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CNRS-UPR 0415 and Université Paris VII (L.B., L.P., P.D.,
A.D.S., R.J.) Institut Cochin de Génétique
Moléculaire F-75014 Paris, France
Institute of Pharmacology (F.R., C.N.)
Vienna University A-1090 Vienna, Austria
CNRS-UPRES-A8068 (M.T.) Institut Cochin de
Génétique Moleculaire Pavillion Gustave
Roussy 75679 Paris Cedex 14, France
Rowett Research Institute (L.B., P.B., P.J.M.)
Bucksburn, Aberdeen AB2 9SB, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit antibodies.
Gi2 and/or Gi3 mediated
adenylyl cyclase inhibition while Gq/11 induced
a transient elevation in cytosolic calcium concentrations in HEK 293
cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic
calcium mobilization via PTX-insensitive G proteins was confirmed in
primary cultures of ovine pars tuberalis cells endogenously expressing
Mel 1a receptors. In conclusion, we report the development of the first
antibody recognizing the cloned human Mel 1a melatonin receptor
protein. We show that Mel 1a receptors functionally couple to both
PTX-sensitive and PTX-insensitive G proteins. The previously unknown
signaling of Mel 1a receptors through Gq/11
widens the spectrum of potential targets for melatonin. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Activated Mel 1a receptors have been shown to mediate the inhibition of
cAMP accumulation via a pertussis toxin (PTX)-sensitive G protein, a
process known to be mediated by Gi/o proteins (8). In
addition, recombinant Mel 1a receptors were shown to activate
Kir3.1/3.2 potassium ion channels, to potentiate PGF2-promoted
stimulation of phospholipase C, and to modulate protein kinase C (PKC)
and phospholipase A2 via
Gß
-subunits liberated during Gi/o protein
activation (9, 10, 11). Other signaling pathways of Mel 1a receptors have
been suggested. Studies on ovine pars tuberalis showed that Mel 1a
receptors regulate their own expression through a cAMP-independent
pathway (12). Melatonin receptors were also shown to stimulate inositol
triphosphate (IP3) production and to modulate
intracellular cGMP levels (13, 14), but it is not clear whether this
applies to Mel 1a receptor signaling. The multiplicity of
pharmacological effects associated with Mel 1a receptor activation
suggests that this receptor may couple to several G proteins.
We have chosen HEK 293 cells, which derive from the human embryonic kidney, to investigate Mel 1a receptor signaling pathways. We developed an antireceptor antibody that coimmunoprecipitated receptor-coupled G proteins from Mel 1a-transfected HEK 293 cells. Using this antibody we set out to investigate Mel 1a receptor signaling pathways in these cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). This
sequence showed little or no homology with the corresponding regions of
other high-affinity melatonin receptors, suggesting little potential
cross-reactivity with the human Mel 1b subtype and high selectivity for
the Mel 1a subtype. Antisera specifically recognized peptide 536 on
enzyme-linked immunosorbent assay (ELISA) with half-maximal binding at
a dilution of approximately 1:7000 while preimmune sera were inactive
(data not shown). Antibodies were purified from sera by affinity
chromatography on a glutaraldehyde-activated Sepharose column coupled
to peptide 536 via the amide group of its lysine residue. Purified
536-antibodies displayed a good affinity for peptide antigen with
half-maximal binding at a concentration of 0.15 µg/ml as determined
by ELISA.
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). To
confirm that this 60-kDa protein corresponds to the Mel 1a receptor,
the receptor was labeled with 2-[125I]-iodomelatonin
(125I-Mel), partially purified, and analyzed with
536-antibodies in immunoblots by the following procedure: membranes of
Mel 1a receptor-expressing COS cells were labeled with
125I-Mel. Of three detergents tested (digitonin, dodecyl
maltoside, Triton X-100), digitonin was most effective in solubilizing
functional receptors (
60% of 125I-Mel-labeled receptors
as assessed by gel filtration on Sephadex G-25). The
digitonin-solubilized receptor was subjected to native gel
electrophoresis, and the radiolabeled protein complex was identified by
autoradiography (Fig. 3A). The labeled
receptor was extracted from the native gel and separated by SDS-PAGE.
