EP0889958A2 - HUNDE Beta-2 UND Beta-3 ADRENERGISCHE REZEPTOREN UND DEREN ANWENDUNGEN - Google Patents

HUNDE Beta-2 UND Beta-3 ADRENERGISCHE REZEPTOREN UND DEREN ANWENDUNGEN

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Publication number
EP0889958A2
EP0889958A2 EP97916504A EP97916504A EP0889958A2 EP 0889958 A2 EP0889958 A2 EP 0889958A2 EP 97916504 A EP97916504 A EP 97916504A EP 97916504 A EP97916504 A EP 97916504A EP 0889958 A2 EP0889958 A2 EP 0889958A2
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Prior art keywords
canine
receptor
adrenergic
vector
cells
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English (en)
French (fr)
Inventor
Gerlinde Lenzen
France Pietri-Rouxel
Marie-Françoise DRUMARE
Arthur Donny Strosberg
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Vetigen
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Vetigen
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    • 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
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • the present invention relates to nucleotide sequences coding for certain canine ⁇ -adrenergic receptors, in particular ⁇ 2- and ⁇ 3- receptors, as well as to the use of these sequences as probes, for the expression of ⁇ - receptors.
  • canine adrenergics to study the tissue localization of the expressed receptor (PCR, Northern blot), to carry out site-directed mutagenesis and for the structure-function study of receptors.
  • the present invention also relates to the use of said canine ⁇ -adrenergic receptors for the preparation of specific antibodies.
  • the present invention also relates to a method for the differential screening of substances, with agonist or antagonist action with respect to canine ⁇ 3-adrenergic receptors and with kits or kits for detecting the degree of affinity of different substances for said substances.
  • canine ⁇ 3-adrenergic receptors Such substances are used in particular for the selective treatment of obesity in dogs or other metabolic disorders linked to obesity or to any other pathology involving canine ⁇ 3 receptors.
  • catecholamines such as adrenaline and noradrenaline
  • synthetic agonists of ⁇ -adrenergic receptors which mimic their biological functions and antagonists, which block these biological functions, exert their effects by binding to recognition sites (membrane receptors) specific, located in the plasma membranes of cells.
  • recognition sites membrane receptors
  • Two main categories of adrenergic receptors have been defined, the ⁇ adrenergic receptors and the ⁇ adrenergic receptors.
  • catecholamine receptors ⁇ 1, ⁇ 2, ⁇ 1, ⁇ 2 and ⁇ 3-RA.
  • Their genes have been recently isolated and identified (S. COTECCHIA et al., 1988, Proc Natl. Acad. Sci. USA, 85, 7159-7163; BK KOBILKA et al., 1987, Science, 238, 650-656; T FRIELLE et al., 1987, Proc. Natl. Acad. Sci. USA 84, 7920-7924; LJ EMORINE et al., 1987, Proc. Natl. Acad.
  • membrane receptors when they have fixed the appropriate ligand (agonist or antagonist), undergo a change in conformation, which induces (agonist) or blocks (antagonist) an intracellular signal, which modifies the behavior of the target cell.
  • agonists bind to ⁇ -adrenergic receptors (RA- ⁇ ) they activate a class of proteins G, which in turn stimulates the activity of adenylyl cyclase, while the antagonists bind to RA- ⁇ , but do not activate adenylyl cyclase.
  • RA- ⁇ ⁇ -adrenergic receptors
  • aenyllyl cyclase When activated, it catalyzes the production of an intracellular mediator or second messenger, in particular cyclic AMP.
  • the inventors participated in the discovery of new ⁇ -adrenergic receptors in humans, called RA-Hu ⁇ 3, in mice (International Application WO 92/12246), called RA-Mu ⁇ 3 and in cattle (International Application WO 94 / 24162), called RA-Bo ⁇ 3, characterized by properties different from those of the ⁇ 1 and ⁇ 2 receptors, in particular in that they behave differently with respect to substances respectively antagonists and agonists of the ⁇ 1 and ⁇ 2 receptors ( International Application WO 90/08775).
  • the RA-Hu ⁇ 3 receptor is more particularly constituted by a sequence of 408 amino acids (VAN SPRONSEN A. et al., Eur. J. Biochem., 1993, 213, 1117-1124) and is considered to contain seven hydrophobic transmembrane regions separated by intra- and extra-cellular hydrophilic loops and the RA-Mu ⁇ 3 receptor consists of a sequence of 400 amino acids (VAN SPRONSEN et al., cited above) while the RA-Bo ⁇ 3 receptor consists of a sequence of 405 amino acids, the latter two sequences also comprising seven transmembrane regions.
  • RA-Hu ⁇ 3, RA-Mu ⁇ 3 and RA-Bo ⁇ 3 has shown that the ⁇ 3-adrenergic receptor is involved in diseases such as diabetes and / or obesity; it is preferentially expressed in adipose tissue which plays an important role in metabolism.
  • RA-Ca ⁇ 3 adrenergic ⁇ 3 receptor in dogs
  • RA- ⁇ 2 ⁇ -adrenergic receptors
  • the subject of the present invention is isolated nucleotide sequences, characterized in that they correspond to a DNA sequence coding for a canine ⁇ -adrenergic receptor selected from the group consisting of cDNA coding for the canine ⁇ 2-adrenergic receptor, of sequence SEQ ID NO: 1 and by DNA coding for the canine ⁇ 3-adrenergic receptor, of sequence SEQ ID NO: 24.
