This work presents the first short orexin A and B analogues, orexin A 23–33 and orexin B 18–28, with high affinity 119 ± 49 and 49 ± 23 nM for OX1receptors expressed on SK-N-MC cells and
Trang 1The SK-N-MC cell line expresses an orexin binding site
different from recombinant orexin 1-type receptor
Heike A Wieland1,*, Richard M So¨ll2,3, Henri N Doods1, Dirk Stenkamp1, Rudolf Hurnaus1,
Ba¨rbel La¨mmle1and Annette G Beck-Sickinger2
1
Division of Preclinical Research, Boehringer Ingelheim Pharma KG, Biberach, Germany;2Institute of Biochemistry,
University of Leipzig, Germany;3Department of Applied Biosciences, Swiss Federal Institute of Technology, Zurich, Switzerland
Orexin A and B (also known as hypocretins), two recently
discovered neuropeptides, play an important role in food
intake, sleep/wake cycle and neuroendocrine functions
Orexins are endogenous ligands of two G-protein-coupled
receptors, termed OX1and OX2 This work presents the first
short orexin A and B analogues, orexin A 23–33 and orexin
B 18–28, with high affinity (119 ± 49 and 49 ± 23 nM) for
OX1receptors expressed on SK-N-MC cells and indicates
the importance of the C-terminal part of the orexin peptides
for this ligand–receptor interaction However, these
C-ter-minal fragments of orexin did not displace the
125I-labelled orexin B from the recombinant orexin 1
receptor stably expressed in Chinese hamster ovary cells To
examine the role of the shortened orexin A 23–33 in feeding,
its effects in mimicking or antagonizing the effects of orexin
A were studied in rats after administration via the lateral
hypothalamus In contrast with orexin A, which potently
induced feeding up to 4 h after administration, orexin A
23–33 neither induced feeding nor inhibited orexin A-induced feeding Modafinil (VigilÒ), which was shown earlier to activate orexin neurons, displayed binding neither
to the orexin receptor expressed on SK-N-MC cells nor to the recombinant orexin 1 receptor, which indicates that modafinil displays its antinarcoleptic action via another yet unknown mechanism PCR and subsequent sequencing revealed expression of the full-length orexin 1 receptor mRNA in SK-N-MC and NT-2 cells Interestingly, sequencing of several cDNA clones derived from RNA of both SK-N-MC and NT-2 cells differed from the published nucleotide sequence at position 1375 Amino acid prediction
of this AfiG change results in an isoleucinefivaline sub-stitution at the protein level, which may provide evidence for
an editing process
Keywords: food intake; hypocretin; ligand–receptor inter-action; obesity; orexin
Two novel neuropeptides, orexin A and B, were recently
discovered independently by two groups and identified as
potent stimulators of food intake after
intracerebroventric-ular administration [1–4] Further investigations revealed a
broad involvement of these peptides in the regulation of
many physiological and behavioural activities that are
associated with feeding behaviour [4–7], in the modulation
of neuroendocrine function and the sleep/wake cycle
[8–12] Both peptide amides derive from prepro-orexin, a
precursor protein produced in defined regions of the lateral
and perifornical hypothalamus, whose mRNA is
up-regu-lated upon fasting Orexin immunoreactive neurons are,
however, distributed widely in the brain, including regions
of the cerebral cortex, the medial groups of the thalamus,
the circumventricular organs, the limbic system and the
brain stem [13–15] A key role for orexins in narcolepsy has been described [10,16–18] It was shown recently that the anti-narcoleptic drug Modafinil (VigilÒ), the mechanism of action of which is unknown, might act through the orexin pathway [10]
Orexin A consists of 33 amino acids, is C-terminally amidated and contains two intramolecular disulfide bonds, that connect cysteine residues from positions 6–12 and 7–14, respectively Orexin B consists of 28 residues and shares 46% identity with orexin A, mainly at the C terminus The three-dimensional solution structure of orexin B was recently determined by two-dimensional NMR and shows two a helices, connected by a short linker sequence at position 20–23 [19] The structure of orexin A is conserved among human, rat, mouse and cow, whereas rodent orexin B contains two amino acid substitutions compared with the human sequence: proline instead of serine in position two and asparagine instead of serine in position 18 Xenopus laevis has orexins that differ slightly from the human sequence, but the C-terminal decapeptide of orexin A and B and the positions next to the disulfide bonds in orexin A remain conserved (Fig 1), which suggests some importance in biological activity of these peptide regions [20]
Orexin A and B are endogenous ligands of two closely related (64% amino acid identity [1]) heptahelical G-protein-coupled receptors, termed OX1and OX2 They induce an intracellular increase in free Ca2+concentration after activation of the receptors [1,21] Orexin A shows
Correspondence to A G Beck-Sickinger, Institute of Biochemistry,
University of Leipzig, Talstr 33, 04103 Leipzig, Germany.
Fax: +49 341 9736 998, Tel.:+ 49 341 9735 901,
E-mail: beck-sickinger@uni-leipzig.de
Abbreviations: OX 1 receptor, orexin 1 receptor; OX 2 receptor, orexin 2
receptor; NPY, neuropeptide Y; HOBt, N-hydroxybenzotriazole;
CHO, Chinese hamster ovary; IC 50 , 50% inhibitory concentration.
*Present address: Aventis Pharma Deutschland GmbH, DG
Thrombotic Diseases/Degenerative Joint Diseases, H811, D-65926
Frankfurt, Germany.
