ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 1, pp. 24–29. © Pleiades Publishing, Inc., 2008. Original Russian Text © Yu.A. Kovalitskaya, A.A. Kolobov, E.A. Kampe-Nemm, Yu.A. Zolotarev, V.V. Yurovskii, V.B. Sadovnikov, V.M. Lipkin, E.V. Navolotskaya, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 1, pp. 29–35.
Synthetic Peptide KKRR Corresponding to the Human ACTH Fragment 15–18 is an Antagonist of the ACTH Receptor Yu. A. Kovalitskayaa, A. A. Kolobovb, E. A. Kampe-Nemmb, Yu. A. Zolotarevc, V. V. Yurovskiid, V. B. Sadovnikova, V. M. Lipkina, and E. V. Navolotskayaa,1 a
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Pushchino Branch, Russian Academy of Sciences, pr. Nauki 6, Pushchino, Moscow oblast, 142290 Russia b State Research Center, Institute of Extrapure Biopreparations, FMBA of the Russian Federation, St. Petersburg, 197110 Russia c Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova 2, Moscow, 123182 Russia d Faculty of Neurosurgery, University of Maryland, Baltimore, USA Received December 8, 2006; in final form, January 25, 2007
Abstract—Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [3H]ACTH (11–24) and [3H]ACTH (15–18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [3H]ACTH-(11–24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (Kd 1.8 ± 0.1 nM). Twenty nine fragments of ACTH (11–24) were synthesized, and their ability to inhibit the specific binding of [3H]ACTH (11–24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15–18) (KKRR) (Ki 2.3 ± 0.2 nM), whose [3H] labeled derivative binds to rat adrenocortical membranes (Kd 2.1 ± 0.1 nM) with a high affinity. The specific binding of [3H]ACTH-(15–18) was inhibited by 100% by unlabeled ACTH (11–24) (Ki 2.0 ± 0.1 nM). ACTH (15–18) in the concentration range of 1–1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor. Key words: adrenocorticotropic hormone (ACTH), peptides, receptors, adenylate cyclase, adrenal cortex DOI: 10.1134/S1068162008010020
INTRODUCTION
The goal of this work was to study the ability of unlabeled synthetic fragments of the ACTH (11–24) peptide to inhibit the specific binding of [3H]ACTH (11–24) to membranes of the rat adrenal cortex, to determine the shortest fragment capable of binding with high affinity to the ACTH receptor, and to examine the effect of the corresponding peptide on the adenylate cyclase activity of adrenocortical membranes.
The major function of the adrenocorticotropic hormone is to stimulate the synthesis and secretion of glucocorticoids by the cells of the zona fasciculata and zona reticularis of the adrenal cortex.2 The sensitivity of cortical cells to ACTH depends on the expression and function of the G protein–coupled receptor MC2R, which belongs to the subfamily of melanocortin receptors [1–4]. The binding of the hormone to MC2R leads to an increase in the adenylate cyclase activity and thus the activation of protein kinase A [5–7]. Kapas et al. studied the binding of ACTH fragments to the cloned murine hormone receptor expressed in human HeLa cells [8]. According to their data, fragment ACTH (11– 24) competes actively with 125I-labeled ACTH for binding (IC50 ∼ 1 nM) but, as distinct from the full-size hormone, is unable to activate adenylate cyclase, i.e., exhibits the properties of an antagonist.
RESULTS AND DISCUSSION ACTH (1–24) is the smallest fragment of ACTH required for the hormone to fully manifest its activity; its sequence without ten C-terminal amino acid residues corresponds to the primary structure of the α-melanocyte-stimulating hormone (α-MSH) (Fig. 1). A comparative analysis of the primary structure of vertebrate ACTH shows that region 15–24 of the molecule is evolutionarily highly conservative. Costa et al. synthesized the analogues of ACTH (1–24) with amino acid substitutions in region 15–24 and estimated their activity in vitro and in vivo [9] (Fig. 2). Based on the results obtained, the authors concluded that region 15–18 (KKRR) in the ACTH molecule is necessary for both
1
Corresponding author; phone: (4967) 73-6668; fax: (4967) 330527; e-mail:
[email protected]. 2 Abbreviations: ACTH, adrenocorticotropic hormone; MC2R, melanocortin–2 receptor; PAM, phenacylamidomethyl; SEM, standard error of the mean.
