Dual Signaling of Human Mel1a Melatonin Receptors via Gi2, Gi3, and Gq/11 Proteins
Lena Brydon,
Florian Roka,
Laurence Petit,
Pierre de Coppet,
Michèle Tissot,
Perry Barrett,
Peter J. Morgan,
Christian Nanoff,
A. Donny Strosberg and
Ralf Jockers
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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Mel 1a melatonin receptors belong to the
superfamily of guanine nucleotide-binding regulatory protein (G
protein)-coupled receptors. So far, interest in Mel 1a receptor
signaling has focused mainly on the modulation of the adenylyl cyclase
pathway via pertussis toxin (PTX)-sensitive G proteins. To further
investigate signaling of the human Mel 1a receptor, we have developed
an antibody directed against the C terminus of this receptor. This
antibody detected the Mel 1a receptor as a protein with an apparent
molecular mass of approximately 60 kDa in immunoblots after separation
by SDS-PAGE. It also specifically precipitated the
2-[125I]iodomelatonin
(125I-Mel)-labeled receptor from Mel
1a-transfected HEK 293 cells. Coprecipitation experiments showed that
Gi2, Gi3, and
Gq/11 proteins couple to the Mel 1a receptor in
an agonist-dependent and guanine nucleotide-sensitive manner. Coupling
was selective since other G proteins present in HEK 293 cells,
(Gi1, Go,
Gs, Gz, and
G12) were not detected in receptor complexes.
Coupling of the Mel 1a receptor to Gi and
Gq was confirmed by inhibition of high-affinity
125I-Mel binding to receptors with
subtype-selective G protein
-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.
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INTRODUCTION
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Melatonin plays a key role in a variety of important physiological
responses including regulation of circadian rhythms, sleep,
reproduction in seasonally breeding mammals, and vision (1). Melatonin
acts principally via high-affinity receptors coupled to heterotrimeric
guanine nucleotide-binding regulatory proteins (G proteins). Three
high-affinity melatonin receptor subtypes have been cloned so far and
classified as Mel 1a (also called mt1), Mel 1b (or
MT2), and Mel 1c (2). The Mel 1a receptor subtype is
believed to mediate major neurobiological functions of melatonin in
mammals including the modulation of circadian rhythms generated by a
master clock located in the suprachiasmatic nuclei (SCN) (3). Targeted
disruption of the mouse Mel 1a receptor confirmed that this receptor
subtype is indeed involved in the regulation of SCN functions (4).
Human Mel 1a receptors are also expressed in other regions of the
brain, including the cerebellum (5, 6), and in peripheral tissues such
as the fetal kidney (7). The functional significance of receptors
expressed at these sites has yet to be established.
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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Development of 536-Antibodies
Antireceptor antibodies have provided useful information about
signal transduction pathways of G protein-coupled receptors (GPCRs)
(15, 16, 17, 18). To raise antibodies specific for the human Mel 1a receptor, a
peptide corresponding to a sequence found in the C-terminal region of
this receptor (Ac-YKWKPSPLMTNNNVVKVDSV-COO-, peptide 536)
was selected as immunogen (Fig. 1
). 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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Figure 1. Alignment of Peptide 536 with Carboxy-Terminal
Sequences of Melatonin Receptors
Single-letter code is used to denote amino acids.
Letters in bold indicate conserved amino acid residues
and * indicates carboxy termini. Nucleotide sequences for melatonin
receptors were obtained from Genbank using the following accession
numbers: P48039 (Mel 1a human), Q61184 (Mel 1a mouse), P48040 (Mel 1a
sheep), P49285 (Mel 1a chick), P49286 (Mel 1b human), P49288 (Mel 1c
chick), and U67879 (Mel 1c( ) xenope).