(Received 24 September 2001, revised 7 December 2001, accepted
12 December 2001)
Trang 2higher affinity to the OX1 receptor, whereas the binding
affinity of the two peptides to OX2receptor is in the same
order of magnitude [10]
Up to now, little is known about the structure–activity
relationship, except for the relevance of the C-terminal
segment of orexin A [22] Only recently, a subtype selective
nonpeptide antagonist was described in vitro [23] We
describe here the shortest orexin A and B analogues that
bind to OX-receptors We have also determined that the
orexin type 1 receptor is expressed by SK-N-MC cells, a
human neuroblastoma cell line, although with a
pharma-cological profile different from that of the recombinantly
expressed OX1receptor In addition, we describe an amino
acid position that differs in clones derived from RNA that
has been isolated from SK-N-MC cells
M A T E R I A L S A N D M E T H O D S
Materials
Na-Fmoc-protected amino acids were from Alexis
(La¨ufelfingen, Switzerland) The side-chain protecting
groups were tert-butyl for serine and threonine, and trityl
for asparagine and histidine The
4-(2¢,4¢-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy (Rink Amide) resin was from
Novabiochem (La¨ufelfingen) N-hydroxybenzotriazole
(HOBt), trifluoroacetic acid, thioanisole, p-thiocresol,
tri-methylsilylbromide, 1,2-ethanedithiol, piperidine,
tert-buta-nol, 1,1,1-trifluoroethanol and dimethylformamide were
from Fluka N,N¢-diisopropylcarbodiimide was from
Aldrich Dimethylformamide (pure) and diethylether were
from Scharlau (La Jota, Barcelona, Spain) Acetonitrile was
from Romil (Cambridge, England)
Dulbecco’s modified Eagle’s medium was from
BioWhit-taker; OPTI-MEM and Lipofectamine were from Gibco
BRL; fetal bovine serum was from BioWhittaker; Hepes
was from Fluka; geneticin was from Gibco BRL; Pefabloc
SC was from Merck; 125I-labelled Tyr-human orexin B
(specific activity 2130 CiÆmmol)1) was from Anawa (Zu¨rich,
Switzerland); 125I-labelled Tyr-human orexin A (specific
activity 2130 CiÆmmol)1was from NEN; orexin B was from
Bachem (Heidelberg, Germany)
Modafinil (VigilÒ) was from Laboratoire L Lafon,
Merckle, Blaubeuren (Germany), NT-2 cells were from
Stratagene
Peptide synthesis
The C-terminal undecapeptides of the orexins, orexin A
23–33 and orexin B 18–28, and the analogues of orexin B
and orexin A 23–33 were synthesized by automated
multiple solid-phase peptide synthesis on a peptide
synthe-sizer (Syro, MultiSynTech, Bochum, Germany) using Rink
Amide resin (30 mg, resin loading 0.6 mmolÆg)1) Amino
acids were attached by the Fmoc-strategy in a double coupling procedure, using a 10-fold excess of Fmoc-amino acid, HOBt and N,N¢-diisopropylcarbodiimide in dimethyl-formamide and a reaction time of 40 min per coupling Fmoc-deprotection was accomplished with 40% piperidine
in dimethylformamide for 3 min, 20% piperidine for 7 min and finally 40% piperidine for a further 5 min The orexin A fragment was cleaved from the resin with a mixture of trifluoroacetic acid/thioanisole/p-thiocresol (90 : 5 : 5, v/v), precipitated from ice-cold diethylether, collected by centrifugation and washed four times with diethylether The methionine-containing orexin B fragment was cleaved from the resin using a mixture of trifluoroacetic acid/thioanisol/ethanedithiol (90 : 7 : 3, v/v), precipitated and washed as described Partial oxidation of the methio-nine residue was reduced by dissolving the peptide (15 mg, 0.014 mmol) in 1 mL trifluoroacetic acid, followed by the addition of ethanedithiol (15.7 lL, 0.2 molÆL)1) and trimethylsilylbromide (13 lL, 0.1 molÆL)1) [24] The solu-tion was shaken for 40 min at room temperature and the peptide was precipitated and washed as described Purifica-tion of the peptide was achieved by preparative HPLC on a C18-column (Waters, 5 lm, 25· 300 mm) with a linear gradient of 10–30% A in B; A ¼ 0.08% trifluoroacetic acid in acetonitrile, B ¼ 0.1% trifluoroacetic acid in water) and a flow rate of 15 mLÆmin)1 The peptides were dissolved in tert-butanol/water (1 : 3) and lyophilized Analytical characterization of the peptides was achieved
by electrospray ionization MS (SSQ 710, Finnigan MAT, Bremen, Germany) and by analytical reversed-phase HPLC
on a LiChrospher RP18-column (5 lm, 3· 125 mm, Merck, Darmstadt, Germany) using linear gradients of 5–50% over 30 min (I), 10–60% over 30 min (II), 10–40% over 30 min (III) or 20–40% over 30 min (IV) Analytical data were as expected [orexin A, 23–33: molecular mass (m),
mexpected1036 Da; mfound, 1036.1 ± 0.5 Da; HPLC reten-tion time (I), 17.3 min; [G23] orexin A 23–33: mexpected
1022 Da; mfound1021.5 ± 0.6 Da; HPLC retention time (II), 11.4 min Orexin B 18–28: mexpected1070 Da; mfound 1069.9 ± 0.1 Da; HPLC retention time (III), 12.9 min [L28] orexin B: mexpected, 2881 Da; m, 2881.3 ± 0.4 Da; HPLC retention time (IV) 16.6 min
cDNA subcloning and nucleotide sequence determination
PCR was used to amplify the full-length orexin 1 receptor according to accession number AF041243 [1] Oligonucleo-tides from MWG Biotech (Ebersberg, Germany) were used
as primers: OX1-f, 5¢-GTAGAGCCTAGGATGCCCCT-3¢; OX1-r: 5¢-AGGAAGTGACTTATCCAGAGT-3¢ Total RNA from SK-N-MC cells and NT-2 cells were used as templates Isolation of total RNA was performed with an RNeasy Total RNA Kit (Qiagen) RT-PCR was
Fig 1 Sequence of (A) mature orexin A
peptides of human, bovine and rat origin and (B)
mature orexin-B peptides Deviations from the
human sequences are underlined.