24
SYNTHETIC PEPTIDE KKRR
25 in vivo and in vitro)
ACTH (1−24)
in vivo and ~34% in vitro)
α-MSH
in vivo and ~46% in vitro) in vivo and ~72% in vitro) in vivo and ~70% in vitro)
Fig. 1. Comparison of amino acid sequences of peptide ACTH (1–24) and human α-MSH. The ACTH (15–24) fragment is designated by boldface type.
in vivo and ~91% in vitro) in vivo and ~83% in vitro)
the binding to receptor and the subsequent activation of adenylate cyclase. Surprisingly, the analogue in which Ala residues were substituted for five C-terminal amino acid residues 20–24 (VKVYP) was 1.5 times more effective than peptide ACTH (1–24) with the natural sequence in vivo (but not in vitro). In the opinion of Costa et al., region 20−24 of the ACTH molecule is not involved in the binding of the hormone to the receptor but is essential for the formation of the spatial structure, which provides the optimal stability of ACTH in the blood. According to the data of Kapas et al., ACTH (11– 24) acts as an antagonist of the receptor: it competes with 125I-labeled ACTH for binding to the cloned receptor (IC50 ∼ 1 nM) but, as distinct from ACTH, does not activate adenylate cyclase [8]. We obtained peptide [3H]ACTH (11–24) (specific activity 22 Ci/mol) and studied its binding to membranes isolated from the adrenal cortex of the rat. The experiments showed that, under the conditions chosen (see the Experimental section), [3H]ACTH (11–24) binds specifically to rat adrenocortical membranes (Fig. 3). It follows from the plots of total (1), specific 2, and nonspecific (3) binding of [3H]ACTH (11–24) to membranes versus incubation time that the dynamic equilibrium in the system labeled peptide−receptor was established approximately after 1 h and persisted for at least 2 h. Therefore, for determining the equilibrium dissociation constant (Kd), the reaction of binding of [3H]ACTH (11–24) to membranes was carried out for 1 h. The nonspecific binding of [3H]ACTH (11–24) under these conditions was 6.4 ± 0.8% of the total binding of the peptide. It follows from the Scatchard plot (1) in Fig. 4 that peptide [3H]ACTH (11–24) binds to one type of high– affinity receptors on rat adrenocortical membranes (Kd of complex [3H]ACTH (11–24)–receptor 1.8 ± 0.1 nM and Bmax – 2.8 ± 0.2 pmol/mg protein). To characterize the specificity of binding, the following unlabeled peptides were tested as potential competitors of [3H]ACTH (11–24): ACTH (1–24) (positive control), ACTH (4– 10), somatostatin, β-endorphin, and [Met5]enkephalin (negative control). The results of the experiments presented in Table 1 indicate that only ACTH (1−24) was capable of inhibiting the binding of [3H]ACTH (11–24) to membranes (Ki 1.7 ± 0.1 nM). The other peptides were inactive. Thus, [3H]ACTH (11–24) binds with high RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Fig. 2. Amino acid sequence and the activity (%) of ACTH (1–24) analogues synthesized by Costa et al. (according to the data reported in [9]).
