NH

2

[Ala3,10,12Phe7Ile11]rPTH(1-12)NH

2 peptide(log[M]) c A M P Le v e l (F o ld o f ba sa l)

B

Figure 2. The effect of substituting the 11th residues of the [Ala3,10,12

(Leu7/Phe7)Arg11]rPTH(1-12)NH2 analogues upon cAMP accumulation

in hPTH1R expressing LLC-PK1 cells. cAMP formation was induced

by substituting Arg11 with Met11, Lys11, or Ile11 in

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH2 (A) and [Ala3,10,12Phe7Arg11] rPTH(1-12)NH2 (B). Each experiment was performed in triplicate and repeated twice. The symbols are defined in the figure key, and the curves were fitted to the data points by non-linear regression analysis, as described in Materials and Methods.

The substitution of Ala10 with Asp10 or Gln10 in [Ala3,10,12Leu7Arg11]rPTH

(1-12)NH2 or [Ala3,10,12Phe7Arg11] rPTH(1-12)NH2 exhibited almost the same

cAMP generating activity as the analogues to [Ala3,10,12(Leu7/Phe7)

Arg11]rPTH(1-12)NH2. In contrast, the substitution of Ala10 by Glu10

significantly decreased the activity of the analogues. The order of potency

was Ala10≈ Asp10≈ Gln10> Glu10 in the [Ala3,10,12Leu7Arg11]rPTH(1-12) NH2

analogues (Figure 3A), and Gln10≈ Asp10≈ Ala10> Glu10 in [Ala3,10,12Phe7Arg11]

rPTH(1-12)NH2 (Figure 3B). The lower potencies of the [Ala3,12(Leu7/Phe7)

Glu10Arg11] rPTH(1-12)NH2 analogues versus the [Ala3,12(Leu7/Phe7) Asp10Arg11]

rPTH(1-12)NH2 analogues is attributed to the fact that the Glu residue is

(30)

-9 -8 -7 -6 -5 -4 0 100 200 300 400

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH

2

[Ala3,12Leu7Asp10Arg11]rPTH(1-12)NH 2

[Ala3,12Leu7Gln10Arg11]rPTH(1-12)NH 2

[Ala3,12Leu7Glu10Arg11]rPTH(1-12)NH 2 peptide(log[M]) c A M P Le v e ls ( Fol d of B a sa l)

A

-9 -8 -7 -6 -5 -4 0 100 200 300 400

[Ala3,10,12Phe7Arg11]rPTH(1-12)NH

2

[Ala3,12Phe7Asp10Arg11]rPTH(1-12)NH 2

[Ala3,12Phe7Gln10Arg11]rPTH(1-12)NH 2

[Ala3,12Phe7Glu10Arg11]rPTH(1-12)NH 2 peptide(log[M]) c A M P Le v e ls ( Fol d of ba sa l)

B

Figure 3. The effect of substituting the 10th residues of the [Ala3,10,12(Leu7/

Phe7)Arg11]rPTH(1-12)NH2 analogues upon cAMP accumulation in

hPTH1R expressing LLC-PK1 cells. cAMP formation was induced by substituting Ala10 with Asp10, Gln10, or Glu10 in [Ala3,10,12Leu7Arg11]

rPTH(1-12)NH2 (A) and [Ala3,10,12Phe7Arg11] rPTH(1-12)NH2 (B). Each

experiment was performed in triplicate and repeated twice. The symbols are defined in the figure key, and the curves were fitted to the data points by non-linear regression analysis, as described in Materials and Methods.

5. Effect of substituting with the Glu10 and Ile11 residues

The substitution of Arg11 by Ile11 in [Ala3,10,12(Leu7/Phe7)Arg11]rPTH(1-12)

NH2 led to a remarkable decrease in cAMP formation, even at high oligope-

ptide concentrations (10-4M), and the substitution of Ala10 by Glu10 in [Ala3,10,12

(Leu7/Phe7)Arg11]rPTH(1-12)NH2 also decreased cAMP formation. In particular,

the Glu10 and Ile11 substituted analogues, [Ala3,12Leu7Glu10Ile11]rPTH(1-12)NH2

and [Ala3,12Phe7Glu10Ile11]rPTH(1-12)NH2, almost failed to form cAMP (Figure 4).

To determine why these substitutions were not better tolerated and why cAMP-generating activity was reduced so remarkably, I determined their solution structures by CD and NMR spectroscopy later.