U ¼ pyroglutamic acid.
Trang 3performed using the Superscript Preamplification System
(Gibco/BRL) After 3 min at 94°C, the reactions were
subjected to 35 cycles of: denaturation, 1 min at 94°C;
annealing, 2 min at 60°C; elongation 2 min at 72 °C in a
primus plus cycler (MWG Biotech) PCR products of the
expected size were cloned in pCR2.1TOPO using the TOPO
TA Cloning Kit from Invitrogen The sequence was
confirmed using the BigDye Terminator Cycle Sequencing
with an ABI 377 Sequencer using the M13 Forward ()20)
and Reverse primers (Invitrogen)
cDNA from orexin type 1 receptor was from Receptor
Biology (Beltsville, MD, USA), sequenced and OX1
R-cDNA was subcloned into pR-cDNA3.1/HisA vector from
Invitrogen
Cell culture
Transfection into Chinese hamster ovary (CHO) cells was
performed using the lipofectamine PLUS method according
to the manufacturer’s protocol (Gibco/BRL) using
expres-sion plasmids encoding the orexin 1 receptor
Binding assays with transfected cells
CHO cells were grown in nutrient mixture Ham’s F12
medium with 10% fetal bovine serum from BioWhittaker
(Boehringer Ingelheim Bioproducts Partnership, Verviers,
Belgium), nonessential amino acids, hygromycin B, 2 mM
L-glutamine and 1% geneticin (Gibco/BRL) at 37°C and
5% CO2until they were confluent in a 24-well plate The
medium was aspirated The cells were washed twice with
0.25 mL NaCl/Pi Incubation buffer [0.2 mL; 84.7 mM
NaCl, 30 mM KCl, 1.2 mM MgSO4Æ7H20, 11.2 mM
NaH2PO4, buffered with Hepes, 15 mM
(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, pH 7.5; from SERVA,
Heidelberg, Germany)] and at the day of the experiment
5.5 mMglucose, 0.1% BSA, 0.05 mgÆmL)1bacitracin was
added The total volume (0.25 mL) contained 100 pMfinal
concentration of 125I-labelled Tyr-human (h) orexin B or
125I-labelled Tyr-human orexin or125I-labelled h
nueropep-tide Y (NPY)-Tyr36 (specific activity: 2000 CiÆmmol)1;
Amersham) and increasing concentrations of the cold
ligand orexin B or increasing concentrations of test
compounds After 120 min of gentle shaking at room
temperature the supernatant was removed followed by two
washes with 0.25 mL NaCl/Pi Lysis buffer was added
(NaCl/Picontaining 2% Triton· 100) and after one wash
with 0.5 mL radioactivity was counted
Membrane preparation and binding assay
on SK-N-MC cells
For membrane preparation of SK-N-MC cells, the cells
were grown in MEM (MEM with Earl’s salt, 10% fetal
bovine serum, 1 mM sodium pyruvate, 1% nonessential
amino acids, 4 mM glutamine) Confluent cells were
removed with 0.02% EDTA/ NaCl/Piand resuspended in
10 mL incubation buffer (MEM/25 mMHepes containing
0.5% BSA, 50 lM phenylmethanesulfonyl fluoride, 0.1%
bacitracin, 3.75 mMCaCl2), then washed twice with 10 mL
NaCl/Pi After addition of 5 mL preparation buffer (5 mM
Hepes, 0.32M sucrose, 50 lM pefabloc, pH 7.0) the cells
were removed with a rubber policeman After centrifugation
at 4°C, 10 min, 48 200 g the supernatant was decanted and centrifuged at 4°C, 30 min, 48 200 g The pellet was resuspended in 15 mL NaCl/Pi The sample was recentri-fuged at 4°C, 50 min, 150 g and the pellet was resuspended
in incubation buffer (84.7 mMNaCl, 30 mMKCl, 1.2 mM MgSO4Æ7H2O, 11.2 mM NaH2PO4 buffered with Hepes) After counting, the cells were diluted to a final concentration
of 1.0· 106 cellsÆmL)1and homogenized using an Ultra-Thurrax After the addition of 5.5 mMglucose, 0.1% BSA and 50 lg bacitracin, 200 ll of this cell suspension was incubated for 2 h at room temperature with 100 pM
125I-labelled orexin B and increasing concentrations of orexin, orexin analogues or NPY (Neosyste`me, Strasbourg, France) in a total volume of 0.25 mL Unbound radio-activity was separated by filtration through Whatman GF/
C filters presoaked in 0.5% polyethylenimine The filters were washed three times with ice-cold 0.9% NaCl All tips and vials were siliconized
Competition binding experiments were analysed by a nonlinear least-squares fitting method with a one- or two-binding site model, respectively (RS/1 software package, BBN Research Systems, Cambridge, MA, USA) The maximum specific radioligand binding was set to 100% All data (n¼ 3) are expressed as mean ± SEM
Circular dichromism Conformational properties of the peptides were investigated
by CD spectroscopy using a JASCO model J720 spectro-polarimeter over 190–250 nm at 20°C in a N2atmosphere The peptides were dissolved in 20 mMNaCl/Piat neutral
pH containing 0%, 30%, 50% or 70% trifluoroethanol and
in pure trifluoroethanol, in a concentration range of 200–300 lM Each measurement was repeated three times using a thermostatable sample cell with a path of 0.02 cm and the following parameters: response time, 2 s; scan speed, 20 nmÆmin)1; sensitivity of 10 mdeg; step resolution, 0.2 nm; band width, 2 nm The CD spectrum of the solvent was subtracted from the CD spectra of the peptide solutions
to eliminate the interference from cell, solvent and optical equipment High frequency noise was reduced by means of