  • the canine adrenergic ⁇ 3 and ⁇ 2 receptors have been effectively isolated, which gives the possibility either of developing selective treatments for dogs after defining a pharmacological profile distinct from that defined for the receptors ⁇ 3 human, murine and bovine, either to have specific products of a receptor subtype: specific canine ⁇ 3 or specific canine ⁇ 2.
  • the present invention also relates to nucleotide probes, characterized in that they consist of a nucleotide sequence as defined above or a fragment thereof, labeled with the aid of a marker such as a radioactive isotope, a suitable enzyme or a fluorochrome.
  • a marker such as a radioactive isotope, a suitable enzyme or a fluorochrome.
  • the hybridization conditions of the probes are defined as follows:
  • suitable hybridization conditions are as follows:
  • Washes are carried out in a solution containing 3 mM NaCl; 0.3 mM sodium citrate;
  • Washings are carried out in a solution containing 15 mM NaCl; 1.5 mM sodium citrate;
  • suitable hybridization conditions are those indicated above for shorter probes, but in which the above-defined medium contains 40 to 50% formamide instead of 10 to 25%.
  • the probes from SEQ ID N ° 1 hybridize only with RA-Ca ⁇ 2 receptors, while the probes from SEQ ID N ° 24 hybridize only with the RA-Ca ⁇ 3 receptor.
  • the present invention also relates to proteins ( ⁇ 2- and ⁇ 3-adrenergic receptors), characterized in that they are coded by a nucleotide sequence as defined above and are selected from the group consisting of SEQ ID NO: 2 (RA-Ca ⁇ 2 receptor) and SEQ ID NO: 25 (RA-Ca ⁇ 3).
  • Canine ⁇ 2 and ⁇ 3 receptors activate adenylyl cyclase; however, the coupling between receptors and adenylyl cyclase via G proteins is deficient in murine CHO-Kl cells (ref .: ATCC CRL 9618, ATCC Catalog of Cell Unes & Hybridomas, 7th edition, 1992) and CHW, for the canine ⁇ 2 receptor and CHO-K1, for the canine ⁇ 3 receptor. This implies a loss of activity of canine ⁇ 2- and ⁇ 3-adrenergic receptors in these cells, although they are effectively expressed there.
  • the activity of the canine ⁇ 3 receptor expressed in CHO-K1 cells is distinguished from the ⁇ 3 activity of other species (man, beef), expressed in the same cells, in that it is zero when the receptor is cloned and expressed in CHO-K1 cells, while it is strong, when it is expressed in monkey COS-1 cells (ref .: ATCC CRL 1650, ATCC Ca talogue of Cell Unes & Hybridomas, 7th edition, 1992).
  • Said receptor presents, in COS-1 cells, in which - there is both correct expression and coupling, the activities of ⁇ 3-adrenergic receptors, namely, it activates adenylyl cyclase in the presence of one of the agonists following: (-) - isoprotere- nol, (-) - epinephrine, (-) - norepinephrine, CGP12177A, CL316,243.
  • the present invention also relates to fragments of said proteins of at least 6 amino acids, corresponding to an epitope capable of producing antibodies under suitable conditions, as described in GUILLAUME JL. et al. (Eur. J. Biochem., 1994, 224, 761-770)
  • the present invention also relates to antibodies directed specifically against the canine ⁇ 3-adrenergic receptor or against one of its epitopes, which antibodies recognize only said canine ⁇ 3-adrenergic receptor or one of its epitopes and neither recognize nor ⁇ 1- receptors, nor canine ⁇ 2- adrenergic receptors.
  • 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 an appropriate recombinant vector, comprising in particular an origin of replication in a suitable host microorganism, in particular a bacterium or a eukaryotic cell, at least one gene of which the expression allows the selection either of bacteria or of eukaryotic cells having received said vector, an appropriate regulatory sequence, in particular a promoter allowing the expression of genes in said bacs series or eukaryotic cells, and into which vector is inserted a nucleotide sequence or a fragment of a sequence as defined above, which vector is a vector for expression of a canine ⁇ 2- or ⁇ 3- adrenergic receptor.
  • said vector consists of a recombinant expression plasmid into which is inserted, at the level of a multisite linker, the sequence coding for the ⁇ 2- receptor or the canine ⁇ 3 -adrenergic receptor ;
  • a plasmid containing the sequence coding for the canine ⁇ 3 -adrenergic receptor was named pcDNA3 / canine adrenergic r ⁇ 3 or "canine Beta3-ADR" and was deposited with the National Collection of Cultures of Microorganisms (CNCM) maintained by the INSTITUT PASTEUR, dated February 9, 1996 under No. I-1672;
  • a plasmid containing the sequence coding for the canine ⁇ 2-adrenergic receptor was designated pcDNA3 / r ⁇ 2-adrenergic.
  • the present invention also relates to an appropriate host cell, obtained by genetic transformation, characterized in that it is transformed by a recombinant expression plasmid, in accordance with the invention.
  • Such a cell is capable of expressing a protein, of canine origin, having a ⁇ 2- or ⁇ 3-adrenergic receptor activity.
  • the host cell is in particular constituted by the CHO-K1 cells (stable expression not coupled to adenylyl cyclase activity) and COS-1 (transient expression and coupling to adenylyl cyclase).