(31)

-9 -8 -7 -6 -5 -4 0 25 50 75 100

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH

2

[Ala3,10,12Leu7Ile11]rPTH(1-12)NH

2 [Ala3,12Leu7Glu10Arg11]rPTH(1-12)NH

2

[Ala3,12Leu7Glu10Ile11]rPTH(1-12)NH 2 pe ptide (log[M ]) % S ti m ul a ti on of c A M P

A

-9 -8 -7 -6 -5 -4 0 25 50 75 100

[Ala3,10,12Phe7Arg11]rP TH(1-12)NH

2

[Ala3,10,12Phe7Ile11]rPTH(1-12)NH

2 [Ala3,12Phe7Glu10Arg11]rPTH(1-12)NH

2

[Ala3,12Phe7Glu10Ile11]rPTH(1-12)NH 2 pe ptide (log[M ]) % S ti m ul a ti on of c A M P

B

Figure 4. Comparison of the biological activity of [Ala3,10,12Leu7Arg11]

rPTH(1-12)NH2 (A) and [Ala3,10,12Phe7Arg11]rPTH(1-12)NH2 (B)

analogues obtained by substituting Ala10 and Arg11 with Glu10 and

Ile11, respectively. Each experiment was performed in duplicate and

repeated three times.

6. Effect of substituting the 7th and 8th residues

Substitution of Phe7 by Leu7 in [Ala3,10Phe7Arg11]rPTH(1-11)NH2 preserved

cAMP production; however, the substitution of Phe7 by dPhe, Val, or Ile

remarkably reduced cAMP accumulation (Figure 5A). Because Met8 is vulnerable

to oxidation, Met8 was substituted by Leu8 or norleucine (Nle8). Substituting

Met8 with Nle had little effect upon cAMP activity; however, substituting it

with Leu reduced cAMP activity 10 fold (Figure 5B), which may be due to the smaller size of the Leu residue.

7. Effect of substituting the 1st and 7th residues in rPTH(1-11)NH2

and rPTH(1-12)NH2

(32)

rPTH(1-12)NH2 into either Leu7 or Phe7 analogues. The induction of cAMP

formation by [Ala3,10Phe7Arg11]rPTH(1-11)NH2 was found to be comparable to that of

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH2, though the substitution of Leu7 by Phe in

most of the PTH(1-12) analogues reduced cAMP generating activity.

Surprisingly, when Ser was introduced as the first residue of [Ser1Ala3,10Leu7

Arg11]rPTH(1-11)NH2, cAMP generating activity was almost undetectable,

but the substitution of the Ala1 of [Ala3,10Phe7Arg11]rPTH(1-11)NH2 by Ser1

restored cAMP activity (Figure 6). This result reconfirms that the 1st∼3rd or

5th residues are important for receptor activation by the N-terminal of short

length PTH, and also demonstrate the criticality of Ala1.

-9 -8 -7 -6 -5 -4 0 50 100 150 200 250 300

[Ala3,10dPhe7Arg11]rPTH(1-11)NH 2 [Ala3,10Val7Arg11]rPTH(1-11)NH

2 [Ala3,10Ile7Arg11]rPTH(1-11)NH

2 [Ala3,10Leu7Arg11]rPTH(1-11)NH 2 [Ala3,10Phe7Arg11]rPTH(1-11)NH 2 Peptide (log[M]) cA M P Le ve ls ( Fol d of B a sa l)

A

-9 -8 -7 -6 -5 -4 0 50 100 150 200 250 300

[Ala3,10Phe7Leu8Arg11]rPTH(1-11)NH 2

[Ala3,10Phe7Nle8Arg11]rPTH(1-11)NH 2

[Ala3,10Phe7Arg11]rPTH(1-11)NH 2 Peptide (log[M]) c A M P Le v e ls ( Fol d of B a sa l)

B

Figure 5. cAMP formation of various PTH analogues in LLC-PK1 cells stably

transfected with hPTH1R. (A) The effect of substituting Phe7 in

[Ala3,10Phe7Arg11]rPTH(1-11)NH2 with dPhe, Val, Ile, or Leu on cAMP

activity. (B) The effect of substituting Met8 in the [Ala3,10Phe7

Arg11]rPTH(1-11)NH2 analogue with Leu, Nle or Ala. Each

experiment was performed in duplicate and repeated three times. The symbols are defined in the figure key, and the curves were fitted to the data points by non-linear regression analysis, as described in

