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Department of Molecular Pharmacology and Biological Chemistry, Northwestern Drug Discovery Program, Northwestern University Institute for Neuroscience, Northwestern University Medical School, Chicago, Illinois 60611
Address all correspondence and requests for reprints to: Margarita L. Dubocovich, Ph.D., Department of Molecular Pharmacology and Biological Chemistry (S215), Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: dubo{at}nwu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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200% of control cells). This effect of melatonin was dependent on
the presence of the h mt1 melatonin receptor, as no
potentiation of forskolin-induced cAMP formation was observed in CHO
cells transfected only with the neomycin resistance plasmid. The
time-dependent increase in forskolin-stimulated cAMP levels resulted in
a potentiation of cAMP-dependent protein kinase activity 1 h after
withdrawal (130% of control cells; P < 0.05)
and in the number of cells containing the phosphorylated form of cAMP
response element-binding protein (75% of cells at 1 and 16 h
compared with 30% in control cells; P < 0.05). An
increase in the undissociated state (G
ß
) of Gi
proteins may underlie this phenomenon as demonstrated by the increase
in pertussis toxin-catalyzed ADP-ribosylation of G proteins (217
± 48% of control; P < 0.05) after melatonin
withdrawal. This increase in the ribosylation was not due to an
up-regulation of Gi protein, as no significant change in
Gi protein levels occurred at this time. We demonstrated
that activation of the h mt1 melatonin receptor in a manner
that resembles the physiological pattern of melatonin exposure alters
signaling, as potentiation of cAMP-mediated signal transduction events
is observed after hormone withdrawal. The CHO cells expressing the
human melatonin receptor may provide an in vitro
cellular model in which to investigate the putative signaling
mechanisms leading to gene regulation by melatonin. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prolonged exposure of pars tuberalis cells maintained in culture to
melatonin leads to desensitization of melatonin receptors and
potentiation of forskolin-mediated cAMP accumulation (4). The
recombinant human mt1 (h
mt1)3 melatonin
receptor expressed in cell lines, which also signals through inhibition
of forskolin-stimulated cAMP formation (7, 8), offers an in
vitro model in which to study the regulation of signaling upon
physiological exposure to melatonin, i.e. concentration and
time. The synthesis and secretion of melatonin occur in a rhythmic
fashion, with blood levels of melatonin being highest during the night
(100 pg/ml) and lowest during the day (10 pg/ml) in humans (9)
and many other species (10). Here, we report the effects of
physiological exposure of mt1 melatonin receptors expressed
in Chinese hamster ovary (CHO) cells to melatonin at different points
along the cAMP-dependent signal transduction cascade, that is on G
protein function, cAMP formation, PKA activation, and phosphorylation
of the transcription factor CREB. We conclude that the physiological
activation of the human mt1 melatonin receptor potentiates
cAMP-mediated signal transduction events after hormone withdrawal.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Melatonin pretreatment and withdrawal
pSV-Neo CHO and mt1-CHO cells were grown to
confluence in the presence of 10% FBS. Cells were refed with Ham’s
F-12 medium without serum and maintained in culture in the absence
(vehicle, 0.000001% ethanol) or presence of melatonin (in vehicle, 400
pM) for 8 h. At the end of this incubation period, the
F-12 medium from control or melatonin-treated cells was removed, and
the cells were refed with fresh F-12 media. At various times after
melatonin withdrawal (0, 1, 2, 4, 8, 12, and 16 h) or 16 h
after vehicle removal (control cells), the following measurements were
performed: ADP ribosylation and immunoblot analyses of G proteins,
forskolin-induced cAMP accumulation, cAMP-dependent protein kinase
activity, and immunocytochemical measurements of CREB
phosphorylation.
cAMP accumulation assays
cAMP accumulation assays were carried out as previously
described (7). Briefly, forskolin-induced [3H]cAMP
accumulation was measured in pSV-Neo CHO cells and mt1-CHO
cells. Cells, grown to confluence, were reincubated with F-12 medium
containing 2 µCi/ml [3H]adenine (26.9 Ci/mmol; New
England Nuclear-DuPont, Boston, MA) in the absence (containing vehicle)
or presence of melatonin (400 pM) for 8 h. The medium
containing melatonin was aspirated, and the cells were reincubated with
F-12 medium containing 1 µCi/ml [3H]adenine. At various
times after melatonin withdrawal, cAMP formation was stimulated by the
addition of F-12 medium containing 100 µM forskolin
(Sigma Chemical Co., St. Louis, MO) and 30 µM rolipram
(RBI, Natick, MA). The plates were incubated for 10 min at 37 C, and
the reaction was terminated by aspiration of the medium and addition of
1 ml ice-cold (5%) trichloroacetic acid (16 h at 4 C) to release
[3H]cAMP into the solution. [3H]cAMP was
separated from [3H]ATP using Dowex (AG50W-X4, Bio-Rad,
Hercules, CA) and alumina (Sigma) column chromatography.
