Western blotting Proteins obtained from both crude nuclear extract and solubilized material were loaded and separated by 12.5% SDS/PAGE, and Western blot analysis was performed according
Trang 1An unexpected relationship
Manuel Macı´as1, Germaine Escames1, Josefa Leon1, Ana Coto3, Younes Sbihi2, Antonio Osuna2
and Darı´o Acun˜a-Castroviejo1
1
Departamento de Fisiologı´a and2Instituto de Biotecnologı´a, Universidad de Granada, Spain;3Departamento de Morfologı´a
y Biologı´a Celular, Facultad de Medicina, Universidad de Oviedo, Spain
Increasing evidence suggests that melatonin can exert some
effect at nuclear level Previous experiments using binding
techniques clearly showed the existence of specific melatonin
binding sites in cell nucleus of rat liver To further identify
these sites, nuclear extracts from rat hepatocytes were treated
with different percentages of ammonium sulfate and purified
by affinity chromatography Subsequent ligand blot analysis
shows the presence of two polypeptides of 60 and
74 kDa that bind specifically to melatonin N-Terminal
sequence analysis showed that the 60 kDa protein shares a
high homology with rat calreticulin, whereas the 74 kDa
protein shows no homology with any known protein The
binding of melatonin to calreticulin was further
charac-terized incubating 2-[125I]melatonin with recombinant
calreticulin Binding kinetics show a Kd¼ 1.08 ± 0.2 nM and Bmax¼ 290 ± 34 fmolÆmg protein)1, compatible with other binding sites of melatonin in the cell The presence of calreticulin was further identified by Western blot analysis, and the lack of endoplasmic reticulum contamination in our material was assessed by Western blot and immunostaining with anti-calnexin Ig The results suggest that calreticulin may represent a new class of high-affinity melatonin binding sites involved in some functions of the indoleamine including genomic regulation
Keywords: affinity chromatography; calreticulin; melatonin; nuclear receptor purification; receptor binding
Melatonin is a highly preserved molecule throughout
phylogeny It appears in very ancient unicellular organisms
[1], remaining unchanged in multicellular species including
humans [2] In mammals, the circadian rhythm of melatonin
is produced through a photoperiodic-dependent synthesis
by the pineal gland [3] In turn, melatonin translates
photoperiodic information from clock and calendar
mes-sages, acting as an endogenous synchronizer of several
endocrine and nonendocrine rhythms [3] This indoleamine
is also produced by a variety of other tissues [4] Melatonin
exerts important regulatory influences on reproduction [5],
and on neuroendocrine [6] and immune systems [7]
Moreover, it also controls cellular proliferation through
regulatory effects on cell cycle kinetics [8], and prevents
apoptosis in several tissues [9] Recent studies have also
focused on the antioxidant and free radical scavenging
properties of melatonin [10–13]
Except for the antioxidant, nonreceptor-mediated effects
of melatonin, the actions of the indoleamine suggest the existence of specific receptors in the cell Three related, but distinct high affinity Gi-protein-coupled melatonin receptor subtypes have been cloned [14–17] Membrane receptors for melatonin are now classified as mt1, MT2 and MT3 In addition, biochemical and immunocytochemical studies in different mammalian tissues have shown the presence and accumulation of melatonin in the cell nuclei [18] This nuclear localization of melatonin can be related to its described genomic effects including the regulation of the mRNA levels for antioxidant enzymes and the inducible isoform of nitric oxide synthase (iNOS) [19,20] So far, no responding gene could be directly linked to the activation of membrane receptors by melatonin However, a synergistic effect of S 20098 and CGP 52608, two selective agonists of the membrane and nuclear melatonin receptors, respect-ively, on interleukin (IL)-6 production by human mononu-clear cells has been shown [21] Thus, to explain the numononu-clear actions of the indoleamine it is reasonable to assume the existence of a receptor in the nucleus of the cell
Previous studies with [3H]melatonin showed the existence
of specific nuclear binding sites for melatonin [22] Using 2-[125I]melatonin, the nuclear receptors for melatonin were fully biochemical and pharmacologically characterized [23,24] The identification of melatonin as a ligand for the ROR receptors [25,26] allowed its classification as a nuclear effector This viewpoint was further supported when it was found that the nuclear receptor for melatonin represses 5-lipoxygenase gene expression in human B lymphocytes [27] Thus, it is reasonable to assume that nuclear melatonin
Correspondence to D Acun˜a-Castroviejo, Departamento de
Fisiologı´a, Avenida de Madrid 11, E-18012 Granada, Spain.