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536-Antibodies Recognize Recombinant Mel 1a Receptors as a 60-kDa
Protein
Purified 536-antibodies were tested for their ability to recognize
the recombinant Mel 1a receptor expressed in COS cells. In immunoblot
experiments on membranes from Mel 1a-transfected COS cells, a major
protein band of approximately 60 kDa was detected. This protein was not
detected in membranes prepared from nontransfected cells, and
immunoreactivity was significantly diminished in the presence of
antigenic peptide 536 (Fig. 2
). 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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Figure 2. Immunoblot Analysis of Cell Membranes Expressing
the Human Mel 1a Receptor
Immunoblots of membrane proteins (15 µg/lane) from nontransfected COS
cells (lanes a and c) and COS cells expressing the human Mel 1a
receptor (lanes b and d) were performed using 536-antibodies (5
µg/ml). The detection was inhibited when antibodies were preincubated
with 5 µg/ml of peptide 536 (lanes c and d). The molecular masses of
standard proteins (kDa) are shown on the left.
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Figure 3. 536-Antibodies Recognize the
125I-Mel-Labeled Mel 1a Receptor as a 60- kDa Protein
Membranes were prepared from Mel 1a receptor-expressing COS cells or
nontransfected cells and receptors labeled with 125I-Mel
(400 pM) as described in Materials and
Methods. Receptors were solubilized with digitonin, separated
by native 410% gradient gel electrophoresis, and visualized by
autoradiography (panel A). The labeled complex and the corresponding
region of the nontransfected sample were extracted from the gel,
denatured, subjected to 12% SDS-PAGE, and then transferred to
nitrocellulose and detected using 536-antibodies (5 µg/ml) (panel B).
Results are representative of two additional experiments.
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Antibody Recognition of Native Mel 1a Receptors
We then tested whether 536-antibodies were able to
immunoprecipitate the Mel 1a receptor. Melatonin receptors expressed in
COS cells were labeled with 125I-Mel, solubilized, and
incubated with 536-antibodies. After Protein A-agarose addition,
40 ± 3.8% (n = 3) of labeled Mel 1a receptor complexes
were precipitated (Fig. 4A
). 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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Figure 4. 536-Antibodies Recognize the Native Mel 1a Receptor
A, Immunoprecipitation: COS cells were transfected with human Mel 1a
receptor cDNA, and cell membranes were prepared. Mel 1a receptors were
labeled with 125I-Mel (400 pM) in the presence
(lane 3) or absence (lanes 12, 4) of melatonin (10 µM).
Receptors were solubilized and incubated with 536-antibodies (AS536) or
preimmune serum (PI) (lane 4) in the presence (lane 2) or absence
(lanes 1, 3, and 4) of peptide 536 (P536) for 16 h at 4 C. Immune
complexes were precipitated with Protein A-agarose and quantified using
a counter. Data are means ± SEM of three
independent experiments. B, Visualization of immunoprecipitated
receptor complexes: 125I-Mel-labeled Mel 1a receptors were
solubilized from COS cell membranes as described and incubated with or
without 536-antibodies for 16 h at 4 C. Samples were separated by
410% native gel electrophoresis and visualized by autoradiography.
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Physical interaction between antibodies and native Mel 1a receptors was
further confirmed by an antibody-induced mobility shift of the
125I-Mel-labeled receptor complex in native gel
electrophoresis. The 125I-Mel-labeled receptor was
solubilized and incubated with 536-antibodies as described in
immunoprecipitation experiments, and then separated by native gel
electrophoresis and visualized by autoradiography. Free radioligand
persisted at the loading well of the gel, and a significant portion
(3540%) of the receptor complex was shifted in the presence of
antibodies, indicating the formation of an antibody-receptor-ligand
complex (Fig. 4B
).