536-Antibodies specifically recognized a protein migrating at
approximately 60 kDa on immunoblots (Fig. 3B). No such protein was
detected when the experiment was performed with nontransfected cells,
indicating the specificity of the immune recognition of the Mel 1a
receptor. The migration of the 60-kDa protein as a diffuse band is
characteristic of highly hydrophobic and glycosylated proteins and is
in good agreement with observations for other members of the seven
transmembrane-spanning receptor family (19, 20). Upon treatment of COS
cells with tunicamycin (5 µg/ml, 24 h), a further protein band
with an apparent molecular mass of 40 kDa, which most likely
corresponds to the deglycosylated form of the receptor, was detected by
536-antibodies (not shown). Collectively, these results confirm that
536-antibodies specifically recognize the recombinant,
125I-Mel-labeled Mel 1a receptor that migrates at an
apparent molecular mass of 60 kDa on SDS-PAGE.
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). Control
experiments confirmed the specificity of precipitation: in the presence
of peptide 536, precipitation was completely inhibited; precipitation
of 125I-Mel-labeled receptor was also largely reduced in
the presence of excess cold melatonin (1 h at room temperature);
preimmune serum was inactive (Fig. 4A). Similar results were obtained
with membranes prepared from HEK 293 cells stably expressing the Mel 1a
receptor (data not shown). No detectable immunoprecipitation was
observed when experiments were repeated with HEK 293 cells transfected
with the human Mel 1b receptor, confirming that 536-antibodies are
specific for the Mel 1a receptor subtype (data not shown).
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).
G Proteins Are Functionally Associated with Solubilized Mel 1a
Receptors
To determine whether G proteins were functionally associated with
the solubilized complex, membranes were prepared from HEK 293 cells
stably expressing the Mel 1a receptor. Receptors were labeled with
125I-Mel and solublilized, and the solubilized complex was
incubated with guanosine 5'-[ß,-imido] triphosphate (GppNHp), a
nonhydrolyzable GTP analog that activates G proteins and thus induces
dissociation of the ternary complex (ligand-receptor-G protein).
Dissociation can be determined by loss of high-affinity agonist
(125I-Mel) binding. Treated complexes were divided into two
parts and either analyzed by immunoprecipitation with 536-antibody or
by autoradiography of the 125I-Mel-labeled Mel 1a receptor,
after separation by native gel electrophoresis (Fig. 5). GppNHp treatment decreased the amount
of precipitated radiolabeled receptor by 48% ± 8 (Fig. 5A) compared
with nontreated samples. The effect of GppNHp treatment was even more
striking when 125I-Mel-labeled receptors were separated by
native gel electrophoresis (Fig. 5B). The majority of
radioligand-labeled receptor complexes disappeared while the amount of
free 125I-Mel running at the start of the gel increased.
Together, these results indicate, in agreement with previous
observations (21, 22), that G proteins remain functionally associated
with the solubilized 125I-Mel-labeled Mel 1a receptor.
Furthermore, this association is maintained after precipitation with
536-antibodies.
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protein subunits
tested. Gi2, Gi3, Go,
Gz, Gq, Gs, and
G12 were readily detected in HEK 293 cells whereas
Gi1 was less abundant (Fig. 6). Expression of a wide range of G
proteins makes HEK 293 cells a useful expression system for studying
receptor-G protein coupling preferences.
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). In the presence of agonist,
Gi2, Gi3, and Gq/11
proteins were detected in Mel 1a receptor complexes whereas
Gi1, Gz, Go,
Gs, and G12 proteins were not detected
despite the fact that these G proteins were present in HEK 293
cells.
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2, Gi3, and
Gq/11 proteins in complexes was receptor-dependent since
they were not detected when experiments were performed with membranes
from nontransfected cells (Fig. 7). No G protein was detected in
complexes when experiments were performed in the absence of agonist
(Fig. 7) or in the presence of the melatonin receptor-specific
antagonist S20928 (data not shown), demonstrating that
receptor-G protein interaction was specific for
agonist-activated Mel 1a receptors (Fig. 7).
The specificity of receptor-G protein coupling may also be examined
using subtype-selective G protein antibodies directed against the
carboxy terminus of the -subunit. The carboxy-terminal domain of the
-subunit comprises major determinants for receptor recognition (23).