Fax: + 34 958 246295, Tel.: + 34 958 246631,
E-mail: dacuna@ugr.es
Abbreviations: ER, endoplasmic reticulum; ERa, estrogen receptor
alpha; IL, interleukin; iNOS, inducible isoform of nitric oxide
synthase; NAS, N-acetylserotonin; PAP, peroxidase–
antiperoxidase; 4-P-PDOT, 4-phenyl 2-propionamidotetraline;
GST, glutathione S-transferase.
(Received 4 September 2002, revised 19 November 2002,
accepted 16 December 2002)
Trang 2signaling is a basic mechanism for the various control
functions of the indoleamine
The present work describes the purification and
charac-terization of two proteins from nuclei extracts of rat
hepatocytes that may represent a new class of melatonin
receptors These polypeptides, with molecular masses of 74
and 60 kDa, were purified by ammonium sulfate
precipi-tation and affinity chromatography; characterized by SDS/
PAGE and Western blotting, and identified by their
N-terminal amino acid sequence The search for sequence
similarities in protein databanks showed that the 60 kDa
protein is highly homologous to calreticulin, whereas the
74 kDa protein is a novel, unidentified protein
Materials and methods
Materials
All reagents were of the highest purity available Antibody
against melatonin (G/S/7048483) was obtained from
Stock-grand Ltd (UK) Tris, sucrose, deoxyribonuclease (DNase),
ribonuclease (RNase), ammonium sulfate, EDTA/Na2,
phenylmethylsufonyl fluoride, leupeptin, pepstatin A,
Triton X-100, Tween 20, melatonin
(N-acetyl-5-methoxy-tryptamine), 6-hydroxymelatonin, N-acetylserotonin (NAS),
goat anti-rabbit immunoglobulin,
peroxidase–antiperoxi-dase complex, 3,3¢-diaminobenzidine tetrahydrochloride,
and all other chemicals and dyes were purchased from
Sigma-Aldrich Quı´mica (Madrid, Spain) 4-P-PDOT
(4-phenyl 2-propionamidotetraline) and luzindole
(2-benzyl-N-acetyptryptoamine) were obtained from Tocris Cookson
Ltd (Bristol, UK)
Isolation of liver nuclei and nuclear protein
fractionation
Nuclei were isolated from rat liver by the procedure described
elsewhere [28], with some modifications [24] Briefly, rat livers
(0.9–1.2 g) were individually homogenized in 3 mL of buffer
A (10 mMTris/HCl, 0.3M sucrose, pH 7.4) with a glass/
Teflon homogenizer (10 strokes) and layered over 3 mL of
buffer A containing 0.4Msucrose The samples were then
centrifuged at 2500 g for 10 min The pellet was gently
resuspended without vortexing in 1 mL of buffer B (50 mM
Tris/HCl, pH 7.4) and centrifuged again All procedures
were carried out at 4C The pellet containing pure, intact
nuclei was checked by electron microscopy for the quality of
the isolated nuclei (data not shown) [47]
The purified nuclei were resuspended in 1 mL of buffer B
and disrupted with a Polytron homogenizer (7 s, set point 9)
to obtain a crude nuclear extract The crude nuclear extract
was further purified by resuspension in buffer B containing
protease inhibitors (5 mM EDTA-Na2, 0.1 mM
phenyl-methylsufonyl fluoride, 20 lMleupeptin and 2 lMpepstatin
A) and 0.1% Triton X-100 The homogenate was incubated
with DNase and RNase (25 lgÆmL)1), for 1 h at 37C and
centrifuged to 48 000 g for 20 min at 4C The proteins in
the supernatant (Triton X-100 soluble fraction) were
preci-pitated with different concentrations of ammonium sulfate
solutions (0–25, 25–45, and 45–65%) [29] After gentle
stirring in an ice bath for 30 min, precipitate was collected by
centrifugation at 48 000 g for 30 min and resuspended in
buffer B The resuspended sample was desalted in a Sephadex G-25 gel filtration column (Amersham Pharmacia Biotech Europe GmbH, Barcelona, Spain) pre-equilibrated with buffer B containing 50% glycerol, and stored at) 20 C Affinity chromatography by melatonin–agarose The hydroxyl group of 6-hydroxymelatonin was coupled to the epoxide group of epoxy-activated Sepharose 6B (Amer-sham Pharmacia Biotech Europe GmbH) [30] to yield a resin, designated melatonin–agarose (Fig 1) Briefly, epoxy-activated Sepharose 6B was swelled and washed extensively with deionized water, and 6-hydroxymelatonin was dis-solved in freshly prepared 50% dioxane/50% 0.1Msodium phosphate buffer, pH 8.7 (at higher pH values, 6-hydroxy-melatonin was not stable) The 6-hydroxy6-hydroxy-melatonin solu-tion was then mixed with the activated resin and sealed under nitrogen The mixture was agitated in darkness for 48–72 h at 32C Ligand excess was eliminated washing the gel with 50% dioxane, followed by bicarbonate (0.1M,