  • the receptors according to the invention constitute a tool for the detection of specific ligands involved in the activation or inhibition of these receptors and make it possible to identify and select ⁇ -adrenergic ligands specific for canine ⁇ 3 receptors, by comparing the results obtained using both the sequences coding for the ⁇ 2- receptor and sequences coding for the canine ⁇ 3-adrenergic receptor.
  • the differential screening method for the study of the affinity in binding of substances and the selection and identification of substances capable of behaving as a specific ligand with respect to a ⁇ 3 receptor - canine adrenergic comprises:
  • Such a method makes it possible to define the pharmacological profile of the receptor, namely canine ⁇ 3 (when the substance studied is brought into contact with the receptor ⁇ 3), or canine ⁇ 2 (when the substance studied is brought into contact with the ⁇ 2 receptor).
  • canine ⁇ 3 when the substance studied is brought into contact with the receptor ⁇ 3
  • canine ⁇ 2 when the substance studied is brought into contact with the ⁇ 2 receptor.
  • the present invention further relates to a method for studying the efficacy of a substance having either an agonist or an antagonist activity with respect to the receptor studied, which method comprises:
  • the culture of the transformed host cell under conditions allowing the expression of the ⁇ 3 receptor coded by the nucleotide sequence, and the transfer of the expressed ⁇ 3 receptor to the membrane of said cell, so that the transmembrane sequences of the ⁇ 3 receptor are exposed on the surface of the transformed host cell;
  • the invention further relates to a kit for the detection of the possible binding affinity of a ligand for a receptor in accordance with the invention and / or for the detection of the activity of said ligand with respect to screw of the receiver studied, which kit includes:
  • control ligands having determined affinities for said ⁇ 3 receptor
  • the affinity of said substance for the receptor is measured by competition in the binding of the radioligand by increasing concentrations of said substance;
  • Figure 1 schematically represents the sequence coding for the canine ⁇ 3 -adrenergic receptor and the location of human primers for the implementation of a PCR;
  • FIGS. 2A and 2B represent the visualization of the amplification product, from genomic DNA of a fragment of sequence coding for the ⁇ 3 -adrenergic receptor;
  • FIGS. 3A and 3B represent Southern blots of dog genomic DNA, hybridized with a canine RA- ⁇ 3 probe
  • FIG. 4 shows a restriction map of canine genomic DNA comprising the canine ⁇ 3- adrenergic gene (darkened area);
  • FIG. 5 shows an agarose gel obtained from canine genomic DNA cut by EcoRI and fractionated in different sizes between 8 and 4.8 Kb;
  • - Figure 6 shows a Sou thern blot of the agarose gel of Figure 5 and shows that the gene coding for canine RA- ⁇ 3 is mainly contained in fraction No. 2;
  • FIGS. 7 and 8 show the unique restriction sites contained in the 2649 bp fragment containing the sequence coding for canine RA- ⁇ 3;
  • FIG. 10 shows an experiment carried out with one of the two stable CHO-K1 clones selected, expressing the canine ⁇ 3 receptor;
  • FIG. 11 represents an experiment carried out with one of the two stable HEK293 clones selected, expressing the ⁇ 3-canine receptor;
  • FIGS. 12 and 13 show the unique restriction sites contained in the fragment of 2679 bp, containing the sequence coding for canine RA- ⁇ 2;
  • FIG. 15 represents a typical experiment carried out with two subclones expressing the canine ⁇ 2 receptor and a clone expressing the human ⁇ 2 receptor.
  • Example 1 Demonstration of a ⁇ 3-adrenergic receptor in dogs.
  • Amplification experiments by PCR were carried out on canine genomic DNA, using as primers two oligonucleotides, synthesized from the human ⁇ 3-adrenergic sequence.
  • the first, called 1269 (SEQ ID NO: 26) - corresponds to the start of the coding part of the canine gene.
  • the second, designated 1263 (SEQ ID NO: 9) and also comprising 18 nucleotides, corresponds to a part coding for the fifth transmembrane segment of the receptor, as illustrated in FIG. 1, in which the location of the oligonucleotides
  • the PCR reaction carried out under the conditions as defined in 2) below makes it possible to obtain an amplified fragment of approximately 643 base pairs, that is to say of the same order of magnitude as the corresponding human fragment.
  • This fragment is visualized by detection of a fluorescence, resulting from the fixation of ethidium bromide after electrophoretic migration in agarose gel.
  • the size of the fragment is determined using known molecular weight markers. In FIG. 2, a fragment with the expected size corresponding to a part of the canine adrenergic ⁇ 3-receptor gene is effectively seen.
  • FIG. 2A corresponds to the visualization by fluorescence in ethidium bromide of the fragments obtained after amplification on human (Hu) and dog (Dg) genomic DNA. Size markers (MW) are multiples of 123 bp (BRL).
  • FIG. 2B corresponds to the results of hybridization with a specific human ⁇ 3 probe.
  • RT-PCR Reverse Transcriptase - Polymerase Chain Reaction
  • RNAs are extracted from brown adipose tissue of puppies, by the method using guanidium thiocyanate, then the poly A + messenger RNAs are purified, using oligo (dT) columns (Pharmacia ref. 27- 9258-A).