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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Figure 5. The Mel 1a Receptor Is Solubilized as a Complex
with Functionally Associated G Proteins
Membranes were prepared from HEK 293 cells stably expressing the human
Mel 1a receptor and receptors labeled with 125I-Mel (400
pM). Solubilized receptors were incubated in the presence
or absence of GppNHp (0.1 mM) for 1 h at 37 C. Labeled
receptors were either immunoprecipitated with 536-antibodies (AS536) or
preimmune serum (PI) (1:40 dilution) (panel A) or separated by native
410% gel electrophoresis and visualized by autoradiography (panel
B). Results are expressed as percent of nontreated control. Data are
means ± SEM of three independent experiments.
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Identification of G Proteins Coupled to the Human Mel 1a
Receptor
A HEK 293 cell clone stably expressing 2 ± 0.1 pmol (n
= 3) of Mel 1a receptor per mg of protein was used for the
identification of G proteins coupled to this receptor by
coimmunoprecipitation. Binding studies with 125I-Mel
showed a dissociation constant (Kd) of 133 ± 15
pM (n = 3), indicating that cells were expressing
high-affinity melatonin receptors. We first determined which G proteins
were expressed in HEK 293 cells and thus available for potential
coupling to Mel 1a receptors. Western blots on rat brain membranes,
used as a positive control, and HEK 293 cell membranes showed the
presence of detectable amounts of all G
protein subunits
tested. Gi
2, Gi
3, Go
,
Gz
, Gq
, Gs
, and
G12
were readily detected in HEK 293 cells whereas
Gi
1 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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Figure 6. G Proteins Endogenously Expressed in HEK 293 Cells
Membranes from HEK 293 (HEK) cells (25 µg protein/lane) or rat brain
(C) (10 µg protein/lane) were denatured as described in
Materials and Methods, separated by 10% SDS-PAGE, and
transferred to nitrocellulose membranes. G proteins were detected with
antisera specific for different G protein subunits. Data are
representative of two further experiments.
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To identify which G proteins were coupled to Mel 1a receptors in HEK
293 cells, membranes from Mel 1a-transfected cells were incubated with
or without melatonin; receptors were then solubilized and incubated
with 536-antibodies. Antibody-receptor complexes were precipitated with
Protein A-agarose, and receptor-coupled G proteins were dissociated by
treatment with GppNHp. Dissociated G proteins were subsequently
separated by SDS-PAGE and identified by Western blot using antisera
specific for different G protein subunits (Fig. 7
). In the presence of agonist,
Gi
2, Gi
3, and Gq/11
proteins were detected in Mel 1a receptor complexes whereas
Gi
1, 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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Figure 7. Detection of G Proteins Coimmunoprecipitating with
the Human Mel 1a Receptor
Membranes from nontransfected HEK 293 cells and HEK 293 cells stably
expressing the Mel 1a receptor were incubated in the presence or
absence of melatonin. For melatonin-stimulated samples, all subsequent
steps were carried out in the continued presence of melatonin.
Receptors were solubilized and incubated with 536-antibodies. Immune
complexes were precipitated with Protein A-agarose, and
receptor-coupled G proteins were dissociated by treatment with GppNHp
(0.1 mM). Dissociated G proteins were precipitated with
trichloroacetic acid, separated by 10% SDS-PAGE, and analyzed by
Western blots using antisera specific for different G protein subunits.
Rat brain membranes (10 µg/lane) were used as positive control for
SDS-PAGE and Western blotting. Data are representative of two
additional experiments.