affinity and high specificity to the ACTH receptor of rat adrenocortical membranes. To assess the affinity of unlabeled ACTH (11–24) fragments to the ACTH receptor of rat adrenocortical membranes (Table 1), we studied the ability of each fragment to inhibit the specific binding of [3H]ACTH (11–24) to membranes. It was found that only ACTH (11–24) fragments containing the sequence KKRR (no. 29, fragment 15–18) and tetrapeptide KRRP (no. 26, fragment 16–19) have a high inhibitory activity. The inhibitory activity of the other peptides was very low (Ki > 1 µM). Tetrapeptides KKRR and KRRP were the shortest ACTH (11–24) fragments that actively inhibited the binding of [3H]ACTH (11–24) to adrenocortical membranes (Ki 2.3 ± 0.2 and 2.0 ± 0.2 nM) (Table 1). To characterize the binding of peptide ACTH (15– 18) to rat adrenocortical membranes in a direct experiment, labeled [3H]ACTH (15–18) (specific activity 26 Ci/mol) was obtained (see the Experimental section). The dependence of total (1), specific (2), and nonspecific (3) binding of [3H]ACTH (15–18) to membranes on incubation time (Fig. 3b) was similar to that for [3H]ACTH (11–24) (Fig. 3a): the dynamic equilibrium in the system labeled peptide–receptor was established approximately after 1 h and persisted for at least 2 h. Therefore, the reaction of binding of [3H]ACTH (15–18) to membranes was carried out for 1 h. The nonspecific binding of [3H]ACTH-(15–18) was 6.4 ± 0.6% of its total binding. An analysis of the specific binding of [3H]ACTH(15–18) to adrenocortical membranes in the Scatchard coordinates (Fig. 4, 2) indicated that the labeled peptide binds with high affinity to one receptor type (Kd 2.1 ± 0.1 nM, Bmax = 2.6 ± 0.2 pmol/mg protein). A study of binding specificity showed that only ACTH (1–24) and ACTH (11–24) are capable of extruding [3H]ACTH (15–18) from the complex with receptor (Ki 1.9 ± 0.1 and 2.0 ± 0.1 nM, Table 2). Unlabeled ACTH (4–10), somatostatin, β-endorphin, and [Met5]enkephalin,
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KOVALITSKAYA et al. B/F 0.30
(‡) Binding of [3H]ACTH (1–24), cpm 30000 1 2
25000
0.25 0.20 1
20000 0.15
2
15000 0.10 10000 0.05 5000 3 0 (b) Binding of [3H]ACTH (15–18), cpm 30000 1 2
25000
0.1
0.2
0.3
0.4
0.5 B, nM
Fig. 4. Analysis of the specific binding of [3H]ACTH (1−24) (1) and [3H]ACTH (15–18) (2) to rat adrenocortical membranes in Scatchard coordinates (B and F are the molar concentrations of bound and labeled peptide, respectively).
EXPERIMENTAL
20000 15000 10000 5000 3 0
20
40
60
80
100 120 Time, min
Fig 3. Dependence of (a) (1) total, (2) specific, and (3) nonspecific binding of peptides [3H]ACTH (11–24) and (b) [3H]ACTH (15–18) to rat adrenocortical membranes on the time of incubation at 4°ë. The specific binding of unlabeled peptide was determined as the difference between its total and nonspecific binding.
tested in parallel experiments, were inactive (Ki > 10 µM). A study of the effect of peptides ACTH (1–24), ACTH (11–24), and ACTH (15–18) on the adenylate cyclase activity in rat adrenocortical membranes showed that, as distinct from ACTH (1–24), which activates the enzyme, its fragments 11–24 and 15–18 in the concentration range of 1–1000 nM did not affect the enzyme activity (Table 3). Thus, ACTH (15–18), similar to ACTH (11–24), binds specifically and with high affinity to the ACTH receptor of the rat adrenal cortex but does not affect the adenylate cyclase activity, i. e., is an antagonist of the ACTH receptor.