  • CDNA is synthesized from 0.5 ⁇ g of said poly A + mRNA, using reverse transcriptase from the Moloney murine leukemia virus (M-MLV Gibco-BRL ref. 510-8025 SA), then this cDNA is amplified by PCR, using the two human primers used in 1) [oligonucleotides 1269 (SEQ ID NO: 26) (sense primer) and 1263 (SEQ ID NO: 9) (antisense primer)] .
  • a solution containing the primers SEQ ID NO: 26 and SEQ ID NO: 9 is added to the neosynthesized cDNA at a concentration of 0.25 ⁇ M each, 10% of dimethyl sulfoxide, 2.5 U of Taq polymerase (CetusR , Perkin Elmer), the various dNTPs (dATP, dGTP, dCTP, dTTP), each at a concentration of 0.25 mM; the reaction buffer used is that recommended by Perkin Elmer.
  • the PCR is carried out on a Perkin Elmer "Gene Amp PCR System 9600" device, under the following conditions:
  • the 643 bp fragment was purified and subcloned into a plasmid vector (Bluescript; subclones R2, R5, R10, R11) and M13 tg 130 and tg 131 for the sequence.
  • a primer specific for the canine ⁇ 3 -adrenergic receptor was synthesized and is located at position 464 relative to the initiation codon ATG (RT1, SEQ ID NO: 3); it is used as a sense primer to clone a second fragment of the canine ⁇ 3-adrenergic gene.
  • RT1, SEQ ID NO: 3 initiation codon ATG
  • antisense primer a human primer (TR2, SEQ ID NO: 47) or, in other words "interspecies”, was chosen because the comparison of the ⁇ 3 -adrenergic sequences shows great similarity in this region.
  • the PCR reaction was carried out as follows:
  • the primers RT1 and TR2 are added at a concentration of 0.25 ⁇ M each, 10% dimethylsulfoxide, 2.5 U of Taq polymerase (Promega), 0.25 mM of dNTP (dATP, dGTP, dCTP, dTTP), the reaction buffer used is that supplied by Promega supplemented with 1.5 mM MgCl 2 .
  • a LEP * Sçientific device, PREM TM is used to carry out this reaction. After an initial denaturation step (5 min. At 92 ° C), 30 cycles are made as follows: 1 min. at -92 ° C; 1 min. at 62 ° C; 1 min. 30 sec. at 72 ° C; and last 1 time 7 min. at 72 ° C.
  • Cloning by PCR makes it possible to obtain the coding part of the gene ranging from methionine in position 1 to cysteine in position 347.
  • These 1041 nucleotides comprise the coding sequence for the 7 transmembrane domains of the receptor.
  • Example 2 Isolation and identification of the canine ⁇ 3- adrenergic gene.
  • enzymes are: Xba I / Hind III
  • the canine RA- ⁇ 3 probes were prepared by
  • the radiolabelled probes are added, at a concentration of approximately 10 "cpm / ml of hybridization medium.
  • Exon 2 generally codes for 6 to 8 amino acids and the following sequence corresponds to the 3 'untranslated part, which makes it possible to conclude that the ⁇ 3 gene proper is contained in the genomic fragment EcoRI and Xba I of 3 , 7 kb, or more broadly framed by 2 EcoRI sites.
  • the genomic library was constructed in a lambda bacteriophage as follows:
  • the size marker used is "Lambda DNA Bst EU digest” (New England Biolabs ref. 301-4S).
  • a and B correspond to 2 digests of genomic DNA by EcoRI and lanes 1, 2, 3 correspond respectively to the following molecular weights: 1: 8 to 7 kb, 2: 7, 2 to 6 kb, 3: 6.3 to 4.8 kb.
  • the DNA was transferred onto a Nylon membrane (Hybond N + see Southern Blot conditions as set out above).
  • Hybond N + see Southern Blot conditions as set out above.
  • the same canine ⁇ 3 probes are used, the labeling having been done by "random priming" using only the dCTP ⁇ 32 P.
  • the hybridization and washing conditions are the same as above.
  • the bacteriophage is used as a vector.
  • the phage particles were packaged using Promega "Packagene R System” in vitro packaging extracts for 3 hours at 22 ° C.
  • the phage particles thus reconstituted are able to infect bacteria of an appropriate strain. This is the strain
  • LE 392 (Genotype: F-, hsdR 574 (r K -, m K + ), supE44, supF58, LacYl or ⁇ (lacIZY) 6, ga1K2, Ga1T22, metB1, trpR55).
  • a colony of these bacteria was cultured to obtain fresh cells to be infected by phages, the aim being to be able to spread the infected bacteria on petri dishes containing a mili nutritious, in order to be able to screen with a radiolabelled probe.
  • the probe was made by PCR on canine genomic DNA with the primers SEQ ID NO: 26 (1269) and SEQ ID NO: 47 (TR2).
  • PCR conditions were as follows: primers at a concentration of 0.25 ⁇ M; buffer comprising 10% dimethyl sulfoxide, 2.5 U of Taq polymerase (Promega), 0.25 mM dNTP (dATP, dGTP, dCTP, dTTP), the reaction buffer used is that supplied by Promega, supplemented with 1.5 mM MgCl 2 .
  • the 1041 nucleotide PCR fragment (ATG) -
  • TM7 was isolated from an agarose gel and then purified by Spin - X TM (Costar 0.22 ⁇ M cellulose acetate).
