Glucagon receptor proteins, peptides, and antibodies

• US 5,770,445 A
• Filed: 05/30/1995
• Issued: 06/23/1998
• Est. Priority Date: 08/28/1992
• Status: Expired due to Term

• Current Assignee: Novo Nordisk A/S
• Original Assignee: Zymogenetics Incorporated (Bristol-Myers Squibb Company)
• Inventors: Kindsvogel, Wayne R., Sheppard, Paul O., Foster, Donald C., Grant, Francis J., Kuijper, Joseph L., Lok, Si, O'Hara, Patrick J., Jelinek, Laura J.
• Primary Examiner(s): Fitzgerald, David L.
• Application Number:
  US08/453,956
• Time in Patent Office: 1,120 Days
• Field of Search: 530/350, 530/324, 530/325, 530/326, 530/327, 530/328, 530/387.2, 530/388.22, 530/389.1, 435/240.27, 435/24, 514/2
• US Class Current: 435/334
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/72 for hormones for neuromediators C07K14/70571
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: G01N 2333/605 Glucagons
• CPC Class Codes: G01N 2333/72 for hormones for neuromediators G01N2333/70571
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

The present invention provides isolated DNA molecules comprising a DNA segment encoding a glucagon receptor. Also provided are DNA constructs comprising a first DNA segment encoding a glucagon receptor operably linked to additional DNA segments required for the expression of the first DNA segment, as well as host cells containing such DNA constructs. The present invention also provides a method for detecting the presence of glucagon antagonists, comprising the steps of (a) exposing a compound in the presence of a glucagon against to a recombinant glucagon receptor coupled to a response pathway under conditions and for time sufficient to allow binding of the compound to the receptor and an associated response through the pathway, and (b) detecting a reduction in the stimulation of the response pathway resulting from the binding of the compound to the glucagon receptor, relative to the stimulation of the response pathway by the glucagon agonist alone and therefrom determining the presence of a glucagon antagonist.

Claims

1. An isolated and purified polypeptide comprising the amino acid sequence of a glucagon receptor having seven transmembrane domains, an N-terminal extracellular domain, a C-terminal intracellular domain, three extracellular loop domains, and three intracellular loop domains,

wherein the receptor is capable of binding glucagon with a K_D no greater than 100 nM and is capable of transducing signal in a suitable host cell in response to such binding,

and wherein the receptor is encoded by DNA which hybridizes under conditions of high stringency to a probe having the nucleotide sequence of the complement of nucleotides 145-1599 of SEQ ID NO:

14 or nucleotides 53-1483 of SEQ ID NO;

24.

2. A polypeptide according to claim 1 comprising the amino acid sequence shown as residues 28-485 of SEQ ID NO:

15, residues 1-485 of SEQ ID NO;

15, r residues 1-477 of SEQ ID NO;

25.

5. A peptide comprising the amino acid sequence of a fragment of a polypeptide according to claim 1, or 3, the fragment comprising at least

(a) the complete sequence of one of the extracellular or intracellular domains of the receptor, or

(b) ten contiguous amino acids from the sequence of the mature receptor.

6. A peptide according to claim 5, wherein the fragment binds glucagon.

7. A peptide according to claim 5, wherein the fragment comprises a receptor domain which is 1ID, 1ELD, 2ID, 2ELD, 3ID, or 3ELD.

8. A peptide according to claim 5, wherein said extracellular or intracellular domain comprises:

(a) residues 169-178 of SEQ ID NO;

15;

(b) residues 303-231 of SEQ ID NO;

15;

(c) residues 259-266 of SEQ ID NO;

15;

(d) residues 293-307 of SEQ ID NO;

15;

(e) residues 334-345 of SEQ ID NO;

15;

or

(f) residues 371-380 of SEQ ID NO;

15.

12. A monospecific antibody which specifically binds to the amino acid sequence of a glucagon receptor according to claim 1, or 3.

13. An antibody according to claim 12, which is a monoclonal antibody.

14. A hybridoma which produces an antibody according to claim 13.

15. A process for making an antibody specific for a glucagon receptor, comprising the steps of:

administering a peptide according to claim 5 to a host animal; and

recovering the antibody from the animal.

17. A process for making a cell which produces an antibody specific for a glucagon receptor, comprising the steps of:

administering a peptide according to claim 5 to a host animal; and

recovering the cell from the animal.

3. An isolated and purified polypeptide comprising the amino acid sequence of a glucagon receptor native to a mammal, the receptor having seven transmembrane domains, an N-terminal extracellular domain, a C-terminal intracellular domain, three extracellular loop domains, and three intracellular loop domains,

wherein the receptor is encoded by DNA which hybridizes under conditions of high stringency to a probe having the nucleotide sequence of the complement of nucleotides 145-1599 of SEQ ID NO:

14 or nucleotides 53-1483 of SEQ ID NO;

24.

4. A polypeptide according to claim 3, wherein the polypeptide is an allelic variant of the sequence shown in SEQ ID NO:

15 or SEQ ID NO;

25.

9. A peptide comprising the amino acid sequence of a fragment of a glucagon receptor polypeptide having the sequence shown as residues 28-485 of SEQ ID NO:

15, residues 1-485 of SEQ ID NO;

15, or residues 1-477 of SEQ ID NO;

25, the fragment comprising at least

(a) the complete sequence of one of the extracellular or intracellular domains of the receptor, or

(b) ten contiguous amino acids from the sequence of the receptor.

10. A peptide according to claim 9, wherein the fragment binds glucagon.

11. A peptide according to claim 9, wherein the fragment comprises a receptor domain which is 1ID, 1ELD, 2ID, 2ELD, 3ID, or 3ELD.

16. A process for making an antibody specific for a glucagon receptor, comprising the steps of:

administering a peptide according to claim 9 to a host animal; and

recovering the antibody from the animal.

18. A process for making a cell which produces an antibody specific for a glucagon receptor, comprising the steps of:

administering a peptide according to claim 9 to a host animal; and

recovering the cell from the animal.