Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones

Definitions

• the present invention relates to nucleic acid sequences coding for xenonope melatonin receptors of the MEL1 A type, also hereinafter called Mel 1c , to oligonucleotides included in said sequences, to their applications as a probe and to the expression of proteins and / or fragments thereof having a functional melatonin receptor activity of the MEL1 A type, to the vectors useful for said expression, to the cellular hosts containing said vectors as well as to a model for studying the receptors melatonin.
• the present invention also relates to a method for screening substances, with agonist or antagonist action against proteins having a melatonin receptor action and with kits or kits for detecting the degree of affinity of different substances for said proteins with melatonin receptor activity.
• primers located in the 3 ′ part of the corresponding nucleic acid sequences did not make it possible to amplify the sequence coding for this protein.
• the subject of the present invention is nucleic acid sequences encoding functional melatonin receptors, of the MEL1 A type and of structure different from that of the receptor described in EBISAWA et al. and having coupling and regulatory properties different from those of the previously described receptor, which sequences are selected from the sequences SEQ ID No 1, SEQ ID No 3, SEQ ID No 5 or SEQ ID No 7, such as presented in the list of sequences included in this Request.
• sequences according to the invention have, in 3 'a more or less long non-coding sequence (long sequences: SEQ ID No. 1 and SEQ ID No. 5; short sequences: SEQ ID N ° 3 and SEQ ID N ° 7), which plays a crucial role in the regulation of RNA: 1 ⁇ 2 life of TARN, modified compared to the sequences of the prior art.
• SEQ ID N ° 1 and SEQ ID N ° 3 code for a protein having 65 fewer amino acids, compared to the sequence described in the prior art, the 2 amino acids in the C-terminal position being, in addition, different.
• This protein corresponds to the melatonin receptor called MEL1 Aa or
• SEQ ID N ° 5 and SEQ ID N ° 7 code for a protein also having 65 amino acids less, compared to the sequence described in the prior art; in addition, 6 amino acid residues are different, with respect to said sequence of the prior art.
• This protein corresponds to the melatonin receptor called MEL1 Ab or Mel 1c ( ⁇ ) .
• Both the sequence coding for the MEL1 Aa or Mel 1c ( ⁇ ) receptor and the sequence coding for the MEL1 Ab or Mel 1c ( ⁇ ) receptor could lead to modifications in RNA regulation, related to the non-coding sequence in 3 '(regulation by an agonist ligand, lifespan of the RNA).
• MEL1 Aa or Mel 1c ( ⁇ ) receptor inhibits adenylyl cyclase; in fact, the MEL1 Ab or Mel 1c ( ⁇ ) receptor lacks this property to inhibit adenylyl cyclase.
• allelic isoforms are capable of modulating the intracellular cGMP (dose-dependent modulation) and in particular of inhibiting the accumulation of cGMP induced by a phosphodiesterase inhibitor.
• the present invention also relates to the fragments of said sequences, useful either for functional expression of the peptide corresponding to the corresponding melatonin receptor, or for the detection of the sequence coding for said receptor.
• the invention includes, among others: - A sequence consisting of a segment of 230 nucleotide base pairs, corresponding to nucleotides 1057-1286 of SEQ ID No. 1 or SEQ ID No. 5;
• the present invention also relates to nucleotide probes, characterized in that they hybridize with the nucleotide sequences as defined above.
• Suitable hybridization conditions are in particular the following: the hybridization is carried out at 42 ° C. in a buffer comprising: 4X SSC, 40% formamide, 0.2% SDS and Denhardt 5X buffer.
• Such probes have the particular advantage of allowing the characterization of the different variants of the functional melatonin receptor of the MEL1 A type (MEL1 Aa or MEL1 Ab).
• a subject of the present invention is also proteins and / or protein fragments, characterized in that they are coded by a nucleotide sequence as defined above and in that they exhibit melatonin receptor activity of the type MEL1 functional.
• said protein is selected from any one of the sequences SEQ LD No. 2 (SEQ ID No. 4 being identical to SEQ ID No. 2) or SEQ ID No. 6 (SEQ LD N ° 8 being identical to SEQ ID N ° 6), as presented in the sequence list included in this Request.