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The presence of Gi
2, Gi
3, 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-Gi
1-3 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-Gi
1-3 and anti-Gq
antibodies were equally effective in decreasing high-affinity binding
(Fig. 8B
). As expected, the combination of anti-Gi
1-3
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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Figure 8. Uncoupling of Mel 1a Receptors by G Protein
Subtype-Selective Antibodies
Membranes were prepared from HEK 293 cells stably expressing Mel 1a
receptors that had been subjected to a mock transient transfection
(panel A) or to transient transfection with a Gq
expression vector (panel B). Affinity-purified antisera (1 µg/assay)
directed against distinct sequences in the carboxy terminus of
Go ( o-C), Gi 1-3
( i1-3-C), Gq ( q-C), and
Gi 1-3 and Gq ( i1-3/q-C),
or the amino terminus of Go ( o-N) were
preincubated with the membranes (10 µg) in buffer (20 mM
HEPES/NaOH, pH 7.6, 1 mM EDTA, 2 mM
MgCl2 containing 4 mM
3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate)
(CHAPS) for 30 min on ice; thereafter, this mixture was diluted with
the same buffer to yield a final CHAPS-concentration of <1
mM, and radioligand binding was initiated by the addition
of 300 pM 125I-Mel. Nonspecific binding was
determined in the presence of 1 µM melatonin and was not
affected by the addition of antisera. Bars represent
means from three to six experiments ± SEM. Binding
data are expressed as percent of specific binding in the absence of
antibody (control) (0.4 pmol/mg of protein); *, P
< 0.001 vs. control.
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Signaling of Mel 1a Receptors in HEK 293 Cells
We tested whether coupling of Mel 1a receptors to Gi2,
Gi3, and Gq/11 proteins could activate
downstream signal transduction pathways in HEK 293 cells.
Gi proteins are known to inhibit adenylyl cyclase.
Accordingly, incubation of HEK 293 cells expressing the human Mel 1a
receptor with melatonin inhibited forskolin-stimulated cAMP
accumulation (Fig. 9
). 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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Figure 9. Modulation of Forskolin-Stimulated cAMP
Accumulation by the Mel 1a Receptor in HEK 293 Cells
HEK 293 cells stably transfected with Mel 1a receptor cDNA were
stimulated with forskolin (10 µM) in the presence of the
indicated concentrations of melatonin. Intracellular cAMP levels were
determined as described in Materials and Methods. Data
represent the means ± SEM of three independent
experiments performed in duplicate. Data are expressed as percent of
mean forskolin-stimulated value (100%).
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Both ß
-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 6090 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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Figure 10. Effect of Mel 1a Receptor Activation on Cytosolic
Ca2+ Levels in HEK 293 Cells
Cells were loaded with 1 µM Fura2/AM, and changes in
[Ca2+]i were measured as described in
Materials and Methods. Cells stably expressing the Mel
1a receptor (panel A) or nontransfected cells (panel B) were stimulated
with melatonin (Mel, 1 µM) or carbachol (Car, 0.1
mM). Melatonin-induced changes in
[Ca2+]i in Mel 1a receptor-transfected HEK
293 cells were dose dependent (panel C). Cells expressing the Mel 1a
receptor were pretreated or not with PTX (18 h, 100 ng/ml) before
measurements of changes in [Ca2+]i (panel D).
Mel 1a receptor-transfected cells were preincubated (5 min) with 100
nM PMA and then treated with melatonin (Mel, 1
µM) or carbachol (Car, 0.1 mM) (panel E).
Cells were pretreated with or without PTX (18 h, 100 ng/ml) and
stimulated with forskolin (10-6 M, F) and/or
melatonin (10-7 M, M). Intracellular cAMP
levels were determined as described in Materials and
Methods (panel F). Results are representative of two additional
experiments.
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The muscarinic receptor-induced increase in
[Ca2+]i is known to be desensitized by the
PKC activator phorbol 12-myristate 13-acetate (PMA) (26). PKC-mediated
phosphorylation of components of the phospholipase C (PLC) pathway such
as PLC itself and/or G protein
-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.
