Recombinant production of glucagon receptors

• US 5,776,725 A
• Filed: 07/01/1993
• Issued: 07/07/1998
• 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:
  US08/086,631
• Time in Patent Office: 1,832 Days
• Field of Search: 536/23.5, 536/24.31, 536/24.32, 435/320.1, 435/240.2, 435/69.1, 435/252.3, 435/254.11, 435/325
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/72 for hormones for neuromediators C07K14/70571
• 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 nucleic acid molecule comprising a nucleotide sequence encoding a glucagon receptor polypeptide having seven transmembrane domains, an N-terminal extracellular domain, three extracellular loop domains, and three intracellular loop domains,

wherein the receptor polypeptide 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 nucleic acid molecule hybridizes under high stringency hybridization conditions to a probe having the nucleotide sequence of the complement of the sequence shown in SEQ ID NO:

14, from nucleotide 145 to 1599, or the sequence shown in SEQ ID NO;

24, from nucleotide 53 to 1483.

2. The nucleic acid molecule of claim 1 wherein said glucagon receptor polypeptide has the amino acid sequence of a rat or a human glucagon receptor.

3. The nucleic acid molecule of claim 1 wherein said molecule is DNA comprising the sequence of nucleotides of SEQ ID NO:

14 from nucleotide 145 to nucleotide 1599 or the sequence of nucleotides of SEQ ID NO;

24 from nucleotide 53 to nucleotide 1483.

4. The nucleic acid molecule of claim 1 wherein said glucagon receptor polypeptide comprises the amino acid sequence shown as residues 28to 485 of SEQ ID NO:

15, residues 1 to 485 of SEQ ID NO;

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

25.

5. The nucleic acid molecule of claim 1 wherein said glucagon receptor polypeptide is an allelic variant of the sequence shown in SEQ ID NO:

15 or SEQ ID NO;

25.

6. The nucleic acid molecule of claim 1, wherein the molecule is DNA.

7. The nucleic acid molecule of claim 6, comprising the coding sequence of the insert in pLJ4 (ATCC 69056) or pLJ6 (ATCC 69183).

8. The nucleic acid molecule of claim 6, comprising the complete sequence of the insert in pLJ4 (ATCC 69056) or pLJ6 (ATCC 69183).

9. The nucleic acid molecule of claim 1, wherein said glucagon receptor polypeptide lacks the native signal peptide sequence.

10. The nucleic acid molecule of claim 9, wherein the mature form of the glucagon receptor polypeptide is a contiguous fragment of SEQ ID NO:

15 or SEQ ID NO;

25.

11. The nucleic acid molecule of claim 10, wherein the molecule is DNA and the nucleotide sequence encoding the amino acid sequence of the mature receptor is a contiguous fragment of the sequence shown in SEQ ID NO:

14 or SEQ ID NO;

24.

12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a fragment of a glucagon receptor polypeptide as defined in claim 1, the fragment comprising at least

(a) the complete sequence of an extracellular or intracellular domain of the receptor, or

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

13. The nucleic acid molecule of claim 12, wherein the fragment binds glucagon.

14. The nucleic acid molecule of claim 12, wherein the fragment comprises a receptor domain which is 1ID, 1ELD, 2ID, 2ELD, 3ID, or 3ELD.

15. The nucleic acid molecule of claim 12, wherein said fragment comprises:

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

15;

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

15;

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

15;

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

15;

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

15;

or

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

15.

16. A DNA construct comprising a first DNA segment encoding a glucagon receptor operably linked to at least one additional DNA segment which facilitates the expression of said first DNA segment in a suitable host cell, wherein said first DNA segment comprises the nucleotide sequence of a DNA molecule according to claim 1.

17. The DNA construct of claim 16 wherein said glucagon receptor polypeptide has the amino acid sequence of a rat or a human glucagon receptor.

