US5288607A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Polypeptides having a β-adrenergic receptor activity in man, implicated in the lipolytic response, nucleic acids coding for these polypeptides and the use of these polypeptides for the screening of a substance active on these polypeptides
Assignee: Centre National de la Recherche Scientifique
•
Filing: 1990-01-25
•
Granted: 1994-02-22
•
Expires: 2010-01-25
Abstract
Abstract PCT No. PCT/FR90/00054 Sec. 371 Date Sep. 3, 1991 Sec. 102(e) Date Sep. 3, 1991 PCT Filed Jan. 25, 1990 PCT Pub. No. WO90/08775 PCT Pub. Date Aug. 9, 1990.Novel polypeptides having a (beta)-adrenergic receptor activity containing the sequence of 402 amino acids, or a fragment of this sequence, said fragment being such that, in particular, either it nonetheless includes the sites contained in said sequence and whose presence is necessary so that, when the fragment is exposed to the surface of a cell, it is capable of participating in the activation of the cyclase adenylate in the presence of an agonist, or it is likely to be recognized by antibodies which also recognize the above succession of 402 amino acids, but fail to recognize the (beta)1 adrenergic receptor and the (beta)2 adrenergic receptor. These polypeptides are useful for screening drugs which act on said polypeptides and for treating obesity, fat diabetes and hyperlipidemias.
Inventors
- 👤 Laurent Emorine
- 👤 Stefano Marullo
- 👤 Donny Strosberg
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 13:20
CPC Classifications
C; C07; C07K; C07K16/00; C07K16/18; C07K16/28; C07K16/286; A; A61; A61P; A61P3/00; A61P3/04; A61P3/08; A61P3/10; A61P7/00; C07K14/00; C07K14/435; C07K14/705; C07K14/70571; A61K; A61K38/00
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
138 words
A substantially pure polypeptide having a β-adrenergic receptor activity comprising: the sequence of 402 amino acids of FIG. 12, or a fragment of said sequence, wherein said fragment contains sites in this sequence such that when this fragment is exposed at the surface of a cell, it is capable of participating in the activation of adenylate cyclase in the presence of an agonist, this activation increasing in the order of the agonists as follows: salbutamol, ##STR4## (1)-isoproterenol or said fragment is capable of being recognized by antibodies which also recognize the above-mentioned sequence of 402 amino acids, but which do not recognize the β1-adrenergic receptor or the β2-adrenergic receptor, or said fragment is capable of generating antibodies which recognize the above-mentioned sequence of 402 amino acids but do not recognize either the β1-adrenergic receptor or the β2-adrenergic receptor.
Claim 2
Dependent Claim
138 words
A substantially pure polypeptide having a β-adrenergic receptor activity comprising: the sequence of 402 amino acids of FIG. 12, or a fragment of said sequence, wherein said fragment contains sites in this sequence such that when this fragment is exposed at the surface of a cell, it is capable of participating in the activation of adenylate cyclase in the presence of an agonist, this activation increasing in the order of the agonists as follows: salbutamol, ##STR4## (1)-isoproterenol or said fragment is capable of being recognized by antibodies which also recognize the above-mentioned sequence of 402 amino acids, but which do not recognize the β1-adrenergic receptor or the β2-adrenergic receptor, or said fragment is capable of generating antibodies which recognize the above-mentioned sequence of 402 amino acids but do not recognize either the β1-adrenergic receptor or the β2-adrenergic receptor.
Claim 3
Dependent Claim
133 words
The polypeptide according to claim 1, wherein said polypeptide has the capacity to stimulate adenylate cyclase and/or accumulate cAMP induced by isoproterenol and is not inhibited by practolol, nadolol, ##STR5## butoxamine, alprenolol, propanolol, pindolol, oxprenolol, at concentrations lower than or equal to 10 -4 M under conditions wherein: cells are cultivated and harvested after treatment with ethylenediamine tetraacetic acid, washed and resuspended in a Hank medium containing 20 mM of Hepes buffered at pH 7.4, 1 mM ascorbic acid and al mM isobutyl-methyl-xanthine; aliquots of 10 6 cells are incubated for 30 minutes with the inhibitors at 10 -4 M before the addition of 5.10 -9 M of isoproterenol; the incubation is continued for a further 30 minutes and the cyclic AMP levels are measured to test for the accumulation of cyclic AMP.
Claim 4
Dependent Claim
109 words
The polypeptide according to claim 2, wherein pindolol and oxprenolol stimulate adenylate cyclase and/or the accumulation of cyclic AMP in CHO cells transfected with said polypeptide under conditions wherein cells are cultivated and harvested after treatment with ethylenediamine tetraacetic acid (Eurobio, Paris), washed and resuspended in a Hank medium containing 20 mM of Hepes buffered at pH 7.4, 1 mM of ascorbic acid and 0.1 mM of isobutyl methyl-xanthine; aliquots of 10 6 cells are incubated for 20 minutes in a total volume of 1 ml with concentrations of pindolol or oxprenolol higher than 10 -11 M and the cAMP levels to test for the accumulation of cyclic AMP.