a low-path Fourier-transform filter The ellipticity was expressed as the mean-residue molar ellipticity [Q]R in degÆcm2Ædmol)1
Rodent model of food intake Adult male Chbb:Thom rats weighing between 300 and
340 g were individually housed and maintained on a
12 light: 12 h dark cycle beginning at 06.00 hours Tap water and standard laboratory chow were available throughout After 1 week of habituation to their new housing conditions, the animals were anaesthetized with sodium pentobarbital (60 mgÆkg)1, intraperitoneally) for the placement of stainless steel guide cannulae Cannulae (26 gauge) were placed 1 mm above the lateral hypothal-amus according to the stereotaxic coordinates: AP : 2.1,
L : 2.0, V : 7.2 (+1 mm injection tip: 8.2) Guide cannulae were maintained in place on the skull with small metal screws and dental acrylic cement Cannulae were closed with a stainless steel stylet when not in use Rats were allowed to recover for at least 1 week and were adapted to the injection procedure On the day of the experiments drugs
Trang 4were injected between 01.00 and 02.00 p.m Injection
cannulae (33 gauge) were inserted 1 mm beyond the tips
of the guide cannulae The injection cannulae were attached
by polyethylene tubing to a Hamilton microsyringe
mount-ed in an infusion pump Injection volume was 0.4 lL given
slowly over 40 s
Groups of six to eight rats received either saline (control),
1.0 nmolÆrat)1orexin A unilaterally, or 1 nmolÆrat)1orexin
A and 3 nmol rat)1 orexin A 23–33 into the lateral
hypothalamus and food intake was monitored for 4 h In
the second set of experiments 1.0 nmol orexin A 23–33 was
given with the injection of 1.0 nmol orexin A in order to
antagonize the effects of orexin A
R E S U L T S
The peptides were synthesized by automated multiple
peptide synthesis on a Rink Amide resin to directly obtain
the peptide amides after cleavage of the peptides from the
resin [25] In addition to the native sequences h-orexin A
and B, we used two C-terminal segments h-orexin A 23–33
and h-orexin B 18–28 Because of the different length of the
natural orexins, these two C-terminal segments are
homo-logous and correspond to the C-terminal undecapeptide of
orexin A and orexin B, respectively Nine amino acids are
identical, whereas orexin B contains a C-terminal
methio-nine in contrast with leucine in orexin A (Fig 1) This led to
the orexin B analogue [L28] orexin B to make sure that any
identified differences are not owing to the different
sequences The second variable position is residue 23
(orexin A, alanine)/18 (orexin B, glycine) To investigate
the role of this exchange we investigated [G23] orexin A
23–33 (Table 1)
The binding affinity of the peptides was tested on
SK-N-MC cells.125I-labelled orexin B binding was inhibited in a
dose-dependent fashion with a Kiof 118 ± 57 nM(Table 1,
Fig 2) and to a similar order of magnitude on NT-2 cells,
another human cell-line (data not shown) All curves
displayed a monophasic shape with slopes close to unity
125I-labelled orexin B could be displaced by the orexin A and
orexin B fragments in the range of human orexin B itself or
with slightly improved affinity (Table 1) Substitution of
orexin A at position 23 did not improve affinity significantly
Several attempts to detect specific125I-labelled orexin A
binding was unsuccessful with SK-N-MC cells whereas
recombinant CHO cells expressing the OX1 receptor revealed a 50% inhibitory concentration (IC50) of
10 ± 6 nMfor inhibition of125I-labelled orexin A binding
by orexin A The C-terminal fragments orexin A 23–33 and orexin B 18–28 do not displace125I-labelled orexin B from the recombinant receptor; neither does [G23] h-orexin A 23–33 displace125I-labelled orexin A The first selective orexin 1 receptor antagonist (SB-334867-A) has been described recently, with a pKi value of 7.17 nM [23,26] We tested a compound related to SB-334867, published earlier by G Chan et al [27], 1-(4-N,N-dimethylaminophenyl)-3-chinolin-4yl-urea), named EXBN8016BS This compound displayed an IC50 of
149 ± 3 nMfor the inhibition of125I-labelled orexin A at the recombinant OX1 receptor whereas it cannot inhibit the125I-labelled orexin B binding to both the recombinant
OX1 receptor or the orexin 1 receptor expressed on SK-N-MC cells Modafinil was shown earlier to activate orexin-responsive neurons Therefore, we examined whether Modafinil acts indirectly via inhibitory orexin autoreceptors Modafinil displayed no significant affinity for the orexin B binding site of SK-N-MC cells or of recombinantly expressed OX1 receptors Sensitivity of orexin B binding to NPY has been observed (Table 1) with an IC of 450 nM
Table 1 Binding affinity of h-orexin A and B, C-terminal orexin A and B fragments and reported antagonists on SK-N-MC cells and CHO cells stably transfected with the human OX 1 receptor (100 p M radioligand).