Membranes from Mel 1a receptor-expressing HEK 293 cells (300 fmol/mg of
protein) were preincubated with affinity-purified antisera followed by
radioligand binding; a decrease in high-affinity 125I-Mel
binding reflected inhibition of the formation of the high-affinity
receptor-G protein complex. The anti-Gi1-3 antibody, as
opposed to other antibodies used (anti-Go,
anti-Gq), clearly reduced 125I-Mel binding
(Fig. 8A). The specificity of this
reaction was confirmed as both nonimmune rabbit IgG (not shown) and an
antibody directed against the N-terminal region of Go
(o-N), which is known not to interfere with receptor-G
protein interaction, were ineffective (Fig. 8, A and B). Interestingly,
in membranes prepared from cells transiently overexpressing
Gq (by 
10-fold as monitored by Western blotting, data
not shown), both anti-Gi1-3 and anti-Gq
antibodies were equally effective in decreasing high-affinity binding
(Fig. 8B). As expected, the combination of anti-Gi1-3
and anti-Gq antibodies was most effective. These results
confirm the interaction of Mel 1a receptors with Gi and
Gq proteins. Coimmunoprecipitation and uncoupling
experiments in HEK 293 cells with the endogenous G protein repertoire
gave apparently discrepant results as to the coupling preference for
Gq. These may be explained by a lower sensitivity of the
uncoupling assay.
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). This inhibition
was dose-dependent with an IC50 value of approximately
10-9 M, which is in good agreement with values
reported for Mel 1a receptors expressed in other cells (3, 11). Thus,
activation of Gi2 and/or Gi3 proteins by Mel 1a
receptors modulates the adenylyl cyclase pathway in HEK 293 cells.
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-subunits of Gi proteins and -subunits of
Gq/11 proteins are known to be involved in the transient
mobilization of Ca2+ via the inositol-specific
phospholipase C pathway (24). Melatonin induced an increase in
intracellular Ca2+ concentrations
([Ca2+]i) in Fura-2/AM-loaded Mel 1a
receptor-expressing HEK 293 cells (Fig. 10A). Maximal increases varied between
30 and 100 nM of [Ca2+]i. The
calcium response was transient and returned to basal levels within
approximately 60–90 sec. Similar observations were made for HEK 293
clones expressing 2 pmol and 0.4 pmol of receptor per mg of protein.
Melatonin had no effect on [Ca2+]i in
nontransfected cells (Fig. 10B), whereas a carbachol-induced increase
in [Ca2+]i via endogenously expressed
Gq-coupled muscarinic acetylcholine receptors (
20 fmol/mg
protein) (25) was observed in both nontransfected and Mel 1a
receptor-transfected cells (Fig. 10, A and B). The effect of melatonin
on [Ca2+]i rise was dose dependent with an
EC50 of approximately 10-9 M (Fig. 10C).
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-subunits has been shown to be
responsible for desensitization (27, 28, 29). Treatment of HEK 293 cells
for 5 min with PMA prevented the rise of
[Ca2+]i induced by melatonin (Fig. 10E) and
confirmed the inhibitory effect on the response induced by
carbachol. In the absence of any indication of PKC-dependent
phosphorylation of the Mel 1a receptor itself (30) that might
contribute to desensitization, these observations suggest that the
melatonin and carbachol-induced rise in
[Ca2+]i are mediated via the PLC pathway.
To determine whether Gi or Gq/11 proteins are
involved in the melatonin-induced rise in
[Ca2+]i, cells were treated with PTX, known
to inactivate Gi/o but not Gq/11 proteins (24).
PTX pretreatment did not blunt the melatonin-induced increase in
[Ca2+]i (Fig. 10D). In contrast,
melatonin-induced inhibition of cAMP accumulation was abolished in
these cells (Fig. 10F). PTX was also without effect on
carbachol-induced [Ca2+]i rise (Fig. 10D).
These data thus indicate that both melatonin and muscarinic receptors
stimulate the PLC pathway through Gq/11 proteins.
We have shown above that Gq overexpression increased
coupling of Mel 1a receptors to Gq proteins as monitored by
disruption of receptor-G protein interaction with anti-Gq
protein antibodies (Fig. 8). We therefore tested whether
Gq overexpression also increased Ca2+
mobilization. Melatonin-induced Ca2+ mobilization was
indeed dependent on the quantity of Gq expressed in HEK
293 cells as shown in Fig. 11. At 1
µg of Gq cDNA, Ca2+ mobilization was
increased by approximately 4.5-fold, and Gq
immunoreactivity (not shown) increased 5- to 10-fold compared with
mock-transfected cells. The carbachol-induced Ca2+ signal
measured using the same cell preparation was independent of
Gq overexpression (Fig. 11). The lack of increased
Ca2+ mobilization by muscarinic receptors might be due to
the fact that intracellular Ca2+ pools were maximally
emptied. However, this seems unlikely since thapsigargin (10
µM), a specific inhibitor of microsomal
Ca2+-ATPases that empties intracellular Ca2+
stores and prevents the subsequent reentry of Ca2+ into
these stores, induced a 4 times higher Ca2+ mobilization
than carbachol, suggesting that Ca2+ pools are not rate
limiting.