The following preparations and reagents were used: peptides ACTH (4–10) and ACTH (1–24), somatostatin, β-endorphin, and [Met5]enkephalin (Sigma, United States); saccharose, BSA, EDTA, EGTA, Tris, phenylmethylsulfonyl fluoride (PMSF), sodium azide (NaN3) (Serva, Germany); N-methylpyrrolidone, diisopropylcarbodiimide, 1-hydroxybenzotriazole, thioanisole (Merck, Germany); and Unisolv 100 scintillator (Amersham, England). Other reagents were of extra purity grade. Distilled water was additionally purified using a Mono-Q system (Millipore, United States). Mature SD male rats weighing 180–210 g were obtained from the nursery of the Institute of Bioorganic Chemistry, Russian Academy of Sciences. Peptide ACTH (11–24) and its fragments (compounds 1–31, Table 1) were synthesized on an Applied Biosystems Model 430A automatic synthesizer (United States) using the Boc/Bzl tactics of peptide chain elongation [10, 11]. The peptides were purified to a homogeneous state by preparative reversed-phase chromatography (Gilson chromatograph, France) on a Delta Pack C18 column, 100 A (39 × 150 mm), 5 µm; flow rate 10 ml/min; eluent 0.1% TFA; gradient of acetonitrile 10–40% in 30 min]. The purity of the peptides after purification was better than 99% for compounds 1−3 (Table 1) and 95% for the other compounds. The molecular masses of the peptides were determined by mass spectrum analysis (Finnigan mass spectrometer, United States). The data of amino acid analysis (hydrolysis by 6 M HCl, 22 h, 110°C; an LKB 4151 Alpha Plus amino acid analyzer, Sweden) corresponded to the structures required.
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Table 1. Inhibition of specific binding of [3H]ACTH (11–24) to rat adrenal cortex membranes by unlabeled peptides IC50 Number
Fragment of the ACTH sequence; structure
Mr (calc. mol. mass)
Ki
nM ± standard deviation 11.0 ± 0.1
1.9 ± 0.1
1
11–24; KPVGKKRRPVKVYP
1652.3 (1652.26)
11.3 ± 0.1
1.7 ± 0.1
2
12–24; PVGKKRRPVKVYP
1523.9 (1523.81)
12.0 ± 0.2
1.9 ± 0.1
3
11–23; KPVGKKRRPVKVY
1555.3 (1555.13)
11.8 ± 0.2
1.8 ± 0.1
4
13–24; VGKKRRPVKVYP
1454.2 (1454.26)
12.2 ± 0.2
1.9 ± 0.1
5
11–22; KPVGKKRRPVKV
1392.1 (1391.94)
12.6 ± 0.2
1.9 ± 0.2
6
14–24; GKKRRPVKVYP
1327.9 (1327.79)
11.9 ± 0.2
1.8 ± 0.2
7
11–21; KPVGKKRRPVK
1292.7 (1292.79)
12.9 ± 0.2
2.0 ± 0.2
8
15–24; KKRRPVKVYP
1270.6 (1270.72)
12.9 ± 0.2
2.0 ± 0.2
9
11–20; KPVGKKRRPV
1164.8 (1164.6)
13.2 ± 0.2
2.0 ± 0.2
10
16–24; KRRPVKVYP
1142.6 (1142.53)
23.8 ± 0.4
3.6 ± 0.3
11
11–19; KPVGKKRRP
1065.6 (1065.45)
12.9 ± 0.3
2.0 ± 0.2
12
17–24; RRPVKVYP
1014.5 (1014.34)
1890 ± 170
286.4 ± 25.8
13
11–18; KPVGKKRR
968.4 (968.32)
13.6 ± 0.3
2.1 ± 0.3
14
18–24; RPVKVYP
858.3 (858.12)
>10000
>10000
15
11–17; KPVGKKR
806.2 (806.12)
>10000
>10000
16
19–24; PVKVYP
702.2 (701.92)
>10000
>10000
17
11–16; KPVGKK
650.3 (649.92)
>10000
>10000
18
20–24; VKVYP
605.2 (604.81)
>10000
>10000
19
11–15; KPVGK
528.0 (527.73)
>10000
>10000
20
21–24; KVYP
505.3 (505.66)
>10000
>10000
21
11–14; KPVG
399.2 (399.54)
>10000
>10000
22
12–23; PVGKKRRPVKVY
1427.2 (1426.94)
12.7 ± 0.1
1.9 ± 0.1
23
13–22; VGKKRRPVKV
1166.8 (1166.62)
12.7 ± 0.2
1.9 ± 0.2
24
14–21; GKKRRPVK
968.1 (968.32)
12.8 ± 0.2
1.9 ± 0.2
25
15–20; KKRRPV
782.8 (783.06)
12.8 ± 0.2
1.9 ± 0.2
26
16–19; KRRP
556.0 (555.72)
12.9 ± 0.2
2.0 ± 0.2
27
14–19; GKKRRP
741.2 (740.98)
12.8 ± 0.2
1.9 ± 0.2
28
15–19; KKRRP
684.1 (683.91)
12.9 ± 0.1
2.0 ± 0.1
29
15–18; KKRR
586.9 (586.78)
15.1 ± 0.2
2.3 ± 0.2
30
16–18; KRR
458.7 (458.59)
7362 ± 589
31
4–10
–
>10000
>10000
1115.5 ± 89.2
Somatostatin
–
–
>10000
>10000
β-Endorphin
–
–
>10000
>10000
[Met5]Enkephalin
–
–
>10000
>10000
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KOVALITSKAYA et al.