• SEQ ID N ° 2 and N ° 4 correspond to the melatonin receptor called MEL1 Aa or Mel 1c ( ⁇ ) ;
• SEQ ID N ° 6 and N ° 8 correspond to the melatonin receptor called MEL1 Ab or Mel 1c ( ⁇ ) .
• the present invention also relates to a recombinant cloning and / or expression vector, characterized in that it comprises a nucleotide sequence in accordance with the invention.
• the term “recombinant vector” means both a plasmid, a cosmid and a phage.
• said vector consists of a recombinant vector into which is inserted a nucleotide sequence as defined above, which vector is an expression vector of a protein having a functional receptor activity of the melatonin type MEL1 A, according to the invention.
• said vector consists of a plasmid pcDNA3 (Invitrogen), comprising a promoter RSV and SEQ LD No. 1.
• Such a plasmid was named X-MEL1a and was deposited under the number I-1583 on June 7, 1995 with the National Collection of Cultures of Microorganisms held by the Institut Pasteur.
• said vector consists of a plasmid pcDNA3 (Invitrogen), comprising a promoter RSV and SEQ ID No. 5.
• Such a plasmid was named X-MEL1b and was deposited under the number 1-1584 on June 7, 1995 with the National Collection of Cultures of Microorganisms held by the Institut Pasteur.
• the present invention also relates to an appropriate host cell, obtained by genetic transformation, characterized in that it is transformed by an expression vector in accordance with the invention.
• Such a cell is capable of expressing a protein, having a functional melatonin receptor activity, of the MEL1 A type.
• the host cell is in particular constituted by cells of the L line.
• the present invention also relates to a process for the expression of a protein in accordance with the invention, characterized in that the host cell, resulting from the transformation by a vector containing a nucleotide sequence that according to the invention coding for a protein having a functional melatonin receptor activity of the MEL1 A type, is cultured so as to produce and transport said expressed protein towards the membrane, so that the transmembrane sequences of said receptor are exposed on the surface of the membrane of the transformed cell host.
• the present invention also relates to a model for studying melatonin receptors of the MEL1 A type, characterized in that it consists of host cells in accordance with the invention, that is to say expressing a receptor functional melatonin type MEL1 A on the surface of their cell membrane.
• Such a model allows the study, from a pharmacological point of view, of melatonin receptors, in particular by allowing the identification of specific ligands of MEL1 A type receptors (agonists and antagonists) and thus the development of drugs. active on the regulation of the biological clock, with particularly interesting applications, in particular in the cardiovascular field (lipolysis) and in oncology (phasing of cells).
• the subject of the invention is also a method for detecting the capacity of a substance to behave as a ligand with respect to a protein according to the invention, which method is characterized in that it comprises:
• the present invention further relates to a method for studying the affinity of a protein according to the invention for one or more determined ligands, which method is characterized in that it comprises:
• the culture of the transformed host cell under conditions allowing the expression of the melatonin receptor of the MEL1 A type, coded by the nucleotide sequence, and the transfer of the expressed melatonin receptor to the membrane of said cell so that the transmembrane sequences of the functional melatonin receptor are exposed on the surface of the transformed host cell;
• a further subject of the invention is a kit for detecting the possible affinity of a ligand for a protein according to the invention, which kit is characterized in that it comprises:
• a protein (melatonin receptor of the MEL1 A type), encoded by a nucleotide sequence in accordance with the invention, contained in a vector whose promoter is inducible;
• the melatonin receptors of the MEL1 A type according to the invention are effectively functional receptors and allow the realization of all the aforementioned applications.