[3H]cAMP was quantified by liquid scintillation
counting.
PKA assays
PKA assays were carried out as described previously (12). After
the melatonin treatment and withdrawal periods, cells were washed twice
with PBS without calcium or magnesium (PBS; 137 mM NaCl,
2.7 mM KCl, 8 mM
Na2HPO4, and 0.62 mM
KH2PO4, pH 7.4), lifted in LIFT buffer (10
mM KPO4, 1 mM EDTA, and 0.25
M sucrose, pH 7.4), and lightly pelleted. Cells were
resuspended in 1 ml F-12 medium containing either 10 or 100
µM forskolin, 30 µM rolipram, and, where
indicated, 400 pM melatonin. The tubes were incubated for
10 min at 37 C, and the reactions were terminated by chilling on ice
and centrifugation in a microcentrifuge. The cells were then
resuspended in 50 µl ice-cold phosphate buffer [10 mM
NaHPO4, 1 mM EDTA, 1 mM
dithiothreitol (DTT), and 250 mM sucrose] containing
phenylmethylsulfonylfluoride (0.2 mM) and leupeptin (10
µg/ml) and lysed by a 10-sec sonication. Cell extract (10 µg) was
added to a 40-µl reaction final concentration mixture containing 30
µM kemptide (Peninsula Laboratories, Belmont, CA), 20
mM Tris-HCl (pH 7.4), 10 mM magnesium acetate,
0.5 mM isobutylmethylxanthine, 10 mM DTT, 5
mM NaF, 200 µM [-32P]ATP
(100–200 cpm/pmol; DuPont-New England Nuclear) in the absence and
presence of 5 µM cAMP. Total cAMP-dependent PKA activity
was determined in the presence of a concentration of cAMP (5
µM) that maximally stimulated PKA. After 5-min incubation
at 37 C, all of the reactions were spotted onto phosphocellulose strips
(P81, Whatman, Clifton, NJ) and washed three times in 75 mM
phosphoric acid and once in 95% ethanol. Filters were then placed in
vials containing scintillation fluid and counted by liquid
scintillation counting.
Immunocytochemical determination of the phosphorylation of CREB
These assays were performed with slight modification as
previously described (13). Pretreatment with melatonin was carried out
exactly as described for adenylyl cyclase assays, except that cells
were grown on coverslips. After melatonin treatments and withdrawal
periods, cells were stimulated with 100 µM forskolin and
30 µM rolipram. After 10-min incubation at 37 C, the
medium was removed, and cells were fixed in 4% paraformaldehyde for 30
min at room temperature and rinsed three times at 5-min intervals with
PBS containing 10 mM glycine (PBS-gly). Cells were then
permeabilized in 0.3% Triton X-100 in PBS-gly containing 2% goat
serum (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA) for 30
min at room temperature to reduce nonspecific staining. After
permeabilization and blocking, cells were incubated in 0.3% Triton
X-100, in PBS-gly containing no primary antibody (nonspecific staining)
or with a 1:500 dilution of antirat phosphorylated CREB antibody
(Upstate Biotechnology, Lake Placid, NY) for 16 h at 4 C. Wells
that received only secondary antibody showed no staining above
background (data not shown). The following day, all coverslips were
rinsed in PBS-gly three times at 5-min intervals and then incubated in
0.3% Triton X-100 in PBS-gly containing antirabbit IgG secondary
antibody (1:1000) for 30 min at 37 C (Vectastain ABC Kit). Coverslips
were rinsed in PBS-gly three times at 5-min intervals and then
incubated with diaminobenzidine-peroxidase (Vector Laboratories). When
color developed, the reaction was terminated by extensive washing in
tap water. Cells were dehydrated, rinsed for 1 h, and mounted
using Permount (Fisher Scientific, Fairlawn, NJ). Cells were imaged
using a light microscope attached to an LCD video camera, projected
onto a computer screen using Metamorph imaging software (Universal
Imaging Corp., WestChester, PA), and then quantitated by NIH Image
software (Wayne Rasband, NIH, Bethesda, MD).