pH 8.0) and acetate (0.1M, pH 4.0) buffers Unreacted epoxide groups were then blocked by incubating the resin with 1M ethanolamine for 16 h at 30C The resin was extensively washed with deionized water, and then with bicarbonate and acetate buffers containing 500 mMNaCl and 0.05% digitonin The resin was resuspended in an equal volume of 0.05% digitonin and stored at 4C
Crude nuclear extracts and solubilized material were separately incubated with melatonin–agarose (15 : 1 v/v) for 16–24 h at 4C under gentle agitation (100 r.p.m on an orbital shaker) The resin was then pelleted (1500 g) and washed with 10 volumes of 0.05% digitonin until protein was
no longer detected in the final washed solution The resin was specifically eluted by incubating it with melatonin (10 lM; one volume) for 6 h to 4C with moderate agitation To remove excess of ligand, the eluate was put over Sephadex G-25 columns pre-equilibrated with 0.05% digitonin The final eluate was lyophilized and stored to )20 C until electrophoresis analysis Protein content in each step of purification was measured using a Bio-Rad protein assay reagent, with bovine serum albumin as protein standard [31] SDS/PAGE and ligand blotting
Samples obtained from affinity column were electropho-resed on 12.5% SDS/PAGE [32] using the PhastSystem (Amersham Pharmacia Biotech GmbH Europe) The gels for protein profiles were stained with silver according to
Fig 1 Schematic representation of the synthesis of melatonin–agarose resin The hydroxyl group of 6-hydroxymelatonin was coupled to the epoxide group of epoxy-activated Sepharose 6B to yield melatonin– agarose.
Trang 3Heukeshoven and Dernick [33] Proteins separated by SDS/
PAGE were transferred to nitrocellulose using the
Phar-macia Semi-dry Transfer kit [34] Briefly, the nitrocellulose
strips were incubated for 1 h at room temperature in
blocking buffer [0.4% gelatin in PBST(150 mM
phosphate-buffered saline, pH 7.4, containing 0.1% (v/v) Tween 20)],
followed by incubation with PBSTbuffer containing 10 lM
melatonin for 1 h at 4C Ligand blot analysis was carried
out using 1 : 800 dilution in PBSTbuffer of a specific
polyclonal antibody against melatonin (G/S/704–8483;
Stockgrand Ltd, Guildford, UK) for 2 h at room
tempera-ture After washing three times in PBST, the nitrocellulose
membranes were incubated for 1 h with sheep peroxidase
conjugated secondary antibody (1 : 800) in PBSTand then
washed again as above The blots were finally developed
with 3,3¢-diaminobenzidine Gels and blots were digitized
and processed by QuantiScan software (Biosoft, UK) and
the molecular masses of the polypeptides were calculated
according to their Rfvalues
Western blotting
Proteins obtained from both crude nuclear extract and
solubilized material were loaded and separated by 12.5%
SDS/PAGE, and Western blot analysis was performed
according to the procedure of Towbin et al [34] Briefly,
separated proteins were transferred onto polyvinylidene
difluoride membranes and blocked for 1 h at room
temperature in 0.4% gelatin in PBST Then, the gels were
incubated for 1 h at room temperature with rabbit
polyclonal antisera to calnexin and calreticulin
(Sigma-Aldrich, Spain) at 1 : 1000 in PBST Blots were washed
three times in PBST, exposed to horseradish
peroxidase-coupled antirabbit immunoglobulin, and detected by ECL
according to the manufacturer’s protocol (Amersham
Pharmacia Biotech, Spain)
Anti-calnexin immunohistochemistry
Isolated nuclei and microsomes were used for the
immu-nohistochemical localization of calnexin in endoplasmic
reticulum (ER) The homogenates were fixed by immersion
in Formaline’s fixative (4%) After dehydration in graded
alcohol, the homogenates were embedded in paraffin and
cut into 10-lm sections The sections were mounted on
gelatin-coated slides and processed by the peroxidase–
antiperoxidase (PAP) technique After three 10-min rinses
in phosphate buffer, endogenous peroxidase within the
homogenates was blocked by a solution of 0.3% hydrogen