  • the radiolabelling was done by random priming, as specified above, using dCTP ⁇ 32 P (Amersham ref. PB 10205).
  • the hybridization conditions are the same as those described above (Southern Blot).
  • the membranes were washed with a solution comprising 3 mM NaCl, 0.3 mM Na-citrate, 0.05% SDS, at 45 ° C for 30 min.
  • the gene was sequenced on the 2 strands (Subclones 4S and 4AS) using primers specifically synthesized.
  • the primers used on the sense strand (4S), in sequential order, are as follows: SEQ ID NO: 14 (SG 2), SEQ ID NO: 15 (RT 3), SEQ ID NO: 3 (RT 1) , SEQ ID NO: 16 (RT 5), SEQ ID NO: 17 (RT 6), SEQ ID NO: 18 (RT 9), SEQ ID NO: 20 (RT 17), SEQ ID NO: 22 (RT 16) , SEQ ID NO: 19 (RT 26), SEQ ID NO: 21 (RT 27) and SEQ ID NO: 23 (RT28).
  • results obtained show the nucleotide sequence of the canine ⁇ 3-adrenergic gene composed of a region coding for the protein (1196 bp in exon 1 plus 22 bp in exon 2), and non-coding regions
  • the unique restriction sites contained in the 2649 bp fragment are positioned in FIGS. 7 and 8.
  • the comparison of the coding regions of the canine and human ⁇ 3 -adrenergic genes indicates a strong homology (82%) (FIG. 9); however, the differences observed, both structural and pharmacological, highlight the importance of the isolation of the canine ⁇ 3 -adrenergic receptor.
  • the canine ⁇ 3-adrenergic sequence codes for a protein of 405 amino acids and carries the structural characteristics of the ⁇ 3 -adrenergic receptors, in particular the seven hydrophobic regions which probably correspond to transmembrane segments.
  • e1 extracellular part
  • NTS and NTS glycosylation sites
  • exon 1 code for the majority of the open phase ranging from the initiation codon "ATG" in position 1, to the sequence GAC GG / in position 1196; the intronic sequence comprises 703 bp.
  • the splicing signals are underlined / gt .......... ag / (see also Maniatis vol. 3 page 16.7 and van Spronsen, Eur. J. Biochem. 213, 1117 - 1124 (1993)).
  • the vector pcDNA 3 (Invitrogene) can transform eukaryotic cells in culture in a way stable and transient and express the cloned genes, dependent on the CMV promoter (Cytomegalovirus).
  • the canine ⁇ 3-adrenergic sequence shows a BspE I site at 54 bases before the ATG codon.
  • the subcloning was done by taking the BspE I sites in 5 'and EcoRI in 3' of the gene.
  • the 2590 bp fragment was ligated into the vector pcDNA 3 as follows:
  • Example 3 Pharmacological properties of the expression product of the canine ⁇ 3 gene.
  • the plasmid pcDNA3 containing the canine adrenergic ⁇ 3 receptor gene was transfected into CHO-K1 cells by a lipofectin transfection method (Gibco); the transfected cells are selected with geneticin (G418).
  • CHO-K1 cells are grown at confluence in a culture medium containing: 50% DMEM medium, 50% Ham's F12 medium, 10% heat-inactivated fetal calf serum and glutamine
  • 1 ⁇ g of DNA from the canine pcDNA3 ⁇ 3 plasmid is mixed with 5 ⁇ l of lipofectin (GIBCO) and 1 ml of the culture medium described, without serum. This mixture is added to the cells in culture, which are incubated for 5 hours at 37 ° C. The medium is then replaced by the above-mentioned culture medium containing serum and the cells are again incubated for 48 hours.
  • GEBCO lipofectin
  • the cells are then diluted and distributed in 96-well plates and incubated at selection pressure, that is to say in the aforementioned culture medium containing geneticin (G418 GIBCO) 400 ⁇ g / ml for approximately 20 days, the medium being changed every other day.
  • selection pressure that is to say in the aforementioned culture medium containing geneticin (G418 GIBCO) 400 ⁇ g / ml for approximately 20 days, the medium being changed every other day.
  • the COS-1 cells are cultivated at confluence in a culture medium containing: 90% of DMEM medium, 10% of heat-inactivated fetal calf serum, 2 mM glutamine and 10 mM HEPES. 48 hours before transfection, the cells are washed, detached with trypsin and seeded at 1.7 ⁇ 10 4 cells / cm in 6-well plates.
  • the COS-1 cells are washed twice in PBS buffer, each well is incubated with 1 ml of DMEM transfection medium containing 1 ⁇ g of pcDNA3 / r ⁇ 3 (miniprep) and 20 ⁇ l of DEAE-Dextran (10 mg / ml) previously mixed and 8 ⁇ l of 10 mM chloroquine, for 4 to 6 hours at 37 ° C.
  • the transfection medium is aspirated and the cells are incubated with 10% DMSO in DMEM medium for exactly 90 seconds.
  • the cells are then washed with PBS and 3 ml of DMEM medium containing 10% serum are added to all the wells.
  • the membrane preparation for the binding tests and the accumulation of cAMP on whole cells are carried out 48 to 72 hours after the transfection.