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). This
effect of melatonin on [Ca2+]i was also
observed in the absence of extracellular Ca2+ in 11.3
± 4.2% (n = 3) of the cells, indicating that an entry of
extracellular Ca2+ is not responsible for the rise in
[Ca2+]i (Fig. 12C). When cells were
pretreated with PTX to inactivate Gi/o, melatonin still
increased [Ca2+]i in 7.9 ± 4.5%
(n = 8) of all recorded cells (Fig. 12B). These data show that
endogenously expressed Mel 1a receptors in ovine pars tuberalis cells
indeed modulate [Ca2+]i in a PTX-insensitive
manner and thus confirm our findings for Mel 1a-transfected HEK 293
cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The starting point of our study was the development of an antibody against a sequence found in the C-terminal region of the Mel 1a receptor. Several lines of evidence indicate that this antibody specifically recognizes the recombinant human Mel 1a receptor in its native and denatured form.
536-antibodies detected the 125I-Mel-labeled Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. The observed size and migration pattern are characteristic of GPCRs, which are highly hydrophobic and nearly always glycosylated, thus migrating as a diffuse band with a molecular mass greater than that predicted from the amino acid sequence (20, 31). The presence of two potential glycosylation sites in the N-terminal domain of the Mel 1a receptor is compatible with receptor glycosylation. Indeed, in the presence of an inhibitor of N glycosylation, a protein of approximately 40 kDa corresponding to the predicted molecular mass of the deglycosylated core protein was detected. Recently, Song et al. (32) reported the development of a polyclonal antibody directed against a peptide sequence located in the third intracellular loop of the human Mel 1a receptor. This antibody detected a protein migrating as a sharp band at 37 kDa in immunoblots of kidney cell membranes. This result is different from what would be expected for typical GPCRs. Furthermore, data concerning the recognition of the cloned Mel 1a receptor by this antibody are not available.
Interaction between 536-antibodies and the digitonin-solubilized Mel 1a receptor has been demonstrated by two experimental approaches: immunoprecipitation of the 125I-Mel-labeled receptor and visualization of the 125I-Mel-labeled receptor-antibody complex after separation by native gel electrophoresis. In both cases, approximately 40% of 125I-Mel-labeled Mel 1a receptor complexes were recognized in Mel 1a-transfected COS and HEK 293 cells. The quantity of immunoprecipitated receptor compares favorably with results observed for other antireceptor antibodies (33, 34).
Coprecipitation of receptor-coupled G proteins was suggested by the
GppNHp sensitivity of high-affinity agonist 125I-Mel
binding to receptor complexes. Indeed, the agonist-activated Mel 1a
receptor was found to couple to Gi2, Gi3,
and Gq/11 proteins in HEK 293 cells. G protein coupling
was strictly agonist dependent and receptor dependent since these
proteins were not detected in the presence of the melatonin
receptor-specific antagonist S20928 (data not shown) or in the absence
of any melatonin receptor ligand or in membranes of nontransfected HEK
293 cells.
Mel 1a receptors have been shown to inhibit adenylyl cyclase in a PTX-sensitive manner indicating coupling with Gi/o proteins (1, 3). Our results now directly confirm that Mel 1a receptors couple to Gi proteins and, in addition, suggest selective coupling among different members of the Gi/o family. Gi2 and Gi3 proteins were coupled to the receptor whereas Gi1 and Go proteins were lacking. Gi1 proteins are less abundantly expressed in HEK 293 cells, which might also influence coupling efficiency to the receptor. However, the example of the somatostatin sst2A receptor, which couples to the extremely low abundant Gi1 protein in Chinese hamster ovary (CHO) cells, shows that G proteins expressed at very low levels may be readily detectable in receptor complexes (15). Selectivity of G protein coupling to Mel 1a receptors is further demonstrated by the lack of coupling to Gz, G12, and Gs, which are all abundantly expressed in HEK 293 cells.