peroxidase in NaCl/Piat room temperature for 30 min This
step was followed by three washes in NaCl/Piand
incuba-tion for 30 min in a soluincuba-tion of 1 : 30 goat serum in NaCl/
Pi Sections were treated with rabbit polyclonal antisera to
calnexin (Stressgen Biotechnologies) in serial dilutions from
1 : 200 to 1 : 5000 in NaCl/Piand incubated for 24 h at
room temperature in a humid atmosphere The slides were
then rinsed in NaCl/Piand incubated with goat antirabbit
immunoglobulin diluted 1 : 100 in NaCl/Pifor 1 h and then
with PAP complex diluted 1 : 100 for 1 h The sites of
peroxidase attachment were demonstrated by incubation in
0.005% 3,3¢-diaminobenzidine tetrahydrochloride solution
in Tris/HCl buffer (50 m , pH 7.6) containing 0.025%
hydrogen peroxide Finally the sections were rinsed in water, dehydrated and coverslipped To ensure method specificity, the usual controls were performed
N-Terminal sequence analysis Proteins separated by SDS/PAGE were transferred to polyvinylidene difluoride and stained with Coomassie blue The polypeptides of 60 and 74 kDa were excised and the first 15 N-terminal amino acid residues of each protein were sequenced by the Protein/Peptide Micro Analytical Labor-atory (California Institute of Technology, USA) Protein sequences comparisons were carried out using the FASTA program [35] Swiss-Prot databanks were accessed though the GeneBank online service
Expression and purification of fusion protein Recombinant fusion protein glutathione S-transferase– calreticulin (GST–calreticulin) or GST were expressed in Escherichia coli strain BL21 (DE3)pysL (Stratagene, La Jolla, CA, USA) using a plasmid encoding rabbit calreti-culin provided by M Michalak (Alberta University, Edmonton, Canada) [36] Rabbit and rat calreticulin share 92.57% homology Cells were grown to late log phase and induced to express the fusion proteins by addition of 0.25 mMisopropyl-1-thio-D-galactopyranoside for 4 h The cells were harvested and lysed in lysis buffer (50 mMTris,
pH 7.8, 0.4MNaCl, 10% glycerol, 0.5 mMEDTA, complete protease inhibitor, 0.1% Nonidet P-40) containing 1% Triton X-100 and 350 lgÆmL)1lysozyme Soluble proteins were separated from the inclusion bodies and bacterial debris by centrifugation at 10 000 g for 20 min at 4C The recombinant proteins were purified from the supernatant
by glutathione-Sepharose (Amersham Pharmacia Biotech Europe GmbH) and extensively washed with NaCl/Pi Matrix bound protein was used for binding assay 2-[125I]Melatonin binding assay
An aliquot of 20 lL of GST-calreticulin or GST proteins were mixed with 50 mMTris-HCl, pH 7.4 (6.96 mMCaCl2,
100 lMdithiothreitol) in a total volume of 100 lL, yielding a final protein concentration of 200 lgÆmL)1 The mixture was incubated at 37C for 2 h in the presence of 100 pM radiolabeled ligand (2-[125I]melatonin, 81.4 TbqÆmmol)1) and the incubation was stopped adding 100 lL of cold 100% trichloroacetic acid followed by centrifugation at
45 000 g for 15 min at 4C The supernatant was discarded
by aspiration, and the radioactivity on the pellets was determined in a gamma-counter Nonspecific binding, cal-culated in the presence of 10 lMmelatonin, was 12–16% of the total binding Kinetic parameters (KDand Bmax) and
IC50values were measured from the displacement curves with the LIGAND-PC program (KELL software, Biosoft, UK) Protein content was determined as described [31] Statistics
Data are expressed as means ± SEM Comparisons among groups were made by a one-way analysis of variance ( ) followed by Student’s t-test
Trang 4Purification by affinity chromatography
The results obtained after purification of the melatonin
receptor present in rat liver nuclei by affinity
chromato-graphy are shown in Table 1 Affinity chromatochromato-graphy of
crude nuclear extract (6000–8000 g) gave 0.14% of protein
(8.4–11.2 lg) The yield of the melatonin–agarose step
significantly increased when solubilized material was used as
source of melatonin receptor A higher percentage of
purified protein was obtained from the 45 to 65%
ammo-nium sulfate fraction This fraction yields 0.45% of protein
(2.7–3.6 lg protein) after its purification by melatonin–
agarose
Electrophoresis analysis
Aliquots of both crude nuclear extract and solubilized