  • HEK293 cells Stable transfection of HEK293 cells: The canine adrenergic pcDNA3 / r ⁇ 3 plasmid was transfected into HEK293 cells by a lipofectin transfection method (Gibco) as described in a); the transfected cells are selected with geneticin (G418) 500 ⁇ g / ml.
  • the cell culture medium is removed, the cells are washed with PBS and incubated with 1 ml / well in lysis buffer (hypotonic shock) containing 10 mM Tris / HCl pH 7.4, 1 mM EDTA and the inhibitors of PMSF proteases (0.5 mM) and leupeptin (5 ⁇ g / ml) for 10 minutes at 4 ° C, then homogenized, transferred to tubes for centrifugation and incubated again for 15 minutes at 4 ° C. The cells are then centrifuged for 60 minutes, at 4 ° C., 20,000 rpm (50,000 g) JA20 rotor. The membrane pellets are taken up in the storage buffer (25 mM Tris / HCl pH 7.4, 1 mM EDTA, 10% glycerol and protease inhibitors) and stored at -80 ° C.
  • lysis buffer hypertonic shock
  • the pre-influent cells (0.5 x 10 6 cells / well) are washed with Ham' ⁇ F12 medium, pH 7.4, containing 1 mM 3-isobutyl-methyl) xanthine (IBMX, Sigma) and HEPES 20 mM.
  • the cells are incubated for 25 min. at 37 ° C in 1 ml of medium, in the absence (basal level) or in the presence of 100 ⁇ M of (-) - isoproterenol
  • the reaction is stopped by centrifugation (3000 xg, 10 min, 4 ° C.) of the incubation medium containing the cells and not by washing ; the supernatant containing the ligand is eliminated and 500 ⁇ l of sodium hydroxide are added to the cell pellet.
  • the lysed cells are added to 1 N acetic acid, pH 7.4 and centrifuged at 3000 x g, 4 ° C, 10 minutes.
  • the binding measurements were carried out on membrane preparations made from COS-1 cells transfected for 72 hours.
  • the membrane aliquots 10-15 ⁇ g of protein per point, are incubated in a buffer containing Hank's saline solution (ref .: 010328 Eurobio catalog), 20 mM HEPES and 0.1% BSA (bovine serum albumin) , with 1 nM of ICYP (2000 Ci / mmol) in the presence or in the absence of increasing concentrations of competitor (1 pM-100 ⁇ M); inhibition of ICYP by (--- bupranonol 10 M makes it possible to estimate the non-specific binding.
  • the saturation curves were carried out in the presence of increasing concentrations of ICYP (1 pM to 4 nM) in the presence of (-) - bupranolol 10 -4 M (non-specific binding).
  • the tubes are incubated for 30 minutes at 37 ° C with shaking.
  • the reaction is stopped by filtration of the aliquots on a glass fiber filter saturated beforehand with 0.3% of polyethyleneimine, followed by 3 washes with 3 ml of ice-cold PBS (phosphate buffered saline) buffer.
  • the radioactivity retained on the filter is measured in a gamma counter (CompuGamma LKB 1282).
  • the canine adrenergic pcDNA3 / r ⁇ 3 plasmid was transfected into CHO-K1 cells as described in Example 3. After 3 weeks, 30 clones resistant to geneticin were selected.
  • FIG. 10 presents a typical experiment carried out with one of the two stable clones expressing the canine ⁇ 3 receptor (canine CHO-K1 ⁇ 3).
  • the accumulation of cAMP was measured simultaneously on this clone and on a stable CHO-K1 clone expressing the human ⁇ 3 receptor as a positive control (human CHO-K1 ⁇ 3).
  • (-) - isoproterenol was used at a concentration of 10 -4 M ( ⁇ so-4) and two ligands specific for the human ⁇ 3-adrenergic receptor, CGP12177A and
  • the dose-response curves made it possible to determine the values of the activation constants (Kact) and the intrinsic activities (IA, taking as reference the maximum activity of 1 '(-) - isoproterenol 10 M), for each ligand tested: (-) - norepinephrine, (-) - epinephrine, (-) - isoproterenol, CGP 12177A and CL 316,243.
  • Agonists specific for the adrenergic ⁇ 3 subtype such as CGP 12177A and CL 316,243 are also described here as agonists of the canine RA- ⁇ 3 receptor.
  • Bupranolol has an antagonistic activity on the canine ⁇ 3 receptor, as previously described for the human and murine ⁇ 3 receptors, whereas it is a partial agonist on the bovine ⁇ 3 receptor.
  • the saturation curves made it possible to determine the value of the dissociation constant of the radioligand ICYP for the canine ⁇ 3 receptor, K D of 4.7513.13 nM.
  • the competition curves were carried out on the same membrane preparations with the ligands: (-) - norepinephrine, (-) - epinephrine, (-) - isoproterenol, CGP 12177A and CL 316.243, for which the inhibition constants (Ki) have been determined.
  • FIG. 11 presents a typical experiment carried out with one of the two stable clones expressing the canine ⁇ 3 receptor.
  • the accumulation of cAMP was measured simultaneously on this clone and on a stable clone HEK293 expressing the human ⁇ 3 receptor as a positive control.
  • Physiological catecholamies, norepinephrine (NE) and epinephrine (PPE), a non-specific agonist (-) - isoproterenol (ISO) and two ligands specific for the human adrenergic ⁇ 3 receptor carazolol (cara) and CL316,243 have been used. All ligands were used at a concentration of 10 -4 M.