ADP ribosylation analysis
All ribosylation assays were performed according to the methods
previously described (14). Cells were grown to confluence on 10-cm
dishes. After the treatment and withdrawal periods, the cells were
washed twice with PBS and centrifuged at 600 x g for
10 min. The cells were then resuspended in buffer [2 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 0.2 M DTT, 10
µg/ml leupeptin, 0.2 mM phenylmethylsulfonylfluoride, and
10 µg/ml aprotinin], homogenized using a Polytron, and pelleted by
centrifugation at 15,000 x g for 30 min. The resulting
pellet was suspended in 500 µl buffer [10 mM Tris-HCl
(pH 7.7), 1 mM MgCl2, and 0.1 mM
EDTA]. Fifty micrograms of membrane protein were incubated at 32 C for
1 h with 0.2 mg/ml thiol-preactivated cholera toxin or 20 µg/ml
thiol-preactivated pertussis toxin (Calbiochem, La Jolla, CA) in buffer
containing 10 µM [-32P]NAD (3.3
Ci/mmol). Labeled membranes were then washed in ice-cold buffer,
solubilized, and subjected to 10% PAGE. After electrophoresis, gels
were dried and exposed to x-ray film. 32P incorporation
into the Mr 46,000 protein band (cholera toxin)
and the Mr 41,000 band (pertussis toxin) was
quantified by integrating densitometric scans.
Immunoblot analysis
Cells collected during the period of melatonin withdrawal were
used to prepare membrane proteins (50 µg), which were separated by
SDS-PAGE, electrotransferred to nitrocellulose filters, and analyzed by
immunoblotting. Nonspecific binding sites on the nitrocellulose filters
were blocked for 1 h at room temperature in 5% nonfat dry milk
and dissolved in Tris-buffered saline. Gi and
Gs proteins were detected by antirabbit
Gi antiserum (1:500), which detects Gi1 =
Gi2 > Gi3 (provided by Dr. David
Manning, University of Pennsylvania School of Medicine, Philadelphia,
PA) (15, 16) or with antirabbit Gs (1:1000; a gift from
Mark Rasenick, University of Illinois, Chicago, IL) in
milk-Tris-buffered saline, respectively. After five rinses in
Tris-buffered saline, the filters were incubated with a 1:5000 dilution
of goat antirabbit IgG-horseradish peroxidase (Pierce Chemical Co.,
Rockford, IL) for 1 h at room temperature. G Protein bands were
detected by enhanced chemiluminescence (Amersham, Arlington Heights,
IL). Filters were exposed to Hyperfilm (Amersham). For quantitation,
films were scanned with a densitometer and quantitated using the
Molecular Analyst System (Bio-Rad, Hercules, CA).
Data analysis
Individual [3H]cAMP levels were normalized by
recovery of [14C]cAMP using a standard amount of
[14C]cAMP (1000 cpm) in each column.
[3H]cAMP values were expressed as a percentage of the
forskolin-induced maximal response in untreated cells as indicated
within each experiment.
PKA activity was expressed as activity ratios where the basal activity or the activity induced by a submaximal concentration of forskolin (10 µM) was divided by the total PKA activity induced by 5 µM cAMP. PKA activity ratios were then expressed as a percentage of the basal response or as a percentage of the forskolin-induced response in control cells for 8-h melatonin treatment experiments.
The intensity of nuclear phosphorylated CREB immunocytochemical staining was assessed in each experiment by measuring the relative OD value above the threshold. A set threshold value was assigned to a nucleus that had no immunocytochemical staining for each set of experiments. Nuclei that had OD values less than the threshold value were rated as having undergone no immunocytochemical staining, and nuclei that had OD values greater than the threshold value were rated as having undergone immunocytochemical staining. Nuclei that had immunocytochemical staining above background were then divided by the total number of nuclei analyzed. Statistical analyses were performed by one-way ANOVA or repeated measures ANOVA using GraphPad Prism (San Diego, CA). Significance is defined as P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Stimulation with forskolin (10
µM) for 10 min increased PKA activity to 139 ± 10%
(n = 3) of the basal value in mt1-CHO cells. This
increase in PKA activity was inhibited by simultaneous exposure to 400
pM melatonin for 10 min (105 ± 7% of basal; n =
3; Fig. 1). To assess whether the increase in protein kinase activity
induced by forskolin in mt1-CHO cells activated the
transcription factor CREB, we determined nuclear anti-phospho-CREB
immunoreactivity by immunocytochemistry (Figs. 1B and 2). In the basal state, 53 ± 6% of
cells (n = 3) showed positive antiphospho-CREB immunoreactivity.