Melatonin Induces Ca2+ Mobilization in
Primary Cultures of Ovine Pars Tuberalis Cells Expressing Endogenous
Mel 1a Receptors
Among several tissues expressing Mel 1a receptors, the
pituitary pars tuberalis is one of the major sites of expression of
this receptor. Therefore, primary cultures of ovine pars tuberalis
cells have become a preferred model system for the study of
endogenously expressed Mel 1a receptors (1). We established primary
cultures of these cells, loaded them with Fura-2/AM, and measured
changes in [Ca2+]i with a single cell
measuring device. Melatonin induced a transient increase in
[Ca2+]i in 11.8 ± 3.0% of all recorded
cells (n = 5) (Fig. 12A
). 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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|
Figure 12. Fura-2 Imaging of Single Ovine Pituitary PT Cells
PT cells were loaded with 2 µM Fura 2/AM, and changes in
cytosolic [Ca2+]i were determined by ratio
imaging of fura-2 fluorescence as described in Materials and
Methods. Cells were pretreated with PTX (18 h, 100 ng/ml)
(panel B) or EGTA (2 min, 5 mM) (panel C) before addition
of KRH buffer containing 1 µM melatonin (panels AC).
Arrows indicate time points of melatonin addition.
Results are representative of three to eight additional experiments.
|
|
 |
DISCUSSION
|
---|
Hormonal activation of a receptor often results in the stimulation
of multiple signal transduction pathways. So far, interest in Mel 1a
receptor signaling has mainly focused on the adenylyl cyclase pathway
(8). We now show that Mel 1a receptors indeed interact with a limited
set of G proteins: Gi2, Gi3, and
Gq/11 proteins but not Gi1, Go,
Gz, Gs, and G12 proteins. Coupling
to PTX-sensitive Gi proteins is responsible for
melatonins inhibitory effect on intracellular cAMP levels. Coupling
to PTX-insensitive Gq/11 proteins activates a second
pathway, the mobilization of Ca2+ from intracellular stores
via PLC in Mel 1a-transfected HEK 293 cells and in primary cultures of
ovine pars tuberalis cells expressing endogenous Mel 1a receptors.
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 Gi
2, Gi
3,
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 melatonins 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 brutons 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
brutons 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.
 |
MATERIALS AND METHODS
|
---|
Materials
Peptide 536 was synthesized and purified by reverse phase HPLC
at the Gustave-Roussy Institute (Villejuif, France). Rabbit polyclonal
anti-536 sera were produced by Eurogentec (Ougrée, Belgium).
2-[125I]iodomelatonin was from DuPont-New England Nuclear
(Boston, MA). S20298 was provided by the Institut de Recherches
Internationales Servier (Courbevoie, France). DMEM, FCS, lamb serum,
PBS, penicillin, streptomycin, Amphotericin, and geneticin
(G418) were from Life Technologies, Inc. (Gaithersburg,
MD).
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
methylsulfate and Protein A-agarose were purchased from
Roche Molecular Biochemicals (Mannheim, Germany).
Digitonin was obtained from Gallard-Schlesinger Industries, Inc. (Carle
Place, NY). Peroxidase-conjugated goat antirabbit IgG was obtained from
Jackson ImmunoResearch Laboratories, Inc. (West Grove,
PA). Polyclonal anti-Gi
1, Gi
1/2,
Gi
3, 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-Gi
1-3, and
Gz
antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The plasmid pCISG
q
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 Freunds 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 (0.42 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 12 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 80120 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 410% 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
|
---|
We are grateful to Dr. Camoin (Institut Cochin de Genetique
Moleculaire ICGM), Paris, France) for his expert advice on choice of
peptide antigen and antibody purification. We thank Dr. Perianin (ICGM)
and Drs. Irving and Brown (University of Aberdeen, Aberdeen, UK) for
valuable advice on Ca2+ measurements. We are grateful to
Dr. Marullo (ICGM) for his help during preparation of the manuscript.
We thank Dr. Milligan (University of Glasgow, Glasgow, UK) and Drs.
Homburger and Guillon (University of Montpellier, Montpellier, France)
for providing polyclonal anti-G protein antisera. We are grateful to
Dr. Simon (Pasadena, CA) for supplying the Gq
cDNA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Ralf Jockers, Laboratoire dImmuno-Pharmacologie Moléculaire, Institut Cochin de Génétique Moléculaire, 22, rue Méchain, F-75014 Paris, France.
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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