Ox1 receptor
125 I-labelled orexin B
IC 50 [n M ]
SK-N-MC cells
125 I-labelled orexin B
K i [n M ]
Ox1 receptor
125 I-labelled orexin A
IC 50 [n M ]
a 1-(4-N,N-dimethyl-aminophenyl)-3-chinolin-4yl-urea) [27].
Fig 2 Receptor binding studies with 125 I-labelled orexin B and orexin B (m) using SK-N-MC cells.
Trang 5The structure of the peptides was investigated by CD
spectroscopy in aqueous solutions at neutral pH,
containing increasing amounts of trifluoroethanol All
peptides adopted mainly random structure Fig 3 shows
the CD spectra of orexin A 23–33 dissolved in water (A),
50% trifluoroethanol in water (B), 70% trifluoroethanol
in water (C) and pure trifluoroethanol (D) in order to
see any stabilizing effects of the solvent All other
peptides showed comparable CD spectra (data not
shown) The negative band at 198 nm in aqueous
solution, an indication of randomly structured peptides,
was shifted to 202 nm in all trifluoroethanol-containing
samples The negative CD value of the water solution at
190 nm was raised to positive values in
trifluoroethanol-containing solutions These shifts indicate partial
forma-tion of an a helix in trifluoroethanol-containing samples
Analysis of the spectra by a secondary structure
estimation program (JASCO, J-700 for Windows) based
on the method of Yang et al [28] revealed a slightly
increasing amount of a helix with increasing amount of
trifluoroethanol, although the maximum amount of helix
was only 11% (Fig 3)
Food intake
Administration of 1 nmol orexin A into the third ventricle
of rats significantly increased food intake after 2 and 4 h
whereas a trend was seen after 6 and 8 h and no effect
was seen after 24 h One nanomole of orexin A also
significantly increased food intake after administration
into the lateral hypothalamus (Fig 4A) Administration
of orexin A 23–33 together with orexin A in order to
evaluate a potential antagonistic property of orexin A 23–
33 did not reveal any effect (Fig 4A) Orexin A 23–33 in
a dose range of 1 and 3 nmol per rat did not induce
feeding (Fig 4B)
Sequencing
To study orexin binding we searched for a suitable cellular system and screened neuronal cell lines of human origin (e.g SK-N-MC and NT-2) for orexin receptor binding sites
Analysis of the cDNA derived from total RNA revealed that these cell lines contain intronless orexin 1 receptor transcripts that seem to be partially edited in the codon for the isoleucine/valine site at position 1375 (amino acid 408) beyond transmembrane region 7 (Fig 5, Table 2) Fig 5 shows that all other amino acids are 100% identical to the published sequence [1]
Analysis of the human genomic DNA revealed that adenosine is present at this position, which leads to an isoleucine at this position in the protein (personal commu-nication, Receptor Biology Inc)
D I S C U S S I O N
The sequence of the C-terminal decapeptide of orexin A and B is conserved throughout all species examined Here
we show that the C-terminal orexin fragments, orexin A 23–33 and orexin B 18–28, bind to the orexin receptor expressed on SK-N-MC cells with an affinity in the same range or with four- to eightfold improved affinity compared
Fig 3 CD spectra and secondary structure according to the calculation
of Yang et al [28] of orexin A 23–33 Solvent: (A) water, pH 7.0;
(B) 50% trifluoroethanol in water; (C) 70% trifluoroethanol in water;
(D) pure trifluoroethanol.
Fig 4 Food intake studies Food intake after administration
of orexin A via the lateral hypothalamus and after administration of orexin A 23–33 and orexin A (A) Food intake after administration of orexin A 23–33 (B).