Materials and Methods. The amino acid sequence of native

(33)

8. Circular dichroism

In order to determine why the biological activity of the rPTH(1-11) and rPTH(1-12) analogues differ according to the presence of Leu or Phe at the

7th residue, I determined the solution structures of [Ala3,10,12Leu7Arg11]rPTH

(1-12)NH2, [Ala3,10,12Phe7Arg11]rPTH(1-12)NH2, [Ser1Ala3,10Leu7Arg11]rPTH(1-11)NH2

and [Ala3,10Phe7Arg11]rPTH(1-11)NH2 by circular dichroism. CD spectra were

acquired for the rPTH analogues under different conditions. (CD) spectra in aqueous solutions were measured at a peptide concentration of 20uM under benign conditions, i.e., in 50mM sodium phosphate, pH 7.0, 25℃. Figure 7 shows that the peptides possess a degree of helical structure in physiological

-9 -8 -7 -6 -5 -4 0 50 100 150 200 250 300

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH

2

[Ala3,10,12Phe7Arg11]rPTH(1-12)NH

2

[Ala3,10Leu7Arg11]rPTH(1-11)NH 2

[Ala3,10Phe7Arg11]rPTH(1-11)NH 2

[Ser1Ala3,10Leu7Arg11]rPTH(1-11)NH 2

[Ser1Ala3,10Phe7Arg11]rPTH(1-11)NH 2 Pe ptide (log[M]) c A M P Le v e ls ( F o ld of B a sa l)

Figure 6. Comparison of cAMP stimulating activity induced by [Ala3,10,12

(Leu7/Phe7)Arg11]rPTH(1-12)NH2, [Ala3,10(Leu7/Phe7)Arg11]rPTH(1-11)NH2, and

[Ser1Ala3,10(Leu7/Phe7)Arg11]rPTH(1-11)NH2 in LLC-PK1 cells stably

transfected with hPTH1R. Each experiment was performed in duplicate and repeated three times. The symbols are defined in the figure key, and the curves were fitted to the data points by non-linear regression analysis, as described in Materials and Methods. The amino acid sequence of native rPTH(1-12) is AVSEIQLMHNLG.

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environments, based on the ellipticity at 222nm, and the characteristic α -helical transition, which was observed in the presence of TFE (Figure 7): i.e., a typical double minimum (n-π* transition) at 208 and 222nm, and a maximum (π-π* transition) at 192nm. The exact mechanism by which TFE stabilizes secondary structure is unknown, but more recent studies suggest that TFE interacts preferentially with the helical conformation of a peptide to shift the structural equilibrium toward this state. In the case of peptides in

50% (v/v) TFE/H2O solution at 25℃, pH 7.0, the CD spectrum showed a clear

double minimum at 208 and 222nm, thus indicating the presence of a Figure 7. Circular dichroism spectra of [Ala3,10,12Leu7Arg11] rPTH(1-12)NH2 (A),

[Ala3,10,12Phe7Arg11]rPTH(1-12)NH2 (B), [Ser1Ala3,10Leu7Arg11] rPTH(1-11)NH2

(C), [Ala3,10Phe7 Arg11]rPTH(1-11)NH2 (D) in different solutions; H2O(◇), 30%

TFE(■), 50% TFE(●) at pH7.0 and 298K. -25 -20 -15 -10 -5 0 5 10 15 20 25 190 2 00 210 22 0 230 24 0 2 50 ]x10 -3deg ree cm 2/d ec im ol e Wavelength (nm)

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH2 ‘ H2O (pH 7.0)

{ TFE30% z TFE50% -25 -20 -15 -10 -5 0 5 10 15 20 25 190 2 00 210 22 0 230 24 0 2 50 ]x10 -3deg ree cm 2/d ec im ol e Wavelength (nm)

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH2 ‘ H2O (pH 7.0)

{ TFE30% z TFE50% -25 -20 -15 -10 -5 0 5 10 15 20 25 190 200 210 220 230 240 250 [Ala3,10,12Phe7Arg11]rPTH(1-12)NH

2 ‘ H2O (pH 7.0) { TFE30% z TFE50% ]x10 -3de gree c m 2/dec im ol e Wavelength (nm) -25 -20 -15 -10 -5 0 5 10 15 20 25 190 200 210 220 230 240 250 [Ala3,10,12Phe7Arg11]rPTH(1-12)NH