Coprecipitation experiments, the uncoupling of Mel 1a receptors by specific anti-G protein antibodies, and Ca2+ measurements indicate that Mel 1a receptors also functionally couple to PTX-insensitive Gq/11 proteins in HEK 293 cells. The physiological relevance of this observation is supported by the fact that PTX-insensitive Ca2+ mobilization has been observed at plasma melatonin concentrations in HEK 293 cells expressing moderate Mel 1a receptor levels (300 fmol/mg of protein). Furthermore, PTX-insensitive Ca2+ mobilization has been confirmed at a major site of endogenous Mel 1a receptor expression, the ovine pars tuberalis. Previous studies have shown that Mel 1a receptors indeed interact in this tissue with both PTX-sensitive and PTX-insensitive G proteins (35). However, the functional relevance remained unclear at that time since no melatonin-induced Ca2+ mobilization was observed measuring the overall changes in [Ca2+]i of a pars tuberalis cell suspension (36). Using the single-cell measuring system, we now show that melatonin clearly increases [Ca2+]i in approximately 10% of all pars tuberalis cells recorded. The restriction of the response to some cells and relative insensitivity of the assay system used may explain why no melatonin-induced Ca2+ mobilization was observed in the previous study. Furthermore, our results suggest that modulation of the Gq/11 pathway by Mel 1a receptors may be associated with a specific subpopulation of cells present in the pars tuberalis. Further studies will be necessary to determine the precise localization of responding cells within this tissue. Coupling of the Mel 1a receptor to Gq/11 proteins may be involved in the regulation of its own expression since activation of PKC, a downstream effector of PLC, has been shown to be involved in regulation of the Mel 1a receptor in the ovine pars tuberalis (30). Association of the Mel 1a receptor with Gq/11 may be present in other cells. For example, activation of PKC by melatonin has been shown to play an important role in melatonin’s phase-shifting effect on SCN cells (10).
In HEK 293 cells, melatonin-induced Ca2+ mobilization
increased when Gq proteins were overexpressed confirming
1) functional coupling to Gq and suggesting 2) that the
quantity of Gq proteins is rate limiting for Mel 1a
receptor signaling. Since Mel 1a receptors couple to Gi and
Gq proteins, this effect might be due to a simple
competition between Gi and Gq. However, this
seems unlikely since Gq overexpression increased the amount
of receptor-bound Gq without modifying the quantity of
receptor-bound Gi proteins as revealed by our uncoupling
assay. Furthermore, experiments performed on PTX-treated cells indicate
that the amount of receptor-coupled Gq proteins is not
enhanced in the absence of functional Gi proteins since
Ca2+ mobilization in PTX-treated cells was unchanged
compared with controls. Rather, the lack of competition points to a
compartmentalization of G protein subtypes into distinct pools (37). A
second possible explanation for the increase in melatonin-induced
Ca2+ mobilization upon Gq overexpression is
a low-affinity binding between Mel 1a receptors and Gq.
When the amount of Gq is significantly increased, a net
increase in interaction, and therefore signaling, would be driven by
mass action. In agreement with this interpretation, Gq
overexpression in HEK 293 cells did not further enhance
Ca2+ signaling of muscarinic receptors, which are known to
couple tightly to Gq proteins. Almost all cells in pars
tuberalis primary cultures are likely to express Mel 1a receptors (38).
However, melatonin increased [Ca2+]i only in
a subpopulation of these cells. This suggests that coupling of Mel 1a
receptors to Gq/11 proteins depends on the cellular
context. Results obtained in transfected HEK 293 cells suggest that
differences in the expression level of Gq/11 proteins might
contribute to these cell type-specific differences.
Coupling of seven-transmembrane-spanning receptors to Gi
and Gq/11 proteins has been observed for several other
receptors, such as the A3 adenosine receptors and the
melanin-concentrating hormone receptor (39, 40). Receptors with dual
signaling properties often stimulate different pathways with different
efficacies. A3 adenosine receptors, for example, interact
with Gi2 and Gi3 proteins and, to a lesser
extent, with Gq/11 proteins in CHO cells (39). These
receptors were shown to inhibit adenylyl cyclase in all cell types
tested, whereas stimulation of PLC was cell type dependent. Likewise,
the PTH /PTH related peptide receptor (PTH/PTHrP receptor) stimulates
adenylyl cyclase and PLC with different efficacies (41). As observed
for Mel 1a receptors, coupling of the PTH/PTHrP receptor to the PLC
pathway was significantly increased by Gq overexpression
in HEK 293 cells.
Gq/11 proteins are involved in several other signaling
pathways, including regulation of ion channels, activation of kinases
such as mitogen-activated protein kinases or bruton’s tyrosine kinase,
and PLC-independent stimulation of phospholipase D (24, 42, 43).