Trang 6with orexin B and orexin A, respectively On the contrary,
initial data with N-terminal fragments, e.g orexin B 1–10 or
orexin B 1–13, showed no significant affinity to this orexin
receptor (data not shown) This indicates an important
sequence motif in the C-terminus of the peptides, that might
play an essential role in binding of the peptides to this
receptor This is in accordance with the recently published
three-dimensional structure of orexin B, solved by
two-dimentional NMR It showed the peptide to consist of two
a-helices, connected by a short linker [19] The C-terminal
helix of the mature human orexin B extended from residue
22 to residue 28 Although these seven residues constitute
the major part of the orexin A and B fragments, a helical
structure, as postulated for the mature orexin B, could be
found neither in orexin A 23–33 nor in orexin B 18–28
Even dissolving the peptides in solutions containing high
amounts of trifluoroethanol, an a helix-inducing solvent,
resulted in peptides with maximal 11% a helix and still 89%
random structure This indicates that besides the
confor-mational properties, the amino acid side chains might also
play an important role in binding of the orexin fragments to the receptors, in particular the trifunctional residues aspar-agine, histidine and threonine and additionally for orexin B 18–28 serine and the C-terminal methionine The confor-mational influence of the C-terminal part of the orexins remains unclear The introduction of a helix inducers into C-terminal peptide fragments might increase binding affin-ity, because the native peptide contains a stable a helix at the C-terminus
Binding studies on SK-N-MC cells, a human neuroblas-toma cell line that is known to express NPY Y1receptors [29], revealed that this cell line also expresses receptors of the orexin family, which, after sequencing, turned out to correlate with the cloned OX1receptor Both neuropeptides, NPY and orexins, are involved in the regulation of food intake Sensitivity of orexin A binding to NPY has been described earlier by studying125I-labelled orexin A binding [30] Interestingly some affinity of NPY could be identified for orexin receptors as well As some cross-reactivity has been reported [31], and orexin-induced food intake seems to involve the NPY pathways [22], this might be one mode of regulation
Our results indicate the importance of the C-terminal part
of the orexins for binding interaction of the ligand and its receptor expressed on SK-N-MC cells The lack of affinity
of the fragments for the recombinant OX1receptor suggests
a different pharmacological profile although a sequence almost identical to the recombinant receptor is expressed in the neuroblastoma cell line This discrepancy might be explained by post-translational modifications, heterodimer-ization (reviewed in [33]) or different accessory proteins of the orexin 1 receptor expressed in SK-N-MC cells, similar
to those found for the CGRP receptor [34] possibly resulting
in different binding profiles The binding to an orexin 2 receptor is possible, but unlikely, because the dual expres-sion of both receptors with a different pharmacological profile should result in a biphasic inhibition curve This was not observed
The different IC50values of ligands, e.g EXBN 8016BS
or orexin A, to inhibit either 125I-labelled orexin A or
Fig 5 Sequence similarity of the orexin 1
receptor cloned both from SK-N-MC and NT-2
cells Position X means G or C at nucleotide
1375 coding for amino acid 408 which is either
translated into isoleucine [1] or valine
(Table 2).
Table 2 Position 1375 is nucleotide 1375 within the OX 1 receptor
cDNA which is either translated to isoleucine or valine.
NT-2-cells
SK-N-MC
Database
(Acc.no AF 041243) [1]
Trang 7125I-labelled orexin B binding at the recombinant OX1
receptor might be due to different binding epitopes of the
receptors as found for other agonist/antagonist systems of
neuropeptides, such as NPY or substance P [35,36] It was
hypothesized earlier [10] that modafinil may promote
wakefulness through orexin neurons Since, however,
mod-afinil has low affinity for orexin 1 receptors, this activation
might involve other orexin receptors or could have another
mechanism
The feeding effects with orexin A are at variance with the
strong and persistent feeding response observed by Sakurai
et al [1], but similar to those described by others in rats [3]
and mice [37] Orexin B showed no effects (data not shown)
which is in agreement with earlier reports [3,4,37] This is
why we chose the shortened orexin A fragment and not
orexin B 18–28 for feeding studies The lack of feeding
effects of orexin A 23–33 indicates that the orexin B binding
site expressed on SK-N-MC cells is not represented in the
lateral hypothalamus and therefore might not be involved in
the regulation of food intake
However, this study has revealed a single nucleotide
mismatch between corresponding cDNAs encoding orexin 1
receptors Human genomic DNA analysis (personal
com-munication, Receptor Biology) indicated that alternative
exons could be excluded as a potential source for this
nucleotide exchange Hence, editing of the RNA transcribed
from these genes best explains our observation which is
similar to the AfiG editing described in glutamate-gated
channels [38,39] and with the G protein-coupled
serotonin-2C receptor [40] Single nucleotide polymorphism cannot be
excluded at this point but the G was not found on a genomic
level Subtle kinds of regulation of G protein-coupled
receptors coupling to G proteins have been described earlier
[33] For example, transcripts encoding the 5-HT2C
recep-tor, a phospholipase C-coupled receprecep-tor, undergo RNA
editing events in which the genomically encoded adenosine
residues are converted to inosines by a double-stranded
RNA adenosine deaminase(s) Seven major 5-HT2C
recep-tor isoforms are predicted, encoded by 11 distinct RNA
species and differing in their second intracellular loops [40]
This post-transcriptional modification leads to a 10- to
15-fold reduction in efficacy of the coupling of 5-HT2Cto
the G protein
We conclude that the structural studies of C-terminal
orexin A and B fragments and their binding affinity to the
orexin receptors as presented in this work, provide another
step in characterization of the receptor binding mode of
orexin A and B and provide important information for
pharmacological and biochemical investigations The
observed A/G exchange in SK-N-MC and NT-2, two
human cell lines, might indicate an editing process which
will be further investigated
A C K N O W L E D G E M E N T S
The financial support of the Deutsche Forschungsgemeinschaft
(Be 1264-3/1) is kindly acknowledged We further acknowledge the
skilled technical expertise of E Liebhardt and S Schacherl-Schmid.
R E F E R E N C E S
1 Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli,
R.M., Tanaka, H., Williams, S.C., Richardson, J.A., Kozlowski,
G.P., Wilson, S., Arch., J.R.S., Buckingham, R.E., Haynes, A.C., Carr, S.A., Annan, R.S., McNulty, D.E., Liu, W.-S., Terrett, J.A., Elshourbagy, N.A., Bergsma, D.J & Yanagisawa, M (1998) Orexins and orexin receptors: a family of hypothalamic neuro-peptides and G protein-coupled receptors that regulate feeding behavior Cell 92, 573–585.