material purified by affinity chromatography were analyzed
by SDS/PAGE gel electrophoresis followed by silver
staining (Fig 2) Crude nuclear extract shows a wide stain
ranging from 94 to 14 kDa (lanes 1 and 2) The protein
pattern was similar in samples either untreated (lane 1) or
treated with b-mercaptoethanol (lane 2) The number of
protein bands decreased with ammonium sulfate
treat-ment in solubilized material, and only two bands were
clearly identified in 45–65% ammonium sulfate samples
(lane 5) These bands, corresponding to polypeptides of 60
and 74 kDa, were also present in 25–45% (lane 4) and
0–25% (lane 3) ammonium sulfate fractions and in crude
nuclear extract samples (lanes 1 and 2) An aliquot of
molecular mass marker solution was applied as reference
(Mrlane)
N-Terminal sequence of the p74 and p60 proteins
To analyze the primary structure of 74 and 60 kDa
polypeptides, protein microsequencing was carried out In
order to transfer a sequenceable quantity of protein onto the
polyvinylidene difluoride, several nanomoles of protein were
loaded onto the gel prior to electrophoresis Electroblotted
polypeptides were located by staining of the polyvinylidene
difluoride with Coomassie blue The sequences obtained
were subjected to similarity searches in the database
network As no sequence identity was found for the
74 kDa protein (SFLEEDRNDQPVEI) after comparing
with known protein sequences, this protein is suggested to
be a novel protein Searching the sequence database of the National Center of Biotechnology Information (NCBI), the protein–protein blast program showed no similarity of the 74 kDa protein with any other known protein to date Besides, there is no information in a GeneBank that may help to identify this protein Interestingly, the N-terminal sequence of the first 12 residues of the 60 kDa protein (DPAIYFKEQFLDGFA) was 100% identity to that of rat calreticulin
Western blotting and anticalnexin immunohistochemistry
To identify the presence of calreticulin and to discard a possible contamination of our preparation with ER, Western blot analyses were performed using the antibodies anticalreticulin and anticalnexin (Fig 3) Lanes 1 and 5 correspond to crude nuclear extract and lanes 2–4 corres-pond to solubilized material treated with 0–25%, 25–45% and 45–65% ammonium sulfate, respectively Lanes 1–4 were incubated in the presence of calreticulin antibody and lane 5 with calnexin antibody A positive control for calnexin antibody is shown in lane 6, that correspond to the microsome sample Only one band corresponding to the polypeptides of 60 kDa was recognized in the presence of anticalreticulin (lanes 1–4), and this band is enriched by 25–65% ammonium sulfate precipitation A lack of immu-noreactive band is apparent in lane 5 The results confirm the identity of the 60 kDa protein as calreticulin, and exclude a contamination from ER
To further assess whether calreticulin was not being copurified from ER during the purification procedure, anticalnexin immunohistochemistry was carried out in preparations from nuclei homogenate A fraction contain-ing ER was also used for control purposes The results show
a lack of immunoreactivity in the nuclei homogenates, whereas positive immunoreactivity for anticalnexin was found in homogenates from microsomes (data not shown) Ligand blotting
Figure 4 demonstrates that the 74 and 60 kDa proteins specifically bind melatonin as determined by ligand blotting using anti-melatonin Ig Lane 3 corresponds to crude nuclear material and lanes 4–6 correspond to solubilized material treated with 0–25, 25–45 and 45–65% ammonium sulfate, respectively Lanes 3, 4, 5 and 6 were incubated with
10 lM melatonin Only the bands corresponding to the
Table 1 Purification of the melatonin receptor Starting material refers to the amount of protein present in samples of crude nuclear extract and in samples of solubilized material treated with different concentrations of ammonium sulfate Affinity chromatography data shows the amount of protein obtained after incubation of these samples with melatonin agarose The percentage of purified proteins in relation to the protein content in the starting material is shown in brackets.