2 ‘ H2O (pH 7.0) { TFE30% z TFE50% ]x10 -3de gree c m 2/dec im ol e Wavelength (nm) -25 -20 -15 -10 -5 0 5 10 15 20 25 190 200 210 220 230 240 250 ]x 10 -3de gr ee cm 2/de cimo le Wavelength (nm) ‘ H2O (pH 7.0) { TFE30% z TFE50% [Ala3,10Phe7Arg11]rPTH(1-11)NH2

-25 -20 -15 -10 -5 0 5 10 15 20 25 190 200 210 220 230 240 250 ]x 10 -3de gr ee cm 2/de cimo le Wavelength (nm) ‘ H2O (pH 7.0) { TFE30% z TFE50% [Ala3,10Phe7Arg11]rPTH(1-11)NH2

-25 -20 -15 -10 -5 0 5 10 15 20 25 190 200 210 220 230 240 250 ]x10 -3 d eg ree cm 2/d ec im ol e Wavelength (nm)

[Ser1Ala3,10Leu7Arg11]rPTH(1-11)NH2 ‘ H2O (pH 7.0) { TFE30% z TFE50% -25 -20 -15 -10 -5 0 5 10 15 20 25 190 200 210 220 230 240 250 ]x10 -3 d eg ree cm 2/d ec im ol e Wavelength (nm)

[Ser1Ala3,10Leu7Arg11]rPTH(1-11)NH2 ‘ H2O (pH 7.0) { TFE30% z TFE50% ]x10 -3 d eg ree cm 2/d ec im ol e Wavelength (nm)

[Ser1Ala3,10Leu7Arg11]rPTH(1-11)NH2 ‘ H2O (pH 7.0) { TFE30% z TFE50%

A

B

C

D

-25 -20 -15 -10 -5 0 5 10 15 20 25 190 2 00 210 22 0 230 24 0 2 50 ]x10 -3deg ree cm 2/d ec im ol e Wavelength (nm)

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH2 ‘ H2O (pH 7.0)

{ TFE30% z TFE50% -25 -20 -15 -10 -5 0 5 10 15 20 25 190 2 00 210 22 0 230 24 0 2 50 ]x10 -3deg ree cm 2/d ec im ol e Wavelength (nm)

[Ala3,10,12Leu7Arg11]rPTH(1-12)NH2 ‘ H2O (pH 7.0)

{ TFE30% z TFE50% -25 -20 -15 -10 -5 0 5 10 15 20 25 190 200 210 220 230 240 250 [Ala3,10,12Phe7Arg11]rPTH(1-12)NH

2 ‘ H2O (pH 7.0) { TFE30% z TFE50% ]x10 -3de gree c m 2/dec im ol e Wavelength (nm) -25 -20 -15 -10 -5 0 5 10 15 20 25 190 200 210 220 230 240 250 [Ala3,10,12Phe7Arg11]rPTH(1-12)NH

2 ‘ H2O (pH 7.0) { TFE30% z TFE50% ]x10 -3de gree c m 2/dec im ol e Wavelength (nm) -25 -20 -15 -10 -5 0 5 10 15 20 25 190 200 210 220 230 240 250 ]x 10 -3de gr ee cm 2/de cimo le Wavelength (nm) ‘ H2O (pH 7.0) { TFE30% z TFE50% [Ala3,10Phe7Arg11]rPTH(1-11)NH2

-25 -20 -15 -10 -5 0 5 10 15 20 25 190 200 210 220 230 240 250 ]x 10 -3de gr ee cm 2/de cimo le Wavelength (nm) ‘ H2O (pH 7.0) { TFE30% z TFE50% [Ala3,10Phe7Arg11]rPTH(1-11)NH2

-25 -20 -15 -10 -5 0 5 10 15 20 25 190 200 210 220 230 240 250 ]x10 -3 d eg ree cm 2/d ec im ol e Wavelength (nm)

[Ser1Ala3,10Leu7Arg11]rPTH(1-11)NH2 ‘ H2O (pH 7.0) { TFE30% z TFE50% -25 -20 -15 -10 -5 0 5 10 15 20 25 190 200 210 220 230 240 250 ]x10 -3 d eg ree cm 2/d ec im ol e Wavelength (nm)

[Ser1Ala3,10Leu7Arg11]rPTH(1-11)NH2 ‘ H2O (pH 7.0) { TFE30% z TFE50% ]x10 -3 d eg ree cm 2/d ec im ol e Wavelength (nm)

[Ser1Ala3,10Leu7Arg11]rPTH(1-11)NH2 ‘ H2O (pH 7.0) { TFE30% z TFE50%

A

B

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significant α-helix population.