18. The DNA construct of claim 16 wherein said first DNA segment comprises the sequence of nucleotides of SEQ ID NO:

14 from nucleotide 145 to nucleotide 1599 or the sequence of nucleotides from SEQ ID NO;

;

24 from nucleotide 53 to nucleotide 1483.

19. The DNA construct of claim 16 wherein said glucagon receptor polypeptide comprises the amino acid sequence shown as residues 28to 485 of SEQ ID NO:

15, residues 1 to 485 of SEQ ID NO;

25, or residues 1 to 477 of SEQ ID NO;

25.

20. A host cell containing a DNA construct according to any one of claims 16-19.

21. A method for producing a glucagon receptor polypeptide, comprising culturing a host cell according to claim 10 under conditions suitable to effect the expression of said first DNA segment.

22. The DNA construct of claim 16, comprising the coding sequence of the insert in pLJ4 (ATCC 69056) or pLJ6 (ATCC 69183).

23. The DNA construct of claim 16, comprising the complete sequence of the insert in pLJ4 (ATCC 69056) or pLJ6 (ATCC 69183).

24. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

15 from glutamine, amino acid 28, to tyrosine, amino acid 142.

25. The nucleic acid molecule of claim 24 wherein said molecule is DNA comprising the sequence of nucleotides of SEQ ID NO:

14 from nucleotide 226 to nucleotide 570.

26. The nucleic acid molecule of claim 24, wherein the molecule is DNA. 570.

27. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a fragment of a glucagon receptor polypeptide having the sequence shown as residues 1 to 485 of SEQ ID NO:

15 or residues 1 to 477 of SEQ ID NO;

25, the fragment comprising at least

(a) the complete sequence of an extracellular or intracellular domain of the receptor, or

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

28. The nucleic acid molecule of claim 27, wherein the fragment binds glucagon.

29. The nucleic acid molecule of claim 27, wherein the fragment comprises a receptor domain which is 1ID, 1ELD, 2ID, 2ELD, 3ID, or 3ELD.

30. The nucleic acid molecule of claim 27, wherein said fragment comprises:

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

15;

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

15;

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

15;

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

15;

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

15;

or

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

15.

31. The nucleic acid molecule of claim 27, wherein said fragment does not include part of the signal peptide sequence.

32. A DNA construct comprising a first DNA segment encoding a glucagon receptor fragment operably linked to at least one additional DNA segment segment which facilitates the expression of said first DNA segment in a suitable host cell, wherein said first DNA segment comprises the nucleotide sequence of a DNA molecule according to claim 27.

33. The DNA construct of claim 32 wherein said glucagon receptor polypeptide has the amino acid sequence of a rat or a human glucagon receptor.

34. The DNA construct of claim 32 wherein said first DNA segment comprises the sequence of nucleotides of SEQ ID NO:

14 from nucleotide 226 to nucleotide 570.

35. The DNA construct of claim 32 wherein said glucagon receptor fragment comprises the amino acid sequence of SEQ ID NO:

15 from glutamine, amino acid 28, to tyrosine, amino acid number 142.

36. A host cell containing a DNA construct according to any one of claims 32-35.

37. A method for producing a fragment of a glucagon receptor polypeptide, comprising culturing a host cell according to claim 36 under conditions suitable to effect the expression of said first DNA segment.

38. A polynucleotide probe which is capable of hybridizing under conditions of high stringency with the coding strand of a cDNA encoding a glucagon receptor or the complement thereof,

wherein the sequence of the coding strand is:

(a) SEQ ID NO;

14;

(b) SEQ ID NO;

24 ;

or

(c) an allelic variant of the coding sequence of (a) or (b);

and wherein the probe comprises at least 18 contiguous nucleotides from the sequence of;

the coding or noncoding strand of the cDNA, or

an RNA molecule complementary to either of said strands;

provided that the at least 18 contiguous nucleotides are not contained entirely within a nucleotide sequence encoding a transmembrane domain of said receptor or a noncoding sequence complementary thereto.