Interestingly, modulation of ion channels such as K+
channels and voltage-dependent Ca2+ channels by
Gq/11 proteins is frequently observed in neurons (24), a
cell type in which Mel 1a receptors are known to be expressed (44). In
addition, G11 proteins specifically activate trpl
(trp-like) Ca2+-permeable cation channels in this cell type
(45). Furthermore, hematopoietic cells constitute a favorable context
for Gq/11-dependent melatonin signaling since both
bruton’s tyrosine kinase and high-affinity melatonin receptors are
known to be functionally expressed in these cells (46).
In conclusion, we have developed an antibody specific for the human Mel 1a receptor. This antibody will be a valuable subtype-selective tool for the characterization of this receptor. Immunoprecipitation of receptor complexes with this antibody enabled us to define the G protein coupling profile of the Mel 1a receptor expressed in HEK 293 cells. We have shown that Mel 1a receptors selectively and functionally coupled to both PTX-sensitive and PTX-insensitive G proteins in HEK 293 cells. Gi2 and Gi3 proteins are responsible for adenylyl cyclase inhibition while Gq/11 proteins induce Ca2+ mobilization. The previously unknown effect of Mel 1a receptors on [Ca2+]i has been confirmed in primary cultures of ovine pars tuberalis cells, one of the major sites of endogenous melatonin receptor expression. These results should now lead to a careful examination of the relationship between PTX-insensitive signaling pathways and Mel 1a receptors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1, Gi1/2,
Gi3, and Go (ON1 and OC1) antibodies were
a generous gift from Dr. Milligan (University of Glasgow, Glasgow, UK)
(47). Polyclonal anti-Go and
Gq/11 were kindly provided by Drs. Homburger
and Guillon (University of Montpellier, Montpellier, France) (48, 49).
Polyclonal anti-Gs antibodies were provided by Dr.
Guillaume (University Paris VII, Paris, France). Polyclonal
anti-G12, anti-Gi1-3, and
Gz antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The plasmid pCISGq
expressing the Gq protein was a gift from Dr. Simon
(Pasadena, CA) (50). All other products, including melatonin,
tunicamycin, carbachol, EGTA, PTX, GppNHp, Pluronic F-127,
thapsigargin, and protease inhibitors, were purchased from
Sigma (St. Louis, MO).
Generation of Antiserum 536
A peptide corresponding to the 19 C-terminal amino acid residues
of the human Mel 1a receptor was selected for the production of
antibodies. A tyrosine residue was added to the N terminus, and the
amino group of this residue was modified by acetylation
(Ac-YKWKPSPLMTNNNVVKVDSV-COO-). The peptide (536) was
conjugated to keyhole limpet hemocyanin via tyrosine by the
bis-diazobenzidine method (51, 52). One milligram of peptide conjugate,
suspended in complete Freund’s adjuvant, was injected intradermally
into rabbits. Boosters were given at 1-month intervals. Animals were
bled monthly beginning 14 days after the second injection. Antipeptide
antibodies were purified from sera by affinity chromatography on a
column bearing the corresponding peptide coupled to
glutaraldehyde-activated Sepharose. Antibody fractions were eluted in
0.1 M glycine-HCl (pH 2.8) and neutralized with Tris Base 3
M (pH 11). Antibody-peptide affinity was determined by
indirect ELISA.
Expression of the Human Mel 1a Receptor in COS M6 and HEK 293
Cells
Human Mel 1a receptor cDNA was obtained from human fetal brain
mRNA by RT-PCR using PCR primers selected from the recently reported
Mel 1a receptor sequence (3). The coding sequence was cloned into the
expression vector pcDNA3 (Invitrogen, San Diego, CA) and
confirmed by DNA sequencing. For transient receptor expression, COS M6
cells were transfected with Mel 1a cDNA using the
diethylaminoethyl-Dextran method as described previously (53).
Cells were cultivated in DMEM supplemented with 10% FCS in an
atmosphere of 95% air/5% CO2 at 37 C and used for
functional assays 3 days after transfection. Human embryonic kidney
(HEK) 293 cells were cultivated in DMEM supplemented with 10% FCS in
an atmosphere of 95% air/5% CO2 at 37 C. For stable
receptor expression HEK 293 cells were transfected with human Mel 1a
receptor cDNA by a liposome-mediated transfection method using the
transfection reagent
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
methylsulfate, according to supplier instructions. Clones were
selected in DMEM supplemented with 10% FCS and 400 µg/ml of
geneticin (G418) and screened for melatonin binding, using
125I-Mel as ligand.
Radioligand Binding Assays
Membranes were prepared as described previously (54).