Besides, substitutions with Glu10 and Ile11 in [Ala3,10,12(Leu7/Phe7)Arg11]rPTH

(1-12)NH2 did not show the typical double minimum at 208 and 222nm even

at high concentrations in TFE solution (Figure 8). Many short and medium

range NOEs were seen for the peptide Ala3-His9 in 30% TFE solution.

Substitutions with Glu10 and Ile11 in [Ala3,10,12(Leu7/Phe7)Arg11]rPTH(1-12)NH2

did not show these NOEs. CD result of decreased helix formation in the peptide.

Figure 8. CD spectra of Ala10→Glu10 and/or Arg11→Ile11 substituted in 30% TFE solutions (A and C) and cd absorbance distribution at 220nm for each peptide represented by bar diagram (B and D); A and B [Ala3,10,12Leu7 Arg11]rPTH(1-12)NH2, [Ala3,10,12Leu7Ile11]rPTH(1-12)NH2, [Ala3,12 Leu7Glu10

Arg11]rPTH(1-12)NH2, and [Ala3,12Leu7Glu10Ile11]rPTH (1-12)NH2, C and D.

[Ala3,10,12Phe7Arg11]rPTH(1-12)NH2, [Ala3,10,12Phe7Ile11]rPTH(1-12)NH2, [Ala3,12

Phe7Glu10Arg11]rPTH(1-12)NH2, and [Ala3,12Leu7Glu10Ile11]rPTH(1-12)NH2.

Wavelength (nm) -30 -20 -10 0 10 20 30 190 200 210 220 230 240 250 Leu7Ala10Arg11 12_13 12_3 12_15 [θ ]x10 -3de gr ee c m 2/d ec im ol e

Leu7Ala10Arg11

Leu7Glu10Arg11

Leu7Ala10Ile11

Leu7Glu10Ile11

Leu7Ala10Arg11

Leu7Glu10Arg11

Leu7Ala10Ile11

Leu7Glu10Ile11

Leu7Ala10Arg11

Leu7Glu10Arg11

Leu7Ala10Ile11

Leu7Glu10Ile11

-14 -12 -10 -8 -6 -4 -2 0

Ala10Arg11 Glu10Arg11 Ala10Ile11 Glu10Ile11

[θ ]x 10 -3de gr ee c m 2/d ec im ol e Leu7 -14 -12 -10 -8 -6 -4 -2 0

Ala10Arg11 Glu10Arg11 Ala10Ile11 Glu10Ile11

[θ ]x 10 -3de gr ee c m 2/d ec im ol e Leu7 -30 -20 -10 0 10 20 30 190 200 210 220 230 240 250 Phe7Ala10Arg11 12_31 -30 -20 -10 0 10 20 30 190 200 210 220 230 240 250 Phe7Ala10Arg11 12_31 [θ ]x10 -3de gr ee cm 2/de cimo le Wavelength (nm)

Phe7Ala10Arg11

Phe7Glu10Arg11

Phe7Ala10Ile11

Phe7Glu10Ile11

Phe7Ala10Arg11

Phe7Glu10Arg11

Phe7Ala10Ile11

Phe7Glu10Ile11

-16 -14 -12 -10 -8 -6 -4 -2 0

Ala10Arg11 G lu 10Arg11 Ala10Ile 11 Glu 10Ile 11

[θ ]x10 -3de gr ee cm 2/dec im ole Phe7 -16 -14 -12 -10 -8 -6 -4 -2 0

Ala10Arg11 G lu 10Arg11 Ala10Ile 11 Glu 10Ile 11

[θ ]x10 -3de gr ee cm 2/dec im ole Phe7

A

B

C

Wavelength (nm)

D

-30 -20 -10 0 10 20 30 190 200 210 220 230 240 250 Leu7Ala10Arg11 12_13 12_3 12_15 [θ ]x10 -3de gr ee c m 2/d ec im ol e