• FIG. 1 shows a diagram of the technique of cloning the cDNA of the melatonin receptor, from Xenopus laevis;
• FIG. 2 illustrates the different cloning fragments used; the localization of the sense (S) ( ⁇ ) and antisense (AS) ( ⁇ ) primers specific for the cDNA sequence coding for the melatonin receptor, previously identified from Xenopus dermis melanophores; the location of the primers which do not correspond to this sequence ( ⁇ ) are also illustrated in this figure;
• FIG. 3 illustrates the steps for cloning and sequencing the cDNA fragments of the melatonin receptor
• FIG. 4 corresponds to a comparison of the sequences coding respectively for the melatonin receptors Mel 1c ( ⁇ ) and Mel 1c ( ⁇ ) : the sequence of the melatonin receptor Mel 1c ( ⁇ ) is illustrated (O) and the substitutions characterizing the melatonin receptor Mel 1c ( ⁇ ) ( ⁇ ) are indicated in FIG. 4A; the long and short 3 'untranslated region DNA sequences are shown in Figure 4B.
• the Mel 1c ( ⁇ ) receptors are preferentially associated with the short 3 'untranslated region
• FIG. 5 illustrates the coding part of cDNA sequence coding for the functional melatonin receptor of type MEL1 A (a or b) in comparison with the sequence EBISAWA et al. (reference cited above);
• FIG. 6 illustrates the characterization of the DNAs coding for the Mel 1c ( ⁇ ) / Mel 1c ( ⁇ ) receptors in different types of Xenopus.
• the PCR amplification used in this test provides fragment 3 of FIG. 2.
• the restriction enzymes used for the digestion of the PCR amplification products obtained are: Afl II (1 site at a time in the sequence Mel 1c ( ⁇ ) and the sequence Mel 1c ( ⁇ ) ), Bsp120 I (1 site in the sequence Mel 1c ( ⁇ ) , no site in the sequence Mel 1c ( ⁇ ) ), Pm1I (1 site in the sequence Mel 1c ( ⁇ ) , no site in the Mel 1c ( ⁇ ) sequence).
• the restriction analysis is carried out using PCR products derived from the following genomic DNAs: 1, X. tropicalis; 2, X. ruwenzoriensis; 3, individual X. homozygous laevis (ff); 4, Mel 1c ( ⁇ ) cDNA clone; 5, cDNA of Mel 1c ( ⁇ ) clone; 6, X. laevis cDNA amplified from a pool of skin mRNAs; 7, individual X. heterozygous laevis (rf); ND, undigested PCR product.
• FIG. 7 illustrates the pharmacological specificity of the 2- ( 125 I) -iodomelatonin / melatonin receptor bond, in the presence of stable L cells expressing a receptor according to the invention (Mel 1c ( ⁇ ) or Mel 1c ( ⁇ ) );
• FIG. 7A shows the saturation isotherm of the 2- ( 125 I) iodomelatonin bond; non-specific binding is measured in the presence of 10 ⁇ M melatonin; this FIG. 7A includes the Scatchard representation of the data in an overlay.
• 7B represents the study of the competitive bonds of 2- ( 125 I) -iodomelatonin (400 ⁇ M) to the receptor, carried out on L cell membranes, expressing either the Mel 1c receptor ( ⁇ ) or the Mel 1c receptor ( ⁇ ) , in the presence of I-melatonin ( ⁇ ), melatonin (D), N-acetyl-5-hydroxy ⁇ tryptamine (NAS) (O), S22153 ( ⁇ ) and S20098 (B).
• FIG. 8 illustrates the modulation of the accumulation of cAMP, stimulated by forskolin, by the receptors Mel 1c ( ⁇ ) and Mel 1c ( ⁇ ) .
• Stable clones of L cells transfected with cDNA encoding either the Mel 1c receptor ( ⁇ ) ( ⁇ ) or the Mel 1c receptor ( ⁇ ) (O) are stimulated by forskolin (FK) (10 ⁇ M) in the presence of the indicated concentrations of melatonin (on the abscissa); on the ordinate, this figure includes the rate of intracellular cAMP in%.
• the value 100% ( ⁇ ) corresponds to the average value of cAMP in the presence of forskolin 10 ⁇ M; ( ⁇ ) indicates the cAMP value, in the presence of melatonin (10 ⁇ M) alone (* P ⁇ 0.05).
• FIG. 9 illustrates the modulation of the accumulation of cAMP stimulated by isoproterenol, by melatonin receptors, in transient transfection trials.