Claim 5
Dependent Claim
17 words
A nucleic acid consisting of a chain of nucleotides coding for the polypeptides according to claim 1.
Claim 6
Dependent Claim
31 words
A nucleic acid according to claim 4, consisting of the chain of nucleotides shown in FIG. 1a, extending from the end constituted by nucleotide 1 to that constituted by nucleotide 2022.
Claim 7
Dependent Claim
21 words
A recombinant vector comprising a nucleic acid according to claim 4 at one of its sites not essential for its replication.
Claim 8
Dependent Claim
32 words
The recombinant vector according to claim 6, wherein said vector is M13mp18-Huβ3, (No. 1085) deposited under the No. I-833 on Jan. 20, 1989 at the CNCM, 25 rue du Docteur Roux, Paris.
Claim 9
Dependent Claim
53 words
The recombinant vector according to claim 6, wherein said vector comprises at one of its sites not essential for its replication elements necessary to promote the expression of one of the amino acid sequences according to claim 1 in a host cell and a promoter recognized by the polymerases of the host cell.
Claim 10
Dependent Claim
28 words
A host cell transformed by a recombinant vector according to claim 6, comprising the elements of regulation allowing the expression of the nucleotide sequence coding for the polypeptide.
Claim 11
Dependent Claim
15 words
The transformed host cell according to claim 9, wherein said host cell is E. coli.
Claim 12
Dependent Claim
16 words
The transformed host cell according to claim 9, wherein said host cell is a CHO cell.
Claim 13
Dependent Claim
63 words
An antibody directed specifically against a polypeptide according to claim 1, wherein said antibody does not recognize the β1-adrenergic receptor nor the β2-adrenergic receptor and does recognize the following amino acid sequence: ______________________________________
1 to 36 178 to 201
64 to 74 223 to 291
101 to 108 314 to 325
133 to 135 345 to 402
______________________________________ set forth in FIG. 1b.
Claim 14
Dependent Claim
197 words
A nucleotide probe wherein said nucleotide probe hybridizes with one of the nucleic acids according to claim 4 or the complementary sequence under conditions of hybridization such that it does not hybridize with the genes or messenger RNA of the β1 or β2 adrenergic receptors, and said probes are selected from the group of nucleotide probes consisting of the nucleic acid sequence shown in FIG. 1b, extending from the end nucleotide at position 1 to the nucleotide at position 637, the nucleic acid sequence shown in FIG. 1b, extending from the end nucleotide at position 638 to the nucleotide at position 745, the nucleic acid sequence shown in FIG. 1b, extending from the end nucleotide at position 1313 to the nucleotide at position 1513, the nucleic acid sequence shown in FIG. 1b, extending from the end nucleotide at position 1679 to the nucleotide at position 1843, the nucleic acid sequence shown in FIG. 1, extending from the end nucleotide at position 1844 to the nucleotide at position 2022, the nucleic acid sequence shown in FIG. 5, extending from the end nucleotide at position 638 to the nucleotide at position 1843, their complementary nucleotide sequence and mixture thereof.
Claim 15
Dependent Claim
52 words
Process for the preparation of a polypeptide according to claim 1, characterized in that a host cell previously transformed by a recombinant vector containing a nucleic acid according to the above is cultivated in a suitable medium and the polypeptide produced by the said transformed cell host is recovered from the culture.
Claim 16
Dependent Claim
102 words
A process for detecting the binding of an agonist or antagonist to a polypeptide according to claim 1 comprising the steps of: (a) placing said agonist or antagonist in contact with a host cell previously transformed by a vector modified by an insert coding for said polypeptide wherein said host cell bears at its surface one or more sites specific for said polypeptide under conditions permitting the formation of a bond between at least one of these specific sites and the said agonist or antagonist; and (b) detecting the formation of a complex formed between said antagonist or agonist and said polypeptide.
Claim 17
Dependent Claim
122 words
A process to study the affinity of a polypeptide according to claim 1, to one or several ligands comprising the steps of: (a) transforming a competent host cell with a vector in which a nucleotide sequence coding for the β-3 receptor has been placed beforehand under the control of regulatory elements which allows the expression of the nucleotide sequence in the host cell; (b) culturing the host cell under conditions allowing the expression of said β-3 receptor and the transport of the β-3 receptor towards the membrane sequences of the β-3 receptor at the surface of said transformed host cell; (c) contacting the transformed host cell with ligands; and (d) detecting an affinity reaction between said transformed host cell and said ligand.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1997-02-21 | 1997-08-23 | 1998-02-22 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2001-02-21 | 2001-08-23 | 2002-02-22 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2005-02-21 | 2005-08-23 | 2006-02-22 | $7700.00 | Window Closed / Lapsed |