Radioligand binding assays were performed in 75 mM Tris (pH
7.4), 12 mM MgCl2, 2 mM EDTA,
protease inhibitors (5 mg/liter soybean trypsin inhibitor, 5 mg/liter
leupeptin, and 10 mg/liter benzamidine) using 125I-Mel as
radioligand. Specific binding was defined as binding displaced by 10
µM melatonin. Assays were carried out for 60 min at 25 C
and terminated by rapid filtration through GF/F glass fiber filters
(Whatman, Clifton, NJ) previously soaked in PBS containing
0.3% polyethylenimine (to reduce nonspecific binding).
Determination of Intracellular cAMP Levels
Intracellular cAMP levels were measured for the Mel
1a-transfected HEK 293 cell clone, as described previously (14).
Calcium Mobilization Assay
Wild-type HEK 293 cells and HEK 293 cells stably transfected
with the human Mel 1a receptor (0.4–2 pmol/mg protein) were suspended
in HEPES (20 mM)-HBSS (H-HBSS) containing 1.2
mM calcium and incubated at a concentration of
107 cells/ml in the presence of 1 µM
Fura-2/AM and 0.02% Pluronic F-127 at 37 C for 45 min (55). Cells were
then washed twice in H-HBSS and resuspended in H-HBSS at 2 x
107 cells/ml and then stored on ice until further use.
Changes in cytosolic calcium were measured with 1–2 x
107 cells in 2 ml H-HBSS at 37 C under continuous stirring
using a Jobin Yvon 3D fluorimeter (Jobin Yvon, Lonjumean,
France). The Fura-2 fluorescence intensity was measured at excitation
and emission wavelengths of 340 and 510 nm, respectively. At the end of
each recording, 0.1% Triton X-100 was added to the cells to determine
the maximum fluorescence ratio (Rmax) and 100
µM manganese was added to determine the minimal
fluorescence ratio (Rmin).
[Ca2+]i was calculated as previously reported
(56).
Preparation of Ovine Pars Tuberalis Cells and
Calcium Measurements
PTs of sheep of mixed breed and sex were collected from a local
abattoir in DMEM. Primary cell cultures were prepared using a method
described previously (56) and seeded into 140-mm cell culture petri
dishes at a density of 80–120 x 106 cells per dish.
The following day, cells were harvested using a cell scraper and
pelleted by centrifugation at 500 x g for 15 min.
Cells were then resuspended in supplemented DMEM (DMEM containing
12.5% lamb serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and
0.25 µg/ml amphotericin, E. R. Squibb & Sons, Inc.,
Princeton, NJ) and counted. For calcium measurements, cells were
aliquoted into 35 mm poly-D-lysine-coated cell culture
petri dishes at a density of 2.5 million cells per dish in supplemented
DMEM. Twenty four hours later, cells were washed three times in
Krebs-Ringer-HEPES (KRH) medium: 120 mM NaCl, 5
mM KCl, 1 mM KH2PO4,
1.44 mM MgSO4, 5 mM
NaHCO3, 1 mM CaCl2, 2.8
mM glucose, 25 mM HEPES. Cells were then loaded
for 60 min at 37 C with 2 µM fura-2-AM in KRH medium.
After washing a further three times in KRH medium, cells were
transferred for Ca2+ imaging to the stage of an upright
water immersion BX50WI fluorescence microscope (Olympus Corp., Lake Success, NY). Solutions were added with continuous
elution by gravity feed. Measurements were performed using an OlymPix
CCD camera with SpectraMaster monochromator and Merlin software, all
supplied by Life Science Resources (Cambridge, UK). Cytosolic
[Ca2+]i was determined by ratio imaging of
fura-2-fluorescence, using excitation wavelengths of 350 and 380 nm and
an emission wavelength of 510 nm. The data sampling was usually a
single ratio measurement every 5 sec. Regions of interest were defined
to cover as much of the area of each single cell as possible, and mean
ratios were calculated for each region using the Merlin software.
Solubilization of the Mel 1a Receptor
Membranes were prepared as described (54) and incubated with or
without ligand for 1 h at 25 C, as detailed above. Membranes were
pelleted by centrifugation at 18,000 x g for 30 min at
4 C. The pellet was washed with ice-cold buffer: 75 mM Tris
(pH 7.4), 12 mM MgCl2, 2 mM EDTA,
and protease inhibitors (5 mg/liter soybean trypsin inhibitor, 5
mg/liter leupeptin, and 10 mg/liter benzamidine), and then resuspended
in the same buffer containing 1% digitonin at a ratio of 1 ml 1%
detergent/mg protein, and agitated for 3 h at 4 C. Nonsolubilized
membrane proteins were removed by centrifugation at 18,000 x
g for 30 min at 4 C.