Leu7Ala10Arg11

Leu7Glu10Arg11

Leu7Ala10Ile11

Leu7Glu10Ile11

Leu7Ala10Arg11

Leu7Glu10Arg11

Leu7Ala10Ile11

Leu7Glu10Ile11

Leu7Ala10Arg11

Leu7Glu10Arg11

Leu7Ala10Ile11

Leu7Glu10Ile11

-14 -12 -10 -8 -6 -4 -2 0

Ala10Arg11 Glu10Arg11 Ala10Ile11 Glu10Ile11

[θ ]x 10 -3de gr ee c m 2/d ec im ol e Leu7 -14 -12 -10 -8 -6 -4 -2 0

Ala10Arg11 Glu10Arg11 Ala10Ile11 Glu10Ile11

[θ ]x 10 -3de gr ee c m 2/d ec im ol e Leu7 -30 -20 -10 0 10 20 30 190 200 210 220 230 240 250 Phe7Ala10Arg11 12_31 -30 -20 -10 0 10 20 30 190 200 210 220 230 240 250 Phe7Ala10Arg11 12_31 [θ ]x10 -3de gr ee cm 2/de cimo le Wavelength (nm)

Phe7Ala10Arg11

Phe7Glu10Arg11

Phe7Ala10Ile11

Phe7Glu10Ile11

Phe7Ala10Arg11

Phe7Glu10Arg11

Phe7Ala10Ile11

Phe7Glu10Ile11

-16 -14 -12 -10 -8 -6 -4 -2 0

Ala10Arg11 G lu 10Arg11 Ala10Ile 11 Glu 10Ile 11

[θ ]x10 -3de gr ee cm 2/dec im ole Phe7 -16 -14 -12 -10 -8 -6 -4 -2 0

Ala10Arg11 G lu 10Arg11 Ala10Ile 11 Glu 10Ile 11

[θ ]x10 -3de gr ee cm 2/dec im ole Phe7

A

B

C

D

(36)

9. Resonance assignment and solution structure

Complete proton resonance assignments were possible using the standard

sequential resonance assignment procedure38. Having classified the individual spin

systems, the backbone sequential resonances were assigned using dN(i,i+1) NOE connectivities in the 2D-nuclear Overhauser effect spectroscopy (NOESY) spectra. A number of well-resolved, intense dNN cross-peaks were also observed, suggesting the formation of an α-helix. Figure 9 summarizes

Figure 9. Summary of the NMR data of [Ala3,10,12Leu7Arg11]rPTH(1-12) NH2 (A),

[Ala3,10,12Phe7Arg11]rPTH(1-12)NH2 (B), [Ser1Ala3,10Leu7Arg11]rPTH(1-11)

NH2 (C), and [Ala3,10Phe7Arg11]rPTH(1-11)NH2 (D) in an aqueous 30%

TFE solution at pH 7.0 and 298K, showing sequential and short-range NOE contacts. The strength of the observed NOEs is represented by the line thicknesses. The dashed line represents NOEs unresolved by resonance

overlapping. Vicinal coupling constants (●; 3JHN <6 Hz) are indicated and

A

1 5 10 12 A V A E I Q L M H A R A dαβ(i,i+3) dβN(i,i+1) dαN(i,i+1) dNN(i,i+1) dαN(i,i+3) 3J HNα * α-HELIX 1 5 10 12 A V A E I Q L M H A R A dαβ(i,i+3) dαβ(i,i+3) dβN(i,i+1) dβN(i,i+1) dαN(i,i+1) dNN(i,i+1) dαN(i,i+3) dαN(i,i+3) 3J HNα * α-HELIX α-HELIX

B

A V A E I Q F M H A R A 1 5 10 12 3J dαβ(i,i+3) dβN(i,i+1) dαN(i,i+1) dNN(i,i+1) dαN(i,i+3) HNα * α-HELIX A V A E I Q F M H A R A 1 5 10 12 3J A V A E I Q F M H A R A 1 5 10 12 3J dαβ(i,i+3) dαβ(i,i+3) dβN(i,i+1) dβN(i,i+1) dαN(i,i+1) dNN(i,i+1) dαN(i,i+3) dαN(i,i+3) HNα * α-HELIX α-HELIX