• the Mel 1c receptor ( ⁇ ) or the Mel 1c receptor ( ⁇ ) is co-expressed with the ⁇ 2 adrenergic receptor ( ⁇ 2-RA), in equal amounts in L cells and stimulated with isoproterenol (ISO, 10 ⁇ M) , in the presence or absence of melatonin (Mel, 10 ⁇ M): cAMP levels are measured; NT means non-transfected.
• the number of specific binding sites to ICYP and to 2-I-iodomelatonin [represented as follows: (specific ICYP binding sites / specific binding sites iodomelatonin) and expressed in fmol / mg of protein], in these transfected cells are respectively: NT (24/0); Mel 1c ( ⁇ ) (5/77); Mel 1c ( ⁇ ) (13/148); ⁇ 2-RA (622/0); ⁇ 2-RA / Mel 1c ( ⁇ ) (386/54), ⁇ 2-RA / Mel 1c ( ⁇ ) (416/151) (* P ⁇ 0.05).
• - Figure 10 illustrates the modulation of the accumulation of cGMP by melatonin receptors. In FIG.
• L cells transiently transfected with a cDNA coding either for the Mel 1c receptor ( ⁇ ) ( ⁇ , O) or for the Mel 1c receptor ( ⁇ ) ( ⁇ , ⁇ ), are incubated with the concentrations indicated on the abscissa of melatonin, in the presence (O, ⁇ ) or in the absence ( ⁇ , ⁇ ) of 1 mM of TMBX.
• a cDNA coding either for the Mel 1c receptor ( ⁇ ) ( ⁇ , O) or for the Mel 1c receptor ( ⁇ ) ( ⁇ , ⁇ )
• the L cells transiently transfected with a cDNA coding either for the Mel 1c receptor ( ⁇ ) or the Mel 1c receptor ( ⁇ ) are preincubated either in a buffer or with Pertussis toxin (PTX, 100 ng / ml) for 18 hours, then incubated in the presence of 1 mM IBMX and in the absence or in the presence of 10 ⁇ M of melatonin.
• the intracellular cGMP levels are determined.
• EXAMPLE 1 Isolation and identification of the 4 functional sequences of the melatonin receptor of the MELl A type.
• RNA of the skin of Xenopus laevis is previously treated with 0.3 U of DNAse I without RNAse (RQ1 DNase, Promega) for 20 min at 37 ° C per mg of nucleic acid.
• the synthesis of the cDNA is carried out by incubation of 200 ng of RNA (heated at 65 ° C. for 5 min before the reaction) with 100 units of MMLV reverse transcriptase "Stratacript RNAse H-" of Stratagene for 30 min at 37 °. vs.
• cDNA is amplified by PCR with different pairs of oligonucleotides specific for the Xenopus laevis melatonin receptor, Pwo DNA polymerase (Boehringer Mannheim) with its own buffer, in the presence of 2 mM MgSO4, in a volume of 50 ⁇ l.
• the cDNA is denatured for 2 min at 94 ° C., amplified 40 times; an extension of 3 min at 72 ° C. is then carried out.
• One cycle corresponds to 15 sec. at 94 ° C; 30 sec. at the specific temperature of the oligonucleotide pair and 90 sec.
• More than 80% of the desired cDNA was amplified using 3 different pairs of primers which include overlapping sequences (fragments 1-3), which correspond to the region coding for the fragment up to Ala 349.
• This remaining part of the melatonin receptor cDNA was amplified using a modified PCR reaction, using the polyadenylation site, at the 3 'end of the cDNA as a primer (principle described in Figure 3).
• Two amplification fragments (estimated at 400 bp (short fragment) and 600 bp (long fragment), respectively) were obtained using this approach.
• the short 400 bp fragment contains 250 non-coding bp (3 'short) and 150 bp coding; the 600 bp fragment contains 450 bp non-coding (3 'long) and 150 bp coding.
• the amplification products were cloned separately into a vector and characterized by enzymatic digestion and by sequencing by the dideoxy method (4-6 clones / fragment).
• Fragment 1 corresponds to the sequence 28-241: 3 clones have been sequenced, which are identical with the published sequence (EBISAWA et al.).
• Both the short and long forms of the 4 fragments have a strong homology with the published sequence of the melatonin receptor up to methionine at position 353.