After forskolin (100 µM) stimulation, 75 ± 7%
(n = 3; P < 0.05) of the cells contained the
phosphorylated form of CREB protein. Melatonin (400 pM)
added in combination with forskolin decreased the number of cells
containing the phosphorylated form of CREB protein in the nucleus to
42 ± 5% (n = 3; P < 0.05; Fig. 1B). No
melatonin-mediated inhibition of the phosphorylation of CREB occurred
in pSV-Neo CHO cells [basal, 40 ± 6%; forskolin (100
µM), 78 ± 9%; forskolin plus melatonin (400
pM), 66 ± 8%].
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). This increase in forskolin-induced
cAMP formation after melatonin withdrawal was mediated through the
human mt1 melatonin receptor. Pretreatment of pSV-Neo CHO
cells with 400 pM melatonin followed by 16-h withdrawal did
not affect forskolin-induced cAMP formation, whereas the same treatment
increased cAMP formation in mt1-CHO cells to 244% of that
in untreated cells (P < 0.05; Fig. 3B).
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) with no change in
total PKA activity that was induced by 5 µM cAMP (Fig. 4B). One hour after withdrawal, forskolin (10
µM)-stimulated PKA activity increased to 141 ±
8.1% of the activity observed in untreated cells (n = 4;
P < 0.05). The effect decreased to 128 ± 13% of
that in untreated cells 16 h after melatonin withdrawal (Fig. 4A).
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and 5). In untreated cells, stimulation by
forskolin resulted in 33 ± 6% of the cells containing the
phosphorylated form of CREB. However, during melatonin withdrawal, the
number of cells containing nuclear phospho-CREB immunoreactivity
increased in a time-dependent manner, where levels of significance were
reached after 1 h of melatonin withdrawal (74 ± 9% of
cells; n = 4; P < 0.05) and remained elevated up
to 16 h after withdrawal (77 ± 13%; n = 4;
P < 0.05; Fig. 4C).
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i/Go proteins is
increased after withdrawal of melatonin). These increases in pertussis
toxin-induced ribosylation of Gi/Go were
not the result of increases in Gi1, Gi2,
and Gi3 protein levels. No change in levels occurred
after pretreatment and withdrawal of melatonin (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In mt1-CHO cells, short melatonin exposure inhibited the
cAMP-dependent signal transduction pathway, whereas after withdrawal
from physiological melatonin exposure, a supersensitization of
forskolin-induced cAMP formation, PKA activation, and phosphorylation
of CREB were observed. Supersensitization of cAMP-dependent signaling
cascades after agonist exposure has been shown for other inhibitory G
protein-coupled receptors as well. Prolonged activation of somatostatin
receptors in anterior pituitary tumor cells by somatostatin for 8
h results in an increase in forskolin-induced cAMP formation with no
change in basal cAMP levels, basal adenylyl cyclase activity, or
phosphodiesterase activity (18). Pretreatment of D2
dopamine receptors in mouse fibroblast Ltk- cells with a D2
agonist (quinpirole) for 24 h results in increases in basal and
forskolin-induced cAMP levels, and in basal and forskolin-induced
adenylyl cyclase activity (19). In addition, agonist exposure of
D2 dopamine receptors expressed in HEK293 or C6 cells
increases forskolin-induced cAMP accumulation (20). Finally,
pretreatment of 2-adrenoceptors in HT29 human colonic
adenocarcinoma cells with an 2-adrenoceptor agonist
(UK14,304) for up to 1 h results in an increase in
forskolin-stimulated cAMP formation with no change in phosphodiesterase
activity (21).
Desensitization of the h mt1 melatonin receptors by
melatonin exposure may mediate the increase in forskolin-induced
cAMP formation after melatonin withdrawal. We previously demonstrated
that the h mt1 melatonin receptor expressed in CHO cells is
tightly coupled to Gi proteins (7). However, although
desensitization of the h mt1 melatonin receptor correlated
with increases in forskolin-induced cAMP formation in these cells
(manuscript in preparation), the mechanisms underlying
supersensitization of the adenylyl cyclase signal transduction cascade
and receptor desensitization may be independent of one another.