Immunoprecipitation of the Human Mel 1a Receptor
125I-Mel-labeled Mel 1a receptors were solubilized
as described above. The digitonin concentration was adjusted to 0.2%,
and 536-antibodies (crude serum) were added to a final dilution of
1:40. Extracts were incubated for 18 h at 4 C with continuous
gentle mixing. During the last 6 h, 50 µl of a 50% (vol/vol)
Protein A-agarose suspension were added. After centrifugation for 1 min
at 5000 x g, supernatants were decanted and the
agarose beads washed five times with 1 ml of cold buffer: 75
mM Tris (pH 7.4), 12 mM MgCl2, 2
mM EDTA, protease inhibitors (as above), and 0.05%
digitonin. Immunoprecipitated complexes were counted using a
counter.
Identification of G Proteins Coupled to the Human Mel 1a
Receptor
Membranes (2 mg protein) from HEK 293 cells expressing
approximately 2 pmol of the human Mel 1a receptor per mg of protein
were incubated with or without ligand for 1 h at 25 C. For
ligand-stimulated samples, all subsequent steps were performed in the
continued presence of ligand. Receptors were solubilized and
precipitated with 536-antibodies as described above. G proteins were
dissociated from immune complexes by treatment with GppNHp (0.1
mM) for 1 h at 37 C. Supernatants were harvested and
reincubated with Protein A-agarose to remove residual IgG. Proteins in
the supernatant were precipitated with trichloroacetic acid (15% final
concentration, 5 min at 4 C). The precipitate was centrifuged at 12,
000 x g for 10 min at 4 C. Pellets were washed with
ice-cold acetone, dried, and denatured in 70 mM Tris/HCl
(pH 6.8), 2% SDS, 4 M urea, 40 mM
dithiothreitol, 10% glycerol, and 0.05% bromophenol blue for 5 min at
80 C. Samples were subjected to 10% SDS-PAGE, transferred to
nitrocellulose, and immunoblotted using antisera specific for different
G protein subunits (1:1000 dilution).
SDS-PAGE/Immunoblotting
Cell membranes obtained from Mel 1a receptor-expressing cells
were denatured in 62.5 mM Tris/HCl (pH 6.8), 5% SDS, 3%
2-mercaptoethanol, 10% glycerol, and 0.05% bromophenol blue for
2 h at 37 C. Denatured proteins were separated by 12% SDS-PAGE
and transferred to nitrocellulose. Immunoblot analysis was carried out
in PBS containing 5% skimmed milk powder and 0.2% Tween 20 with
purified 536-antibodies (5 µg/ml). Reactive bands were visualized
using enhanced chemiluminescence.
Native Gel Electrophoresis of Solubilized Receptor
Complexes
Membranes from cells expressing Mel 1a receptors were labeled
with 125I-Mel and solubilized as described above. The
digitonin concentration of the sample was adjusted to 0.05% with cold
buffer: 75 mM Tris (pH 7.4), 12 mM
MgCl2, 2 mM EDTA and protease inhibitors (as
above), and samples were separated by 4–10% gradient native gel
electrophoresis as described previously (21).
125I-Mel-labeled receptor complexes were visualized by
autoradiography. In some experiments 125I-Mel-labeled
proteins were extracted from the gel for further analysis. The
125I-Mel-labeled region was dissected from the gel and
finely minced, before incubation with 100 mM Tris/HCl (pH
8) and 0.1% SDS for 18 h at 37 C. The sample was centrifuged for
10 min at 18,000 x g, and the supernatant was filtered
using a 0.45 µM centrifuge filter unit. The filtrate was
then concentrated using a Centricon 30 unit, before analysis by
SDS-PAGE and immunoblotting with 536-antibodies.
|
ACKNOWLEDGMENTS |
|---|
cDNA. |
FOOTNOTES |
|---|
This work was supported by grants from Centre Nationale de la Recherche Scientifique, the Université de Paris, Institut de Recherches Internationales Servier, the Austrian Science Foundation (FWF-P-12125-GEN to C.N.) and the European Union-sponsored European Network on Biological Signal Transduction. During this work R.J. was supported by the Société de Secours des Amis des Sciences and the Fondation pour la Recherche Médicale.
Received for publication February 12, 1999. Revision received July 23, 1999. Accepted for publication August 27, 1999.
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