2 De Lecea, L., Kilduff, T.S., Peyron, C., Gao, X.-B., Foye, P.E., Danielson, P.E., Fukuhara, C., Battenberg, E.L.F., Gautvik, V.T., Bartlett,F.S., I.I.Frankel, W.N., Van den Pol, A.N., Bloom, F.E., Gautvik, K.M & Sutcliffe, J.G (1998) The hypocretins: hypo-thalamus-specific peptides with neuroexcitatory activity Proc Natl Acad Sci USA 95, 322–327.
3 Haynes, A.C., Jackson, B., Overend, P., Buckingham, R.E., Wilson, S., Tadayyon, M & Arch., J.R.S (1999) Effects of single and chronic intracerebroventricular administration of the orexins
on feeding in the rat Peptides 20, 1099–1105.
4 Ida, T., Nakahara, K., Katayama, T., Murakami, N & Nakazato, M (1999) Effect of lateral cerebroventricular injection
of the appetite-stimulating neuropeptide, orexin and neuropeptide
Y, on the various behavioral activities of rats Brain Res 821, 526–529.
5 Takahashi, N., Okumura, T., Yamada, H & Kohgo, Y (1999) Stimulation of gastric acid secretion by centrally administered orexin-A in conscious rats Biochem Biophys Res Commun 254, 623–627.
6 Griffond, B., Risold, P.Y., Jacquemard, C., Colard, C & Fellmann, D (1999) Insulin-induced hypoglycemia increases preprohypocretin (orexin) mRNA in the rat lateral hypothalamic area Neurosci Lett 262, 77–80.
7 Dyer, C.J., Touchette, K.J., Carroll, J.A., Allee, G.L & Matteri, R.L (1999) Cloning of porcine prepro-orexin cDNA and effects of an intramuscular injection of synthetic porcine orexin-B on food intake in young pigs Domestic Anim Endocrinol.
16, 145–148.
8 Van den Pol, A.N., Gao, X.B., Obrietan, K., Kilduff, T.S & Belousov, A.B (1998) Presynaptic and postsynaptic actions and modulation of neuroendocrine neurons by a new hypothalamic peptide, hypocretin/orexin J Neurosci 18, 7962–7971.
9 Date, Y., Mondal, M.S., Matsukura, S., Ueta, Y., Yamashita, H., Kaiya, H., Kangawa, K & Nakazato, M (2000) Distribution of orexin/hypocretin in the rat median eminence and pituitary Brain Res Mol Brain Res 76, 1–6.
10 Chemelli, R.M., Willie, J.T., Sinton, C.M., Elmquist, J.K., Scammell, T., Lee, C., Richardson, J.A., Williams, S.C., Xiong, Y., Kisanuki, Y., Fitch, T.E., Nakazato, M., Hammer, R.E., Saper, C.B & Yanagisawa, M (1999) Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation Cell 98, 437–451.
11 Singer, C.M & Lewy, A.J (1999) Does our DNA determine when
we sleep? Nat Med 5, 983.
12 Hagan, J.J., Leslie, R.A., Patel, S., Evans, M.L., Wattam, T.A., Holmes, S., Benham, C.D., Taylor, S.G., Routledge, C., Hemmati, P., Munton, R.P., Ashmeade, T.E., Shah, A.S., Hatcher, J.P., Hatcher, P.D., Jones, D.N.C., Smith, M.I., Piper, D.C., Hunter, A.J., Porter, R.A & Upton, N (1999) Orexin A activates locus coeruleus cell firing and increases arousal in the rat Proc Natl Acad Sci USA 96, 10911–10916.
13 Nambu, T., Sakurai, T., Mizukami, K., Hosoya, Y., Yanagisawa,
M & Goto, K (1999) Distribution of orexin neurons in the adult rat brain Brain Res 827, 243–260.
14 Dube, M.G., Kalra, S.P & Kalra, P.S (1999) Food intake elicited
by central administration of orexins/hypocretins: identification of hypothalamic sites of action Brain Res 842, 473–477.
15 Peyron, C., Tighe, D.K., van den Pol, A.N., de Lecea, L., Heller, H.C., Sutcliffe, J.G & Kilduff, T.S (1998) Neurons containing hypocretin (orexin) project to multiple neuronal systems.
J Neurosci 18, 9996–10015.
Trang 816 Nishino, S., Ripley, B., Overeem, S., Lammers, G.J & Mignot, E.
(2000) Hypocretin (orexin) deficiency in human narcolepsy.
Lancet 355, 39–40.
17 Lin, L., Faraco, J., Li, R., Kadotani, H., Rogers, W., Lin, X., Qiu,
X., de Jong, P.J., Nishino, S & Mignot, E (1999) the sleep
dis-order canine narcolepsy is caused by a mutation in the hypocretin
(orexin) receptor 2 gene Cell 98, 365–376.
18 Siegel, J.M (1999) Narcolepsy: a key role for hypocretins
(orexins) Cell 98, 409–412.
19 Lee, J.H., Bang, E., Chae, K.J., Kim, J.Y., Lee, D.W & Lee, W.
(1999) Solution structure of a new hypothalamic neuropeptide,
human hypocretin-2/orexin-B Eur J Biochem 266, 831–839.