Evidence suggests that pretreatment of other Gi-coupled
receptors with their respective agonists results in supersensitization
of adenylyl cyclase signal transduction; however, desensitization of
the receptors occurred for some [e.g. somatostatin (18) and
D2 dopamine receptors (19, 20)], but not all
[e.g. 2-adrenergic receptor (21)], of the
receptors.
Decreases in the levels of Gi proteins may underlie the
potentiation of forskolin-induced cAMP formation in mt1-CHO
after melatonin withdrawal (22). This phenomenon is observed in primary
cultures of rat striatal neurons (23) and in rat cardiomyocytes (24)
after agonist exposure. In the present study melatonin withdrawal did
not alter Gs or Gi1, Gi2,
and Gi3 protein levels in CHO cells as determined using
antibodies that recognize Gs, Gi1 =
Gi2 > Gi3. We analyzed all these G
proteins even though previous reports demonstrate the existence of only
Gi2 and Gi3 in CHO cells (25, 26).
Pertussis toxin and cholera toxin catalyze ADP ribosylation of the
heteromeric, undissociated forms of Gi and Gs,
respectively, and do not affect the free -subunit (27, 28).
Therefore, an increase in ribosylation of
Gi/Go with no concomitant change in
Gi1, Gi2, and Gi3 protein
levels may suggest an increase in the undissociated (Gß) state
of Gi proteins after melatonin withdrawal. A decrease in
the dissociated form of Gi protein (Gi) may
attenuate the inhibitory influence of Gi on adenylyl
cyclase, resulting in a supersensitization of the cAMP-dependent signal
transduction cascade. In reconstituted preparations, Gi
subunit can inhibit adenylyl cyclases V and VI (29). The identity of
the adenylyl cyclase present in CHO cells, however, has not been
determined. Chronic antidepressant treatment increases G protein
activation of adenylyl cyclase with no change in levels of total
Gi protein in the rat cerebral cortex. These results
could be also explained by an increase in ternary Gß protein
complexes (30).
A direct effect of melatonin treatment on adenylyl cyclase activity could also underlie the increases in forskolin-induced cAMP formation after withdrawal. To date, evidence suggests that eight isoforms of adenylyl cyclase may exist even though cloning has revealed the existence of only six isoforms (31, 32). After agonist exposure, the potency of forskolin to activate specifically adenylyl cyclases I and II is increased in HEK293 cells expressing the D2 (short and long) dopamine receptor compared with that in untreated cells (20). In contrast, no changes in forskolin-induced activation of adenylyl cyclase occur in rat cerebral cortex slices after serotonin exposure even though GppNHp increases adenylyl cyclase (33). Another mechanism that may underlie the increases in adenylyl cyclase activity is enzyme up-regulation. However, even though a diurnal regulation of adenylyl cyclase I occurs in the rat pineal gland, perhaps through fluctuating cAMP levels (34), this mechanism seems unlikely in mt1-CHO cells because melatonin treatment does not increase basal levels of cAMP compared with those in untreated cells after withdrawal (data not shown).
PKA activation is initiated by cAMP binding to the regulatory subunits of the tetrameric enzyme PKA, leading to dissociation of the regulatory subunits from the catalytic subunits (35). In our system, short exposure of mt1-CHO cells to melatonin reduces basal and forskolin-induced activation of PKA, suggesting that melatonin can inhibit cAMP formation, resulting in inhibition of PKA activity. The potentiation of forskolin-stimulated PKA activity observed after melatonin withdrawal is likely to result from the intracellular increases in cAMP induced by forskolin under these conditions. Evidence suggests that the regulatory subunits of PKA exert an inhibitory influence on PKA activity (36) and that cAMP analogs can down-regulate the regulatory subunits of PKA (37). It is conceivable that treatment with melatonin results in down-regulation of the regulatory subunits of this kinase and decreases the inhibitory influence that these regulatory subunits have on PKA, leading to increased PKA activity. Increases in PKA activity may not result from up-regulation of PKA in our model, as total PKA activity was of similar magnitude in both control and melatonin-treated cells.