• fragment 4 With regard to fragment 4 short (see SEQ TD No. 5 and No. 7) (fragment C), beyond methionine 353, two different amino acids have been detected, i.e. tyrosine at instead of leucine (position 354) and valine in place of glycine (position 355), followed by a stop codon, causing the loss of 65 amino acids, if compared with the published sequence of the melatonin receptor of Xenopus laevis ( Figure 4 A).
• fragment 4 long fragment L
• fragment L see SEQ ID N ° 1 and N ° 3
• 4 clones have been sequenced, one of them corresponds to the sequence published up to methionine 353, 3 of them show the same change as that observed in the short fragment, up to methionine 353.
• amino acids 354 and 355 are modified compared to the published sequence and have a stop codon at position 356, as seen in the short fragment.
• the 3 'non-coding region of the long fragment is identical to that of the short segment, up to the polyadenylation tail; it also comprises 160 bp, followed by its own polyadenylation tail (see list of sequences, SEQ ID N ° 1 and N ° 3) (see Figure 2).
• the cDNA coding for the complete melatonin receptor can be amplified from the DNA of xenopus skin, by PCR, using the primers 3 S and 12 AS, located in the 3 'non-coding region ( see figure 2).
• the 2 mRNAs code for proteins of 354 amino acids which are very similar to the Mel lc chicken receptor (78% homology).
• Fragment C (short fragment) was therefore also called Mel 1c ( ⁇ ) and fragment L (long fragment) (comprising many substitutions) was called Mel 1c ( ⁇ ).
• EXAMPLE 2 Construction of a vector for the expression of the functional melatonin receptor of the MELl A type.
• the Xenopus laevis melatonin receptor was cloned according to the RT-PCR method (see Figure 1).
• RNA is isolated from the skin of Xenopus and transcribed into cDNA using reverse transcriptase.
• the amplified receptor is cloned into the expression vector pcDNA3-RSV derived from the plasmid pcDNA3 (Invitrogen) and its identity has been verified by sequencing, as follows:
• the highly homologous Mel 1c ( ⁇ ) (or Mell Aa) and Mel 1c ( ⁇ ) (or Mell Ab) receptors can represent either 2 different alleles at the same genomic locus, or 2 isoforms encoded by different genes.
• fragment 3 coding for the Mel 1c receptors were amplified by PCR, from genomic DNA of 43 Xenopus laevis.
• the vector pcDNA3-RSV containing the coding region and the non-coding region 3 'of the melatonin receptor of Xenopus laevis according to Example 2, is transfected into the L cells of mice.
• a coprecipitation of DNA and CaPO 4 is prepared as follows: in a final volume of 500 ⁇ l, 30 ⁇ g of carrier DNA (pGEM3Z from Promega), 2 ⁇ g of the vector pcDNA3-RSV containing the region are mixed coding and the 3 'non-coding region of the melatonin receptor of Xenopus laevis and CaCl 2 (250 mM final) (Solution A).
• the cells are washed twice with DMEM and left in 6 ml of DMEM / FCS, supplemented with 5 mM of nabutyrate at 37 ° C overnight. The following day, the medium is removed, the cells are again washed with DMEM / FCS then incubated overnight in DMEM / FCS.
• the cells are distributed in 96-well plates at different dilutions (1/2, 1/4, 1/8, 1/16, 1/32) in DMEM medium supplemented with: 10% FBS ( v / v), 4.5 g / l glucose, 100 U / ml penicillin, 100 mg / ml streptomycin, 1 mM glutamine and 400 ⁇ g / ml G418 (geneticin from Gibco Life Technologies).
• the L cells are spread either on plates comprising 6 wells (for cAMP tests, 0.5 ⁇ 10 6 cells / well), or in dishes 10 cm in diameter (cGMP tests, 2.10 6 cells / dish) and transfected by the DEAE-dextran method (LOPOTA et al., NAR, 1984, 12, 5707-5717), the following day. After washing with PB S, DMEM without serum, containing Hepes 50 mM, DEAE-dextran at 200 ⁇ g / ml and 1 ⁇ g / ml of plasmid DNA plasmid pcDNA3-RSV), is added.