20 Shibahara, M., Sakurai, T., Nambu, T., Takenouchi, T., Iwaasa,
H., Egashira, S.I., Ihara, M & Goto, K (1999) Structure, tissue
distribution, and pharmacological characterization of Xenopus
orexins Peptides 20, 1169–1176.
21 Smart, D., Jerman, J.C., Brough, S.J., Rushton, S.L., Murdoch,
P.R., Jewitt, F., Elshourbagy, N.A., Ellis, C.E., Middlemiss, D.N.
& Brown, F (1999) Characterization of recombinant human
orexin receptor pharmacology in a Chinese hamster ovary cell-line
using FLIPR Br J Pharmacol 128, 1–3.
22 Darker, J.G., Porter, R.A., Eggleston, D.S., Smart, D., Brough,
S.J., Sabido-David, C & Jerman, J.C (2001) Structure–activity
analysis of truncated orexin-A analogues at the orexin-1 receptor.
Bioorg Med Chem Lett 11, 737–740.
23 Smart, D., Sabido-David, C., Brough, S.J., Jewitt, F., Johns, A.,
Porter, R.A & Jerman, J.C (2001) SB-334867-A: the first
selective orexin-1 receptor antagonist Br J Pharmacol 132,
1179–1182.
24 Beck, W & Jung, G (1994) Convenient reduction of S-oxidesin
synthetic peptides, lipopeptides and peptide libraries Lett Pept.
Sci 1, 31–37.
25 So¨ll, R & Beck-Sickinger, A.G (2000) On the synthesis of orexin
A: a novel protocol to obtain peptides with two intramolecular
disulfide bonds J Pept Sci 6, 387.
26 Rodgers, R.J., Halford, J.C., Nunes de Souza, R.L., Canto de
Souza, A.L., Piper, D.C., Arch., J.R., Upton, N., Porter, R.A.,
Johns, A & Blundell, J.E (2001) SB-334867, a selective orexin-1
receptor antagonist, enhances behavioural satiety and blocks the
hyperphagic effect of orexin-A in rats Eur J Neurosci 13, 1444–
1452.
27 Chan, G., Johns, A., Jurewicz, A., Porter, R & Widdowson, K.
Phenyl urea and phenyl thiourea derivatives as HFGAN72
antagonists WO 99/09024.
28 Yang, J.T., Wu, C.S & Martinez, H.M., (1986) Calculation of
protein conformation from circular dichromism Methods
Enzy-mol 130, 208.
29 Beck-Sickinger, A.G., Wieland, H.A., Wittneben, H., Willim, K.D., Rudolf, K & Jung, G (1994) Complete L -alanine scan of neuropeptide Y reveals ligands binding to Y1 and Y2 recep-tors with distinguished conformations Eur J Biochem 225, 947–958.
30 Kane, J.K., Tanaka, H., Parker, S.L., Yanagisawa, M & Li, M.D (2000) Sensitivity of orexin-A binding to phospholipase C inhibi-tors, neuropeptide Y, and secretin Biochem Biophys Res Commun 271, 959–965.
31 Jaszberenyi, M., Bujdoso, E & Telegdy, G (2001) The role of neuropeptide Y in orexin-induced hypothalamic-pituitary-adrenal activation J Neuroendocrinol 13, 438–441.
32 Yamanaka, A., Kunii, K., Nambu, T., Tsujino, N., Sakai, A., Matsuzaki, I., Miwa, Y., Goto, K & Sakurai, T (2000) Orexin-induced food intake involves neuropeptide Y pathway Brain Res.
859, 404–409.
33 Bockaert, J & Pin, J.P (1999) Molecular tinkering of G protein-coupled receptors: an evolutionary process EMBO J 18, 1723– 1729.
34 McLatchie, L., Fraser, N., Main, M., Wise, A., Brown, J., Thompson, N., Solari, R., Lee, M & Foord, S (1998) RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor Nature 393, 333–339.
35 Sautel, M., Rudolf, K., Wittneben, H., Herzog, H., Martinez, R., Munoz, M., Eberlein, W., Engel, W., Walker, Ph & Beck-Sick-inger, A.G (1996) Neuropeptide Y and the non peptide antagonist BIBP 3226 share a large overlapping binding site at the human Y 1
receptor Mol Pharmacol 50, 285–292.
36 Rosenkilde, M.M., Cahir, M., Gether, U., Hjorth, S.A & Schwartz, T.W (1994) Mutations along transmembrane segment
II of the NK-1 receptor affect substance P competition with non-peptide antagonists but not substance P binding J Biol Chem 269, 28160–28164.
37 Lubkin, M & Stricker-Krongrad, A (1998) Independent feeding and metabolic actions of Orexins in mice Biochem Biophys Res Commun 253, 241–245.
38 Sommer, B., Ko¨hler, M., Sprengel, R & Seeburg, P.H (1991) RNA editing in brain controls a determinant of ion flow in glutamate gated channels Cell 67, 11–19.
39 Higushi, M., Single, F., Ko¨hler, M., Sommer, B., Sprengel, R & Seeburg, P.H (1993) RNA editing of AMPA receptor subunit GluR-B: a base-paired intron-exon structure determines position and efficiency Cell 75, 1361–1370.
40 Burns, C.M., Chu, H., Rueter, S.M., Hutchinson, L.K., Canton, H., Sanders-Bush, E & Emeson, R.B (1997) Regulation of serotonin-2C receptor G-protein coupling by RNA editing Nature 387, 303–308.