  • the canine ⁇ 2 -adrenergic gene was isolated from a cDNA library of brown adipose tissue from newborn puppies, built in the bacteriophage ⁇ gt 11.
  • RNAs were obtained from brown adipose tissue of puppies by the method using guanidium thiocyanate. Then, the poly A + messenger RNAs were purified using oligo (dT) columns (Pharmacia ref. 27-9258-A).
  • the next step was to synthesize the cDNA using the purified poly A + messenger RNA as a template, and as a primer for first strand synthesis an oligo (dT) 15 primer from the "RiboClone cDNA synthesis System" kit (Promega ref . C2100).
  • the synthesis of the first strand of cDNA is done using an enzyme (AMV reverse transcriptase), followed by the synthesis of the second strand using two enzymes acting at the same time (E. coli polymerase I and RNase H). Then the double-stranded cDNA is treated with T4 DNA polymerase, in order to obtain blunt ends.
  • the Promega C2100 kit was used for all these successive reactions.
  • the fractions between 1.3 and 2.3 kilobases (kb) are selected. This selective step is considered an enrichment, but it is not really a purification, in other words the cDNA molecules of neighboring sizes are not completely excluded.
  • the cDNA is deposited on a potassium acetate gradient (5 to 20%) and then centrifuged at 50,000 rpm (corresponds to 170,000-304,000 g in this rotor) for three hours at 22 ° C in a SW 55 rotor (Beckman). Then, 27 cDNA fractions were taken. After depositing a small amount of each fraction on a 0.8% agarose gel, we selected 4 fractions corresponding to the desired sizes. These fractions were mixed and then ligated with the vector ⁇ gt 11.
  • the bacteriophage ⁇ gt 11 used as a vector, comes from the "Protoclone Lambda gt 11 System” kit (Promega ref. T 301 / 0-2).
  • the phage DNA is digested with EcoRI and dephosphorylated. Dephosphorylation prevents the vector from closing in on itself.
  • the vector alone was also ligated and packaged in order to be able to know the background noise of this batch of ⁇ gt 11.
  • each phage dilution was incubated with Y1090 (r-) cells at 37 ° C for 30 minutes, and then these infected bacteria were spread on nutrient medium (LB agar) contained in petri dishes. The dishes are incubated overnight at 37 ° C., and the following day there are lysis plaques, each plaque corresponds to a recombinant phage.
  • LB agar nutrient medium
  • a radiolabelled probe As a radiolabelled probe, a fragment of about 600 base pairs (bp) of the human ⁇ 3-adrenergic gene (L.J. Emorine et al., 1989, Science, 245, 1118-1121) was used.
  • the probe includes the coding region ranging from the initiation codon (ATG) to the transmembrane domain 5 (TM 5).
  • Random Priming (Kit Boehringer ref. 1004 760), incorporating 50 ⁇ Ci of dATP ( ⁇ 32 P) and 50 ⁇ Ci of dCTP ( ⁇ 32 P) (Amersham ref. PB 10204 and ref. PB 10205 respectively).
  • DNA fingerprints of the lysis plaques on Hybond N + membranes were taken. These membranes were then hybridized with the probe in question, then washed and exposed overnight on autoradiography film.
  • ⁇ D1 The remaining positive clone, called ⁇ D1, was purified by four successive isolations, followed by hybridization with the human ⁇ 3-adrenergic probe.
  • the DNA of phage ⁇ D1 was prepared. This DNA was then cut with the restriction enzyme EcoRI in order to verify the insert size. It should indeed be noted that two fragments were obtained: one of 2.4 kb and one of 0.25 kb, which suggests the presence of an EcoRI site specific to the canine ⁇ 2-adrenergic gene, because the EcoRI site is unique on the vector ⁇ gt 11.
  • the 4 subclones were completely sequenced using the Sequenase version 2.0 kit (Amersham- United States Biochemical ref. 70770). The sequence was carried out using specific primers, which hybridize on the sense strand (H and 16.4) or on the antisense strand (D3 and 16.2). These primers are represented by the following sequences:
  • the canine ⁇ 2-adrenergic sequence codes for a protein of 415 amino acids SEQ ID NO: 2 and carries the structural characteristics of the ⁇ -adrenergic receptors, in particular the seven hydrophobic regions which probably correspond to transmembrane segments. In the extracellular part (el) there are two glycosylation sites (NRS and NGS) common to the other ⁇ 2-adrenergic receptors.
  • the intracellular C-terminal part (i4) is more conserved between the ⁇ 2-adrenergic receptors than between the ⁇ 3-adrenergic receptors.
  • the human sequence reveals an absence of 6 residues in position 360.
  • Three residues are absent in the canine ⁇ 2-adrenergic sequence in position 391 compared to the other ⁇ 2-adrenergic sequences.
  • FIG. 14 shows the comparison in amino acids of the various ⁇ 2-adrenergic receptors.
  • the EcoRI fragment of 2427 bp from clone "H” was purified and used as a probe. This DNA fragment was radiolabeled, as described above, by the Random Priming method. The probe thus prepared was used in genomic DNA hybridization experiments (Southern Blot), to estimate its specificity vis-à-vis the ⁇ 2-adrenergic gene. Canine genomic DNA was cut by the following restriction enzymes: EcoRI, Hind III, Bam HI, Xba I. After separation of the fragments in an electrophoretic field on 0.7% agarose gel, the DNA was transferred to a nylon membrane (Amersham, Hybond N +). This membrane was hybridized with the EcoRI probe then washed at low stringency and then autoradiographed.