• the medium is replaced with 10% DMSO in DMEM without serum for 1.5 min.
• the cells are washed with PB S and incubated in a DMEM medium containing 10% fetal calf serum. The tests are carried out three days after the transfection.
• the monolayers of sub-confluent cells are washed with
• the cell suspensions are incubated with 400 ⁇ M of 2- ( 125 I) -iodomelatonin (for binding tests on melatonin receptors) or 200 ⁇ M ( 125 I) -CYP (for tests of binding to ⁇ 2 adrenergic receptors) in the absence or presence of 10 ⁇ M of cold ligands (melatonin or D / L-propranolol, respectively).
• Protein concentrations are measured on cell homogenates by the Bradford method (Analytical Biochem., 1976, 72, 248-254), using the Bio-Rad protein assay system. Bovine serum albumin is used as standard. * Results
• the dissociation constants Ko of the radiolabelled agonist 2- ( 125 I) - iodomelatonin are 160 ⁇ 32 and 143 ⁇ 25 pM respectively (FIG. 7A), values which are in agreement with those determined for other melatonin receptors of high affinity, including those previously cloned, from melanophores of X. laevis.
• Competitive experiments have shown that the affinity for different ligands is characteristic of the melatonin receptors (FIG. 7B and Table I).
• the cells grown in plates containing 6 wells are washed twice with DMEM without serum, preincubated for 15 min at 37 ° C., then incubated for 15 min at 37 ° C. in DMEM medium containing 1 mM of TBMX ( 3-isobutyl-1-methylxanthine) with or without isoproterenol (10 ⁇ M) (tests on transient transfected cells), or 10 ⁇ M of forskolin (tests on stable cell clones) and increasing amounts of melatonin.
• TBMX 3-isobutyl-1-methylxanthine
• isoproterenol 10 ⁇ M
• tests on transient transfected cells tests on transient transfected cells
• 10 ⁇ M of forskolin tests on stable cell clones
• the cell lysates are centrifuged in a microcentrifuge at 17,000 xg for 5 min.
• the supernatants are tested for their content in cyclic AMP using the 3 H cyclic AMP system (Amersham).
• the tests for measuring cyclic AMP can be carried out in plates containing 24 wells (0.5 ⁇ 10 6 cells in 300 ⁇ l). The cells are then incubated for 15 min and the reaction is stopped by the addition of 100 ⁇ l of 20% trichloroacetic acid and the supernatants are tested for their content in cyclic AMP.
• the IC 50 value of this effect is approximately 6.10 -10 M, which is in agreement with the values obtained previously with melatonin receptors having a high affinity.
• baseline levels of cAMP are not affected by melatonin in any of the cell lines studied, even in the presence of LMBX.
• the inhibition of the accumulation of cAMP by melatonin receptors is coupled to the proteins G i ., Via their ⁇ subunits.
• the L control cells and clones expressing either the Mel 1c ( ⁇ ) or Mel receptors 1c ( ⁇ ) are compared as regards their content in G i ⁇ subunits, in accordance with the following protocol:
• the cells are dissolved in Laemmli buffer containing 2% SDS and 40 mM dithiothreitol and sonicated at 4 ° C. After centrifugation in a microcentrifuge at 17,000 xg, 50 ⁇ l of supernatant is separated into its components in a 12% SDS / polyacrylamide gel. The analysis of the immunoblots is carried out as described in SELZER E. et al. (Proc. Natl. Acad. Sci. USA, 1993, 90, 1609-1613), with 84 antisera which correspond to the 3 subtypes of G i ⁇ .
• L cells are co-transfected with the cDNA of the human ⁇ 2 adrenergic receptor and with either the cDNA of the Mel 1c ( ⁇ ) receptor or of the Mel 1c ( ⁇ ) receptor; the co-expression of these different receptors makes it possible to selectively study the subpopulation of cells which has effectively internalized exogenous DNA.
• No binding site, for any of the receptors, can be measured in wild-type control cells.
• incubation with isoproterenol results in a 5-fold increase in cAMP.