Sample
Starting material (lg protein)
Affinity chromatography (lg protein)
Solubilized material (Triton X-100)
0–25% ammonium sulfate precipitate 900–1200 2.2–2.9 (0.24%)
25 ) 45% ammonium sulfate precipitate 800–900 2.7–3.1 (0.34%) 45–65% ammonium sulfate precipitate 600–800 2.7–3.6 (0.45%)
Trang 5polypeptides of 74 and 60 kDa were recognized in the
presence of melatonin, thus suggesting that these proteins
bind melatonin selectively Lanes corresponding to crude
nuclear extract or solubilized material incubated with
melatonin in the absence of anti-melatonin Ig (lane 1) or
incubated with anti-melatonin Ig in the absence of ligand
(lane 2) served as controls Standards stained with
Coo-massie blue R were included (Mrlane) These molecules do
not correspond to proteins such as histones which are
associated with DNA, as was further assessed by amino acid
sequencing, because they were not recognized by antibodies
against histones (data not shown)
Binding experiments
To further assess the characteristics of melatonin–calreti-culin binding, a series of competitive experiments were carried out Figure 5 (left) shows a typical displacement curve for 2-[125I]melatonin performed with bacterially produced GST-calreticulin fusion proteins The IC50value was 0.97 ± 0.3 nM Scatchard transformation was carried out from competition experiments after recalculating the specific activities (Fig 5, left inset) Kinetic analysis of these data yielded a Kdof 1.08 ± 0.2 nMand a Bmaxof 290 ± 34 fmolÆmg protein)1 Specific binding was undetectable in the absence of Ca2+ Binding experiments in the presence of different doses (1 nMto 10 mM) of NAS, 4-P-PDOTand luzindole showed no competition with the radioligand Moreover, the binding was specific for calreticulin, as no specific binding to bacterially produced GSTprotein was detected
Discussion
The objective of this work was to purify the nuclear melatonin receptor elsewhere characterized in liver nuclei [23,24], by classical protein purification approaches From this work, two polypeptides of 74 and 60 kDa that specifically bind to melatonin were obtained Luzindole and 4-P-PDOT, two selective antagonists for the mt1/MT2 subtypes of the melatonin membrane receptors [37] do not compete with 2-[125I]melatonin binding to calreticulin, the
60 kDa protein These results, together with the sequencing
Fig 4 Ligandblotting (SDS/PAGE) of nuclear extracts purifiedby affinity chromatography Crude nuclear extract (lane 3) and solubilized material pretreated with 0–25, 25–45 and 45–65% (lanes 4, 5 and 6, respectively) ammonium sulfate The blots were preincubated with
10 l M melatonin (lane 3, 4, 5 and 6) as described in Material and methods Lane 1 and 2 served as controls The crude nuclear extract was incubated with 10 l M melatonin in absence of anti-melatonin Ig (lane 1), or incubated with the primary antibody in absence of ligand (lane 2) Molecular mass markers stained with Coomassie blue R are indicated in the M r lane.
Fig 2 Silver-stainedSDS/PAGE gels (12.5% homogenous media) of
purifiedmaterial by affinity chromatography The gels show the
dena-tured polypeptide composition of crude nuclear extract either
untreated (lane 1) or treated with 1-mercaptoethanol (lane 2), and
solubilized material treated with ammonium sulfate at 0–25% (lane 3),
25–45% (lane 4) and 45–65% (lane 5) Molecular mass markers are
indicated in the M r lane.
Fig 3 Western blotting of crude nuclear extract (lanes 1 and 5) and
solubilizedmaterial pretreatedwith 0–25, 25–45 and45–65% (lanes 2, 3
and4, respectively) ammonium sulfate The blots were incubated with
anti-calreticulin (lanes 1–4) and anti-calnexin Igs (lane 5) as described
in Material and methods Lane 6 corresponds to a positive control for
calnexin antibody in microsomes.