  • the results indicate, for the EcoRI cut, a fragment of 8 to 8.5 kb, for the BamHI cut a fragment of about 20 kb, for the Xbal cut a fragment of 7.5 to 8 kb and for the Hind III cut. a 4 kb fragment.
  • the presence of a single band for each cut indicates that there are no other sequences highly homologous in the canine genome to the ⁇ 2-adrenergic gene.
  • Example 5 Construction of a vector for the expression of the canine ⁇ 2-adrenergic receptor.
  • the restriction map of the canine ⁇ 2-adrenergic gene indicates the presence of a site of cleavage by the enzyme Nae I, at position 154, ie
  • the DNA of the clone M13-H was digested with the enzymes Nae I and EcoRI, to release the fragment of 2273 bp containing the coding region of the gene ⁇ 2- adrenergic canine and part of the 3 'region not translated. This DNA fragment was purified, then inserted into the expression vector pcDNA 3 (Invitrogene), at the Bam HI and EcoRI cleavage sites.
  • the Bam HI ends of the vector were treated with the Klenow fragment of polymerase I, so as to obtain blunt ends (Maniatis and al., Molecular Cloning, 2nd edition, pages 5.40 to 5.43).
  • the Nae I cut generates blunt ends and after ligation, the recombinant canine pcDNA 3 / r ⁇ 2- canine adrenergic plasmid (subclone 41) was thus obtained.
  • the canine ⁇ 2-adrenergic receptor gene is then dependent on the strong promoter CMV (cytomegalovirus), which ensures overexpression of the gene once introduced into mammalian cells.
  • CMV cytomegalovirus
  • Example 6 Study of the expression of the canine ⁇ 2 receptor in CHO-K1 cells.
  • the canine adrenergic pcDNA3 / r ⁇ 2 plasmid was transfected into CHO-K1 cells as described in Example 3. After approximately 3 weeks, 39 clones resistant to geneticin were selected.
  • FIG. 15 presents a typical experiment carried out with 2 subclones expressing the canine ⁇ 2 receptor (CHO-K1 ⁇ 2 canine 10 and CHO-K1 ⁇ 2 canine 31).

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EP97916504A 1996-03-26 1997-03-26 HUNDE Beta-2 UND Beta-3 ADRENERGISCHE REZEPTOREN UND DEREN ANWENDUNGEN Withdrawn EP0889958A2 (de)

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FR9603730A FR2746813B1 (fr) 1996-03-26 1996-03-26 Sequences codant pour les recepteurs beta2- et beta3- adrenergiques canins et leurs applications en tant que sondes et pour l'expression de peptides
PCT/FR1997/000537 WO1997035973A2 (fr) 1996-03-26 1997-03-26 SEQUENCES CODANT POUR LES RECEPTEURS β2- ET β3-ADRENERGIQUES CANINS ET LEURS APPLICATIONS EN TANT QUE SONDES ET POUR L'EXPRESSION DE PEPTIDES

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US7993756B2 (en) 2005-05-04 2011-08-09 Viance, Llc Long-chain quaternary ammonium compounds as wood treatment agents

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US6861217B1 (en) 1998-11-25 2005-03-01 Genaissance Pharmaceuticals, Inc. Variation in drug response related to polymorphisms in the β2-adrenergic receptor
WO2000031307A1 (en) * 1998-11-25 2000-06-02 University Of Cincinnati POLYMORPHISMS IN THE 5' LEADER CISTRON OF THE β2-ADRENERGIC RECEPTOR
CN106749511A (zh) * 2017-01-09 2017-05-31 常州大学 苯丙氨酸二肽苯乙酰胺衍生物及其制备方法和用途
CN111965367A (zh) * 2020-07-23 2020-11-20 北京大学第三医院（北京大学第三临床医学院） Hip-55在作为gpcr偏向药物靶点中的应用

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FR2642075B1 (fr) * 1989-01-25 1991-04-26 Pasteur Institut Polypeptides ayant une activite de recepteur (beta)-adrenergique chez l'homme, impliques dans la reponse lipolytique, acides nucleiques codant pour ces polypeptides et utilisation de ces polypeptides pour le criblage de substance active sur ces polypeptides
FR2671559B1 (fr) * 1991-01-14 2002-09-27 Ct Nal Rech Scient Sequences codant pour le recepteur beta3-adrenergique murin, leur utilisation comme sondes et pour l'expression de peptides, vecteurs, hotes cellulaires contenant ledit vecteur, et applications desdits peptides.
US5418160A (en) * 1991-11-01 1995-05-23 The United States As Represented By The Secretary Of The Department Of Health And Human Services Cloned cell line expressing rat β3A adrenergic receptor
EP0600136B1 (de) * 1992-12-01 2002-03-13 Centre National De La Recherche Scientifique Intron/Exon Struktur der Genen von den menschlichen und Maus Beta 3 adrenergischen Rezeptoren
FR2704235B1 (fr) * 1993-04-21 1995-06-23 Vetigen Sarl Séquences nucléotidiques codant pour le récepteur beta3-adrénergique (RAbeta3) bovin et leurs applications.

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