Treatment of the mt1-CHO cells with melatonin resulted in an increase in forskolin-induced phosphorylation of CREB protein compared with that in untreated cells after withdrawal. It is well established that cAMP binds to the tetrameric form of PKA, resulting in a translocation of the dissociated PKA catalytic subunits to the nucleus and phosphorylation of CREB on Ser133 (35, 38). The increases in forskolin-induced CREB phosphorylation after melatonin withdrawal may be due to the increase in forskolin-stimulated PKA activity. Up-regulation of CREB protein via cAMP response elements would also explain the potentiation of CREB phosphorylation by forskolin after melatonin withdrawal. However, it is unlikely that this is occurring in mt1-CHO cells because basal levels of phosphorylated CREB were not increased (data not shown).
The supersensitization of the adenylyl cyclase signal transduction cascade by 8-h exposure to melatonin reported in this study is the result of mt1 melatonin receptor-dependent effects. Acute exposure to melatonin (400 pM) did not inhibit phosphorylation of CREB in pSV-Neo CHO cells. After melatonin withdrawal, pSV-Neo CHO cells did not show enhancement of forskolin-induced cAMP accumulation. Additionally, CHO cells transfected with h mt2 melatonin receptor did not show an increase in forskolin-induced cAMP accumulation (data not shown), again arguing for an mt1 receptor-specific effect. Therefore, we conclude that this supersensitization phenomenon is specific for the mt1 receptor transfected into CHO cells and is not the result of intracellular effects of melatonin.
Protein kinase C (PKC) may also be involved in the sensitization
process induced by melatonin treatment. For example, phosphorylation of
CREB protein has been shown to occur through mechanisms independent of
cAMP, i.e. via activation of PKC by serum or PGs (39). The
involvement of PKC in melatonin receptor-mediated signaling has been
demonstrated on the firing rate in slices of the suprachiasmatic
nucleus (40). However, although melatonin can modulate PLC-mediated
pathways, it has been shown that it does not stimulate phospholipases
directly (8, 41). In our hands, only high concentrations of melatonin
(
1 nM) stimulated phosphoinositide hydrolysis in
mt1-CHO cells (data not published). Therefore, because
melatonin concentrations less than 1 nM were used in the
present studies, and all melatonin pretreatments were performed in F-12
medium without serum, PKC is probably not involved in the
supersensitization of the cAMP-dependent cascade in mt1-CHO
cells.
Overall, these results suggest potential functional consequences in h mt1 melatonin receptor-mediated signaling after withdrawal of melatonin in tissues endowed with these receptors. This study demonstrates that melatonin treatment and withdrawal induce sensitization of cAMP-mediated signaling. As the hormone melatonin is released in a circadian fashion, with peak levels occurring during the night and persisting for approximately 8 h, the results of this study are relevant to explain the functional consequences of the circadian rhythm of melatonin production. It can be speculated that circadian exposure of melatonin receptor to melatonin serves to turn off the inhibitory signal, leading to sensitization of the adenylyl cyclase cascade for stimulatory input by an as yet unidentified signal.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Pharmacology and Toxicology, Mylan
School of Pharmacy, Duquesne University, Mellon Hall of Science,
Pittsburgh, Pennsylvania 15282.
3 Here we use the official nomenclature and
classification of melatonin receptors approved by the nomenclature
committee of the International Union of Pharmacology (42 ). The
denomination mt1 corresponds to that of the recombinant
melatonin receptor subtype previously known as Mel1a (11 ),
which does not show well defined functional characteristics. The
mt2 recombinant receptor, previously known as
Mel1b, is referred to here as MT2 because it
has a defined function and was pharmacologically characterized in a
native tissue (43 ). MT3 corresponds to the
pharmacologically defined melatonin receptor subtype, with unknown
molecular structure, previously referred to as ML2
(44 ).
Received November 7, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Mol Endocrinol 11:538–5492-adrenergic receptor-mediated sensitization of
forskolin-stimulated cyclic AMP production. Proc Natl Acad Sci USA 84:1294–1298-subunits. Naunyn-Schmiedeberg Arch Pharmacol 351:27–34[Medline]
-azidoanilido [32P]GTP
labeling of multiple G protein a subunits. J Neurochem 64:2534–2543[Medline]
-opioid receptor expressed in CHO
cells: interaction with multiple G-proteins is not specific for any
individual G subunit and is similar to that of other
opioid receptors. Mol Brain Res 29:336–346[Medline]
-subunit. Biochemistry 33:4865–4871[CrossRef][Medline]
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