Trang 6data, suggest that the proteins purified in this study
represent a new class of specific binding sites for melatonin
The proteins present in the crude nuclear extract,
precipitated with ammonium sulfate, allowed us to increase
the efficiency of the receptor purification by decreasing the
number of proteins present in the sample We found that
maximal calreticulin activity was obtained in samples
precipitated with 45–65% ammonium sulfate, i.e in the
similar precipitating fraction as previously described [38]
However, the fraction obtained with 25–45% ammonium
sulfate precipitation also has high content of calreticulin and
so, the largest yield of melatonin receptors (0.79%) was
obtained after affinity chromatography of samples
precipi-tated with 25–65% ammonium sulfate In addition, the
number of proteins separated by SDS/PAGE and stained
with silver nitrate was significantly reduced after ammonium
sulfate treatment The results suggest that these sample
pretreatments before affinity chromatography not only
increased the percentage of receptor obtained but also
removed a large number of proteins that might interfere
with the purification procedure itself
The development of the melatonin–agarose resin allowed
us to improve the purification procedure of the melatonin
nuclear receptor Criteria used for the validation of other
affinity resins [39] strongly suggest that melatonin–agarose
interacts with solubilized receptors in a specific manner
Purification achieved with melatonin–agarose is similar to
that achieved with affinity resins developed for the
purifi-cation of other receptors [40] The purified proteins obtained
after affinity chromatography show pharmacological
pro-perties compatible with a receptor of melatonin These data
support the utility of the melatonin–agarose resin to purify
the melatonin nuclear receptor and suggest that large-scale
purification may be now feasible Electrophoresis and
Western blot analysis of the samples purified by affinity
chromatography revealed the presence of two proteins
corresponding to molecular weights of 74 and 60 kDa
The N-terminal amino acid sequence of the purified
proteins was searched in protein databases for homology
identity Regarding the 74 kDa protein, no proteins with a
similar sequence were found, suggesting that this
polypep-tide is a novel, formerly unknown protein It was, however,
an unexpected finding that the N-terminal amino acid sequence of the 60 kDa protein showed considerable sequence homology with rat calreticulin It is interesting that calreticulin, a highly acidic protein, moves at about 60–65 kDa on SDS/PAGE [41], although the deduced molecular mass from the amino acids is 46 kDa A 60-kDa polypeptide obtained after SDS/PAGE of a 55–70% ammonium sulfate precipitate of the HeLa cell cytosol was also identified as calreticulin by mass spectrometry [38] Thus, the similarity of our purification methodology compared with that used by these authors, the molecular mass of the polypeptide obtained by SDS/PAGE, and the sequence analysis results, strongly suggest that the 60 kDa protein purified by us is calreticulin
The melatonin binding to calreticulin is highly specific and displays nanomolar affinity The specificity of this biding is also supported because NAS, the metabolic precursor of melatonin with certain degree of affinity for other melatonin binding sites [24], does not interfere with the melatonin binding Moreover, the presence of 4-P-PDOTand luzindole does not interfere with the binding
of melatonin to calreticulin These results suggest a highly specific binding of melatonin to calreticulin, although further experiments with saturation studies should be carried out to assess that these binding sites can be saturated, thus confirming the existence of functional binding of melatonin to calreticulin The data strongly suggests a new class of protein binding site for melatonin and suggests that calreticulin is a target for the intracellular action of melatonin
Calreticulin is a ubiquitous and highly conserved Ca2+ -binding protein of the ER that could be regulated through intracellular signaling pathways involving Ca2+ binding [42] The protein is multifunctional and may play an important role in the modulation of a variety of cellular processes These functions include chaperon activity, con-trol of intracellular Ca2+homeostasis, and regulation of cell adhesiveness by interacting with the integrins at the cytoplasmatic site of the plasma membrane Surprisingly, calreticulin controls the steroid-sensitive gene expression
Fig 5 Binding of 2-[ 125 I]melatonin to
calreti-culin (A) Competition experiments were
per-formed with recombinant calreticulin (GST–
calreticulin) and increasing concentrations of
nonlabeled melatonin Results were expressed
as the percentage of specifically bound
2-[125I]melatonin Inset: Scatchard plot of the
data in (A) showing the binding kinetics of
melatonin to GST–calreticulin (B) Values of
B max of melatonin binding to
GST–calreti-culin in the presence (CRT+ Ca2+) and
absence (CRT ) of Ca 2+ , and to GSTprotein.
Melatonin, d; NAS, s; 4-P-PDOT, ;
luzindole, h *P < 0.001.