C

1 5 10 S V A E I Q L M H A R dαβ(i,i+3) dβN(i,i+1) dαN(i,i+1) dNN(i,i+1) dαN(i,i+3) 3J HNα * * α-HELIX 1 5 10 S V A E I Q L M H A R dαβ(i,i+3) dαβ(i,i+3) dβN(i,i+1) dβN(i,i+1) dαN(i,i+1) dNN(i,i+1) dαN(i,i+3) dαN(i,i+3) 3J HNα * * α-HELIX α-HELIX 1 5 10 A V A E I Q F M H A R dαβ(i,i+3) dβN(i,i+1) dαN(i,i+1) dNN(i,i+1) dαN(i,i+3) 3JHNα * α-HELIX 1 5 10 A V A E I Q F M H A R dαβ(i,i+3) dαβ(i,i+3) dβN(i,i+1) dβN(i,i+1) dαN(i,i+1) dNN(i,i+1) dαN(i,i+3) dαN(i,i+3) 3JHNα * α-HELIX α-HELIX

D

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the sequential and short-range NOE connectivities observed for the rPTH analogues in 30% TFE. The observation of continuous dNN(i,i+1) contacts together with the characteristic dN(i,i+3) and the d(i,i+3) NOEs supports the

existence of α-helices containing the Ala3-His9 residues in all of the rPTH

analogues.

The NMR structures were calculated using the experimental restraints

derived from 2D-NOESY and double-quantum-filtered correlated

spectroscopy (DQF-COSY) spectra. A total of 50 distance geometry (DG) structures served as starting structures for the dynamic simulated-annealing (SA) calculations of the peptides in TFE solutions. The 20 lowest energy

structures (<SA>k) from the 50 simulated-annealing structures were selected

for detailed structural analysis (Table 3). The average structure (<SA>k)

was calculated from the geometrical average of the 20 <SA>k structural

coordinates, and restraint energy minimization (REM) was used to correct covalent bonds and angular distortions. In Figure 10, the final 20 structures

are displayed as averaged structures (<SA>kr) of [Ala3,12Leu7Glu10Arg11]

rPTH(1-12)NH2. In Figure 11(A/B), the final 20 structures are displayed as

averaged structures (<SA>kr) of rPTH(1-12). A Ramachandran plot (data

Figure 10. NMR structure of the [Ala3,12Leu7Glu10Arg11]rPTH(1-12)

NH2 analogue. Superposition of the final 20 <SA>k

structures upon the energy-minimized average

structure (<SA>kr) of Ala3-His9, and ribbon drawings of

the REM structures of [Ala3,12Leu7Glu10Arg11]rPTH

(38)

not shown) for all 20 <SA>k structures showed that the, and angles of the

finally simulated-annealing structures are distributed adequately in

energetically acceptable regions. A best-fit superposition of the 20 <SA>k

structures along with the energy-minimized average structure (<SA>kr) of

rPTH(1-11) are also shown in Figure 11(C/D). The main secondary structural feature of the rPTH analogues is the α-helix spanning residues

of Ala3-His9 in 30% TFE/H2O solution.

Figure 11. NMR structures of the rPTH(1-12)NH2 and rPTH(1-11)NH2 analogues.

Superposition of the final 20 <SA>k structures upon the energy-

minimized average structure (<SA>kr) of Ala3-His9, and ribbon draw-

ings of the REM structures of [Ala3,10,12Leu7Arg11]rPTH(1-12)NH2 (A),

[Ala3,10,12Phe7Arg11]rPTH(1-12)NH2 (B), [Ser1Ala3,10Leu7Arg11]rPTH(1-11)

NH2 (C), and [Ala3,10Phe7Arg11]rPTH(1-11)NH2 (D) in an aqueous 30% TFE

solution at pH 7.0 and 298K.

B

A

수치

Figure 1. Selection  of  the  LLC-PK1  cell  lines  stably  over-  expressing  hPTH1R  by  measuring  the  level  of  PTH(1-34)-stimulated  cAMP  accumulation.
Table  1. Amino-acid  sequence  of  synthetic  PTH  analogues  used  in  this  study  P e p t i d e A m i n o   a c i d   s e q u e n c e s [ L e u 7 ] r P T H ( 1 - 1 2 )   a n a l o g u e s 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 [ A la 3 ,1 0 ,1 2 L e u 7 A r g 1
Table 2. EC 50   values  (means  ±  SEM)  of  the  rPTH(1-12)  and  rPTH(1-11)  analogues  used
Figure 2. The  effect  of  substituting  the  11 th residues  of  the  [Ala 3,10,12
+7

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