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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}-promoted stimulation of phospholipase C, and to modulate protein kinase C (PKC) and phospholipase A2 via Gß{gamma}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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({alpha}) xenope).

 
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. 2Go). 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 ({approx}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. 3AGo). 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. 3BGo). 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 4–10% 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.

 
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. 4AGo). 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. 4AGo). 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 1–2, 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 {gamma} 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 4–10% native gel electrophoresis and visualized by autoradiography.

 
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 (35–40%) of the receptor complex was shifted in the presence of antibodies, indicating the formation of an antibody-receptor-ligand complex (Fig. 4BGo).

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,{gamma}-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. 5Go). GppNHp treatment decreased the amount of precipitated radiolabeled receptor by 48% ± 8 (Fig. 5AGo) 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. 5BGo). 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 4–10% gel electrophoresis and visualized by autoradiography (panel B). Results are expressed as percent of nontreated control. Data are means ± SEM of three independent experiments.

 
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{alpha} protein subunits tested. Gi{alpha}2, Gi{alpha}3, Go{alpha}, Gz{alpha}, Gq{alpha}, Gs{alpha}, and G12{alpha} were readily detected in HEK 293 cells whereas Gi{alpha}1 was less abundant (Fig. 6Go). 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.

 
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. 7Go). In the presence of agonist, Gi{alpha}2, Gi{alpha}3, and Gq/11{alpha} proteins were detected in Mel 1a receptor complexes whereas Gi{alpha}1, Gz{alpha}, Go{alpha}, Gs{alpha}, and G12{alpha} 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.

 
The presence of Gi{alpha}2, Gi{alpha}3, and Gq/11{alpha} proteins in complexes was receptor-dependent since they were not detected when experiments were performed with membranes from nontransfected cells (Fig. 7Go). No G protein was detected in complexes when experiments were performed in the absence of agonist (Fig. 7Go) or in the presence of the melatonin receptor-specific antagonist S20928 (data not shown), demonstrating that receptor-G{alpha} protein interaction was specific for agonist-activated Mel 1a receptors (Fig. 7Go).

The specificity of receptor-G protein coupling may also be examined using subtype-selective G protein antibodies directed against the carboxy terminus of the {alpha}-subunit. The carboxy-terminal domain of the {alpha}-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{alpha}1-3 antibody, as opposed to other antibodies used (anti-Go{alpha}, anti-Gq{alpha}), clearly reduced 125I-Mel binding (Fig. 8AGo). 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{alpha} ({alpha}o-N), which is known not to interfere with receptor-G protein interaction, were ineffective (Fig. 8Go, A and B). Interestingly, in membranes prepared from cells transiently overexpressing Gq{alpha} (by ~10-fold as monitored by Western blotting, data not shown), both anti-Gi{alpha}1-3 and anti-Gq{alpha} antibodies were equally effective in decreasing high-affinity binding (Fig. 8BGo). As expected, the combination of anti-Gi{alpha}1-3 and anti-Gq{alpha} 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{alpha} expression vector (panel B). Affinity-purified antisera (1 µg/assay) directed against distinct sequences in the carboxy terminus of Go{alpha} ({alpha}o-C), Gi{alpha}1-3 ({alpha}i1-3-C), Gq{alpha} ({alpha}q-C), and Gi{alpha}1-3 and Gq{alpha} ({alpha}i1-3/q-C), or the amino terminus of Go{alpha} ({alpha}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.

 
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. 9Go). 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%).

 
Both ß{gamma}-subunits of Gi proteins and {alpha}-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. 10AGo). 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. 10BGo), 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. 10Go, A and B). The effect of melatonin on [Ca2+]i rise was dose dependent with an EC50 of approximately 10-9 M (Fig. 10CGo).



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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.

 
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 {alpha}-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. 10EGo) 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. 10DGo). In contrast, melatonin-induced inhibition of cAMP accumulation was abolished in these cells (Fig. 10FGo). PTX was also without effect on carbachol-induced [Ca2+]i rise (Fig. 10DGo). These data thus indicate that both melatonin and muscarinic receptors stimulate the PLC pathway through Gq/11 proteins.

We have shown above that Gq{alpha} 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. 8Go). We therefore tested whether Gq{alpha} overexpression also increased Ca2+ mobilization. Melatonin-induced Ca2+ mobilization was indeed dependent on the quantity of Gq{alpha} expressed in HEK 293 cells as shown in Fig. 11Go. At 1 µg of Gq cDNA, Ca2+ mobilization was increased by approximately 4.5-fold, and Gq{alpha} 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{alpha} overexpression (Fig. 11Go). 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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Figure 11. Effect of Gq{alpha} Overexpression on Ca2+ Mobilization by Mel 1a Receptors and Muscarinic Receptors

HEK 293 cells stably expressing Mel 1a receptors (0.4 pmol/mg protein) were subjected to mock transient transfection (mock) or to transient transfection with different quantities (0.5 and 1.0 µg/15 million cells) of a Gq{alpha} expression vector. Calcium measurements were carried out as described in Fig. 10Go. Results are expressed as fold increase over melatonin-induced rise in [Ca2+]i in mock transfected cells. Data are means ± SEM of four independent measurements.

 
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. 12AGo). 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. 12CGo). 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. 12BGo). 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 A–C). Arrows indicate time points of melatonin addition. Results are representative of three to eight additional experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 melatonin’s 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{alpha}2, Gi{alpha}3, and Gq/11{alpha} 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{alpha} proteins were overexpressed confirming 1) functional coupling to Gq and suggesting 2) that the quantity of Gq{alpha} 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{alpha} overexpression is a low-affinity binding between Mel 1a receptors and Gq. When the amount of Gq{alpha} is significantly increased, a net increase in interaction, and therefore signaling, would be driven by mass action. In agreement with this interpretation, Gq{alpha} 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{alpha} 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{alpha} 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}1, Gi{alpha}1/2, Gi{alpha}3, and Go{alpha} (ON1 and OC1) antibodies were a generous gift from Dr. Milligan (University of Glasgow, Glasgow, UK) (47). Polyclonal anti-Go{alpha} and Gq/11{alpha} were kindly provided by Drs. Homburger and Guillon (University of Montpellier, Montpellier, France) (48, 49). Polyclonal anti-Gs{alpha} antibodies were provided by Dr. Guillaume (University Paris VII, Paris, France). Polyclonal anti-G12{alpha}, anti-Gi{alpha}1-3, and Gz{alpha} antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The plasmid pCISG{alpha}q expressing the Gq{alpha} 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 {gamma} 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
 
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{alpha} cDNA.


    FOOTNOTES
 
Address requests for reprints to: Dr. Ralf Jockers, Laboratoire d’Immuno-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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