Trang 7[43,44] This was an unexpected finding, as calreticulin is an
ER-resident protein [45] and steroid receptors are found
either in the cytoplasm or in the nucleus
Several pieces of evidence suggest a parallel between
calreticulin and calmodulin in nuclear melatonin signaling
Melatonin binds both calmodulin and calreticulin only in
the presence of Ca2+ [46] Calreticulin mediates nuclear
export of the glucocorticoid receptor, and overexpression of
calreticulin antagonizes nuclear receptor-dependent
tran-scriptional activation [38] Melatonin protects against
glucocorticoid-induced apoptosis regulating glucocorticoid
receptor expression [47] Calreticulin specifically interacts
with the first zinc finger of different nuclear receptors Based
on this feature, it was shown that calreticulin interacts with
amino acids 206–211 of the DNA binding domain region of
estrogen receptor alpha (ERa), reversing ERa inhibition of
invasion in vitro [48] Calmodulin also modulates ERa
interacting with amino acids 290–310 of this receptor [49],
whereas melatonin–calmodulin interaction blocks the
acti-vation of estrogen receptor for DNA binding [50] Thus, the
oncostatic effects of melatonin against ERa activation may
depend on its binding to calmodulin and calreticulin,
preventing both the binding of ERa to DNA and its
proliferative effects
The problem of calreticulin localization into the cell
continues Calreticulin-like immunoreactivity was detected
in the nucleus of some cells, although it seems that it is not a
nuclear resident protein [45,51] An explanation for these
contradictions may depend on the purification
methodo-logy In fact, resident nuclear proteins are associated with
the insoluble nuclear fractions, whereas the
Triton-soluble fractions contain proteins of ER origin [52]
Michalak [45] identified 60-kDa calreticulin in the Triton
X-100 soluble fraction of purified nuclei, which includes the
solubilized outer nuclear membrane containing proteins of
the ER, but not in the Triton-insoluble fraction containing
nuclear material surrounded by the inner nuclear
mem-brane These results suggest that calreticulin is not a resident
nuclear protein Other reports failed to identify calreticulin
in the cytosol, suggesting that the ER, but not the cytosol
form of calreticulin is responsible for inhibition of
gluco-corticoid receptor-mediated gene expression [44,45,53,54], a
proposed function for this protein However, in vitro
DNA-binding assays indicated that recombinant calreticulin could
inhibit DNA binding by steroid receptors, suggesting that
the effect of calreticulin on nuclear hormone receptor
transactivation might be direct [48] Experimental evidence
exists supporting both nuclear [53] and cytosolic [38,54,55]
localization of calreticulin These data provide evidence for
two pools of calreticulin, the first contained within the
lumen of the ER, and the second contained within the
cytosol Our data show that nuclei homogenates are lacking
calnexin, a marker for ER [56], but that they do contain
calreticulin Therefore, our procedure for nuclear protein
purification started from a material lacking ER
contamin-ation, suggesting that the calreticulin found in our material
does not come from this localization and that it was
associated with the nuclei
It seems that, although it is not a nuclear resident protein,
calreticulin may localize in the nucleus The mechanism(s)
by which calreticulin molecules are imported into, and
retained in, the nucleus are unknown It is unclear whether
nuclear localization of calreticulin is determined by its simple exclusion from the ER, possibly due to elimination of its N-terminal signal sequence, or by its retrotranslocation from the endoplasmic reticulum to the cytoplasm The classical view of strict protein compartmentalization has now been challenged, and it is thought that proteins may shuttle between the nuclear and cytoplasmic compartments [57] Calreticulin may have a similar behavior to localize in distinct subcellular compartments, acting independently on compartment-specific targets
These data may suggest that the interaction between melatonin and calreticulin (and calmodulin) could be of physiological importance in regulating the activity of a broad spectrum of nuclear receptors [38] This hypothesis is further supported because the high melatonin-calreticulin binding affinity, which correlates well with the melatonin concentration in nucleus [18,24] Therefore, melatonin might be a mechanism involved in importing and/or retaining calreticulin in the nucleus Melatonin–calreticulin interaction also can be related to the balance of ligand-induced import and calreticulin-dependent export, provi-ding the cell with a nuclear transport-based mechanism Based on these findings, it is necessary to re-evaluate our current understanding of the molecular pathways of mela-tonin actions
Acknowledgements
We thank Dr M Michalak for providing the plasmid which encode GST-calreticulin and Dr M Martı´n for the binding experiments We also thank Dr C Carlberg for helpful suggestions This work was supported by the CICYTgrant SAF98 : 0156 and Junta de Andalucı´a (CTS-101) M Macı´as is a fellow from the Programa de Formacion de Personal Investigador, Ministerio de Educacion y Cultura, Spain.
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