Table 2. Inhibition by unlabeled peptides of specific binding of [3H]ACTH (15–18) to rat adrenocortical membranes Ki
IC50 Peptide
nM ± standard deviation
ACTH (1–24)
11.0 ± 0.1
1.9 ± 0.1
ACTH (11–24)
11.3 ± 0.1
2.0 ± 0.1
ACTH (4–10)
>10000
>10000
Somatostatin
>10000
>10000
β-Endorphin
>10000
>10000
[Met5]Enkephalin
>10000
>10000
Table 3. Effect of ACTH (15–18), ACTH (1–24), and ACTH (11–24) on the adenylate cyclase activity of rat adrenocortical membranes Activity, nmol cAMP per g of protein Peptide in 10 min ± standard deviation concentration, nM ACTH (15–18) ACTH (1–24) ACTH (11–24) 0
1.43 ± 0.12
1.43 ± 0.12
1.43 ± 0.12
0.1
1.46 ± 0.13
1.44 ± 0.15
1.48 ± 0.12
1
1.46 ± 0.15
1.82 ± 0.17
1.54 ± 0.18
10
1.43 ± 0.18
2.23 ± 0.19
1.50 ± 0.14
100
1.52 ± 0.16
2.38 ± 0.21
1.43 ± 0.12
1000
1.41 ± 0.18
2.36 ± 0.17
1.49 ± 0.12
Labeled analogues [3H]ACTH (11–24) and (15–18) were obtained by high-temperature solid-phase catalytic isotope exchange [12]. Aluminum oxide (50 mg) was added to a solution of the peptide (2.0 mg) in water (0.5 ml), and the solution was evaporated on a rotor evaporator. Aluminum oxide with the peptide applied was mixed with 10 mg of catalyzer (5% Rh/Al2O3). The solid mixture obtained was placed in a 10-ml ampoule. The ampoule was evacuated, filled with gaseous tritium to a pressure of 250 mmHg, heated to 170°C, and kept at this temperature for 20 min. Then the ampoule was cooled, evacuated, blown out with hydrogen, and evacuated again. The labeled peptide was extracted from the solid reaction mixture by two portions of 50% aqueous ethanol, 3 ml each, and the combined solution was evaporated. Labile tritium was removed by repeating the procedure twice. The labeled peptide was purified by HPLC using a Beckman spec-
[3H]ACTH
trophotometer at 254 and 280 nm on columns of Kromasil (4 × 150 mm); granulation 5 µm at 20°C. The elution was with 0.1% TFA in a gradient of methanol 42−70% in 20 min; the flow rate was 3 ml/min. The incorporation of tritium into the peptide was calculated by the liquid scintillation counting method. Membranes from rat adrenal cortex were isolated by the method described in [13]. Protein concentration was determined by the method of Lowry [14] with BSA as a reference. Reaction of binding of the peptide [3H]ACTH (11–24) to membranes was performed in 50 mM TrisHCl buffer containing PMSF (0.6 mg/ml), pH 7.5, according to the following scheme: 100 µl of a labeled peptide from a solution with a concentration of 10–10 to 10–7 M (three parallel samples for each concentration), 100 µl of buffer (total binding), or 100 µl of a 10–3 M solution of unlabeled ACTH (11–24) in buffer (nonspecific binding), and 800 µl of a suspension of freshly isolated membranes (0.2 mg of protein) were added to siliconized test tubes. The test tubes were incubated at 4°C for 1 h, and the reaction mixture was filtered through GF/B fiberglass filters (Whatman, England). The filters were washed three times with an ice physiological solution (5 ml). The radioactivity on filters was counted by an LS 5801 liquid scintillation counter (Beckman, United States). The specific binding of [3H]ACTH (11–24) to membranes was determined as the difference between its total and nonspecific binding. The parameters of specific binding of [3H]ACTH (11–24) to membranes (equilibrium dissociation constant Kd and density of receptors Bmax, the maximum binding capacity per 1 mg of protein) were determined from the plots of the ratio of molar concentrations of bound (B) and free (F) labeled peptide versus the molar concentration of bound labeled peptide (B) (Scatchard plot) [15]. Estimation of the ability of unlabeled ACTH (4– 10), ACTH (1–24), ACTH-(11–24), and ACTH (11– 24), somatostatin, and b-endorphin to inhibit the specific binding of [3H]ACTH (11–24) A suspension of membranes (0.2 mg of protein, 800 µl) was incubated with [3H]ACTH (11–24) (5 nM, 100 ml) and one of potential inhibitors (concentration range 10–10 to 10−4 M, three samples for each concentration) as described above. The inhibition constant (Ki) was determined by the formula Ki = [IC]50/(1 + [L]/Kd), where [L] is the molar concentration of [3H]ACTH (11–24); Kd is the equilibrium dissociation constant for [3H]ACTH (11–24)−receptor complex; [IC]50 is the concentration of the unlabeled ligand that causes a 50% inhibition of specific binding of labeled [3H]ACTH (11–24) [16]. The value of IC50 was determined graphically from the curve of inhibition [plot of inhibition (%) versus the molar concentration of inhibitor]. The value of Kd was determined preliminarily as described above. Reaction of binding of [3H]ACTH (15–18) to membranes was carried out as described above for
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[3H]ACTH (11–24). To characterize the specificity of binding of [3H]ACTH (15–18), we tested unlabeled ACTH (11–24), ACTH (4–10), somatostatin, β-endorphin, and [Met5]enkephalin as potential inhibitors. A suspension of membranes (0.2 mg of protein, 800 µl) was incubated with 10 nM [3H]ACTH (15–18) (100 µl) in the absence or presence of one of unlabeled peptides (10–12 to 10–5 M, 100 µl). The inhibition constant was determined as described above. Adenylate cyclase activity was assayed using α[32P]ATP by the method described earlier [17, 18]. The activity of the enzyme was expressed as the amount of cAMP (nmol) formed in 10 min per 1 mg of protein of adrenocortical membranes. ACKNOWLEDGMENTS This work was supported by the Russian Foundation for Basic Research (project no. 05-04-48060), the program Molecular and Cell Biology (head V.M. Lipkin), the program Naukogrady (project no. 04-04-97200), and the International Scientific and Technical Center (project no. 2615). REFERENCES 1. Cone, R.D., Mountjoy, K.G., Robbins, L.S., Nadeau, J.H., Johnson, K.R., Roselli-Rehfuss, L., and Mortrud, M.T., Ann. N. Y. Acad. Sci., 1993, vol. 680, pp. 342–363. 2. Clark, A.J.L., Noon, L., Swords, F.M., Hunyady, L., and King, P., Ann. N. Y. Acad. Sci., 2003, vol. 994, pp. 111– 117. 3. Beuschlein, F., Fassnacht, M., Klink, A., Allolio, B., and Reincke, M., Eur. J. Endocrinol., 2001, vol. 144, pp. 199–206. 4. Voisey, J., Carroll, L., and van Daal, A., Curr. Drug Targets, 2003, vol. 4, pp. 586–597.
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