• in cells co-transfected with identical amounts (1 ⁇ g of plasmid DNA) of cDNA encoding the Mel 1c ( ⁇ ) and adrenergic ⁇ 2 receptors the simultaneous incubation with isoproterenol and melatonin results in a decrease significant of the accumulation of cAMP (65 ⁇ 5% of the control, p ⁇ 0.05) ( Figure 9).
• the Mel 1c ( ⁇ ) receptors are not capable of inhibiting the accumulation of cAMP induced by isoproterenol, despite the expression of an equivalent number of receptor binding sites. Similar results are obtained when the cDNA coding for the Mel 1c receptor ( ⁇ ) is in excess of a factor of 10 compared to the cDNA coding for the adrenergic receptor ⁇ 2.
• Mel 1c ( ⁇ ) receptors inhibit the accumulation of forskolin-stimulated cAMP in a dose-dependent manner with an IC 50 value of approximately 6.10 -10 M, a value consistent with that reported for melatonin receptors previously described.
• the inhibition of the adenylyl cyclase function is mainly coupled to the activated G i proteins.
• the transiently transfected cells grown in dishes having a diameter of 10 cm, are incubated at 37 ° C for 15 min in DMEM medium without serum, in the presence or in the absence of 1 mM of LMBX and of quantities increasing melatonin.
• the medium is then replaced with 1 ml of ice-cold 65% ethanol and the cell extracts are centrifuged at 2000 x g for 15 min at 4 ° C.
• the supernatants are dried; the pellets are resuspended in 250 ⁇ l of an acetylated buffer.
• the cyclic GMP is dosed in accordance with the EIA kit (Amersham). Additional pathways such as modulation of cGMP content have been proposed for the melatonin receptor (VANECEK J. et al., Brain Res., 1989, 505, 157-159).
• the L cells expressing either the Mel 1c ( ⁇ ) receptors or the Meli c ( ⁇ ) receptors are incubated with melatonin and the effects on intracellular cGMP are analyzed.
• Melatonin up to 10 ⁇ M has no effect on baseline cGMP levels in any of the cell lines.
• the intracellular cGMP content depends on the opposite activity of guanylyl cyclases, which synthesize cGMP and phosphodiesterases (PDE), which degrade cGMP.
• PDE phosphodiesterases
• the PDE inhibitor IBMX when added to the medium, blocks the degradation of cGMP by PDE. Under these conditions, the level of cGMP increases by a factor of 3, indicating the presence of basal PDE activity in the unstimulated L cells.
• the stimulation by melatonin of the control cells does not affect the increase in production of cGMP by TBMX.
• Pertussis toxin which catalyzes the inactivating ribosylation of ADP of the ⁇ G i / o subunits of G proteins, blocks the inhibition of the accumulation of cAMP linked to the protein G i and stimulated by the receptors to the melatonin. Preincubating cells with Pertussis toxin completely abolishes the effect of melatonin on cGMP accumulation, suggesting that this pathway is also dependent on the G i / o protein.
• the 2 melatonin receptor cDNAs Mel 1c ( ⁇ ) and Mel 1c ( ⁇ ) are found either in long form or in short form. Most of the Mel 1c receptor ( ⁇ ) cDNA is in the short form (3: 1, short: long), while the Mel 1c receptor cDNA ( ⁇ ) is more abundant in the long form (1 : 3).
• the 3 'untranslated regions of several receptors of the same family, such as the ⁇ 2 adrenergic receptor or the muscarinic receptor contain molecular determinants involved in the regulation of mRNA stability. This suggests that the short and long mRNAs encoding the Mel 1c ( ⁇ ) and Mel 1c ( ⁇ ) receptors are subject to different regulations.
• cGMP As a second messenger in the biological pathway of the melatonin receptor is still controversial. Pigmentary aggregation in Xenopus melanocytes is regulated by melatonin and by cAMP whereas contradictory results exist regarding its regulation by cGMP. The role of cGMP in stimulating biological signals linked to melatonin in other cellular systems has not been studied. A second argument in favor of the modulation of cGMP by melatonin receptors emerges from the tests carried out on neonatal rat pituitary tissue in which a melatonin inhibitory effect on cGMP production is observed.

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