Nucleic sequences encoding an AT2 receptor-interacting protein (ATIP) and their applications

• US 20060040277A1
• Filed: 10/29/2004
• Published: 02/23/2006
• Est. Priority Date: 08/04/1998
• Status: Abandoned Application

• Current Assignee: Clara Nahmias, Donny Strosberg Arthur, Nathalie Elbaz
• Original Assignee: Clara Nahmias, Donny Strosberg Arthur, Nathalie Elbaz
• Inventors: Nahmias, Clara, Strosberg Arthur, Donny, Elbaz, Nathalie
• Application Number:
  US10/976,933
• Publication Number:
  US 20060040277A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

The invention concerns nucleic sequences coding for a protein capable of interacting with the AT2 receptor, oligonucleotides included in said sequences, their application as probes and for expressing said proteins, vectors useful for said expression, host cells containing said vectors, and study model of AT2 receptor. The invention also concerns said proteins and their uses. Said isolated nucleic acid fragment coding for a protein capable of binding with the AT2 receptor is selected among the group consisting of the sequences SEQ ID NO: 1, 3, 5, 7 and 9.

Claims

1-5. -5. (canceled)

6. Purified and isolated protein, which is capable of interacting with the AT2 receptor and which is selected from the group consisting of the sequences SEQ ID NO:

2, 4, 6 or 8, which protein is called ATIP.

8. Antibodies, characterized in that they are directed against a protein or a protein fragment according to claim 6.

15. Method for selecting proteins inhibiting ATIP protein according to claim 6 AT2 receptor interaction, which method comprises:

(a) cotransforming a suitable yeast strain with three vectors which respectively encode (i) a bait corresponding to a fragment containing the C-terminal end of the AT2 receptor fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the said transcription factor, (ii) a fragment containing at least SEQ ID NO;

5 of the ATIP protein according to claim 6, fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the said transcription factor and (iii) a polypeptide corresponding to a sequence contained in a cDNA library, which vectors comprise, in addition, selectable markers,

(b) selecting the clones of cDNA library expressing a polypeptide inhibiting the AT2 receptor-ATIP protein according to claim 6 interaction, on an appropriate selective medium, and

(c) identifying the said polypeptide.

16. Method for screening polypeptides interacting with the ATIP protein according to claim 6, which method comprises:

(a) cotransforming a suitable yeast strain with two vectors as defined above, namely which respectively encode (i) a fragment containing at least SEQ ID NO;

5 of the ATIP protein according to claim 6, fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the said transcription factor and (ii) a polypeptide corresponding to a sequence contained in a cDNA library, fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the said transcription factor, which vectors comprise, in addition, selectable markers, and

(b) selecting the clones expressing a polypeptide interacting with the ATIP protein according to claim 6, on a suitable selective medium.

17. Method for characterizing the domains involved in the ATIP protein-AT2 receptor interaction, characterized in that it comprises:

(a) cotransforming a suitable yeast strain with two vectors, namely (i) a vector encoding a fragment containing at least SEQ ID NO;

5 of the ATIP protein according to claim 6, mutated or not, fused with a protein selected from the group consisting of the DNA- binding domain of a transcription factor and the activation domain of the said transcription factor and (ii) a vector encoding a fragment containing the C-terminal end of the AT2 receptor, mutated or not, fused with a protein selected from the group consisting of the DNA-binding domain of a transcription factor and the activation domain of the said transcription factor, which vectors comprise, in addition, selectable markers, one of the two vectors necessarily encoding a mutated protein, and

(b) visualizing, by selection on a suitable selective medium, the possible loss of the ATIP protein according to claim 6 AT2 receptor interaction.

18. Method for selecting substances capable of influencing the ATIP protein according to claim 6 AT2 receptor interaction, which method comprises:

(a) bringing the ATIP protein according to claim 6, attached to a support, into contact with a fusion protein AT2 receptor-protein tag, optionally in the presence of a substance to be tested,

(b) at least one washing of the said support thus treated with a suitable buffer, and

(c) visualizing the possible ATIP protein according to claim 6 AT2 receptor interaction, in particular in SDS-PAGE, followed by immunoblotting with antibodies directed against the protein tag, fused with the AT2 receptor.

19. Method for selecting substances capable of interacting with the ATIP protein according to claim 6, characterized in that it comprises:

(a) bringing the ATIP protein according to claim 6, attached to a support, into contact with a cell lysate,

(b) at least one washing of the said support thus treated with a suitable buffer,

(c) visualizing the possible protein combined with the ATIP protein, in particular in SDS-PAGE, followed by immunoblotting with appropriate antibodies, and

(d) identifying the protein in the cell lysate interacting with the ATIP protein.

7. Translational product, characterized in that it is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:

1, 3, 5, 7, and 9.

9-14. -14. (canceled)

20. (canceled)