# US 6,296,848 B1 · GRB2 associating polypeptides and nucleic acids encoding therefor

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- Number: US 6,296,848 B1
- Document ID: US6296848B1
- Status: Granted
- Kind: B1
- Application number: 09/418,540
- Filed: 1999-10-14
- Granted: 2001-10-02
- Earliest claimed: 1995-11-17
- Assignees: The Regents of the University of California
- Inventors: David A. Pot; Lewis T. Williams; Anne Bennett Jefferson; Philip W. Majerus
- Web page: https://patentgrep.com/patent/US6296848B1

## CPC

- C12N 9/16: acting on ester bonds (3.1)
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/4702: {Regulators; Modulating activity}
- C07K 16/18: against material from animals or humans
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention generally relates to novel GRB2 associating proteins and nucleic acids which encode these protein. In particular, these novel proteins possess inositol polyphosphate 5-phosphatase and phosphatidylinositol 5-phosphatase activities, important in growth factor mediated signal transduction. As such, the proteins, nucleic acids encoding the proteins, cells capable of expressing these nucleic acids and antibodies specific for these proteins will find a variety of uses in a variety of screening, therapeutic and other applications.

## Claims

### Claim 1

An isolated polypeptide comprising a catalytically active fragment of an amino acid sequence of FIG. 10 (SEQ ID NO:2), wherein said fragment comprises an inositol polyphosphate 5-phosphatase activity such that said fragment hydrolyzes a 5-phosphate from Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3, but not Ins(1,4,5)P3 or Ptdlns(4,5)P2.

### Claim 2 (depends on 1)

The isolated polypeptide of claim 1, wherein said polypeptide consists of a catalytically active fragment of an amino acid sequence shown in FIG. 10 (SEQ ID NO:2).

### Claim 3 (depends on 1)

The isolated polypeptide of claim 1, wherein said polypeptide further comprises a heterologous protein fused to the fragment.

### Claim 4 (depends on 1)

A pharmaceutical composition comprising an effective amount of the polypeptide of claim 1 in combination with a pharmaceutically acceptable carrier.

### Claim 5

A kit for determining whether a test compound is an agonist or antagonist of GA5Ptase activity, comprising:
a catalytically active fragment of a GA5Ptase polypeptide which comprises an amino acid sequence shown in FIG. 10 (SEQ ID NO:2), wherein said fragment has an inositol polyphosphate 5-phosphatase activity such that said fragment hydrolyzes a 5-phosphate from Ins(1,3,4,5)P4 and Ptdlns(3,4,5)P3, but not Ins(1,4,5)P3 or Ptdlns(4,5)P2;
a GA5Ptase substrate; and
instructions for assaying for the presence of said substrate and a product of GA5Ptase.

### Claim 6 (depends on 5)

The kit of claim 5, wherein said GA5Ptase substrate is selected from the group consisting of Ins(1,3,4,5)P4 and Ptdlns(3,4,5)P3.

### Claim 7 (depends on 5)

The kit of claim 5, wherein said GA5Ptase polypeptide and said GA5Ptase substrate are lyophilized, and further comprising a buffer solution for reconstituting said GA5Ptase polypeptide and said GA5Ptase substrate.

### Claim 8

An isolated polypeptide, said polypeptide being immunologically cross-reactive with an antibody to a fragment of a GA5Ptase protein which comprises an amino acid sequence shown in FIG. 10 (SEQ ID NO:2), wherein said fragment has an inositol polyphosphate 5-phosphatase activity such that said fragment hydrolyzes a 5-phosphate from Ins(1,3,4,5)P4 and PtdIns(3,4,5)P3, but not Ins(1,4,5)P3 or PtdIns(4,5)P2.

### Claim 9

An isolated polypeptide comprising a GRB2-associating fragment of amino acid sequence of FIG. 10 (SEQ ID NO:2).

### Claim 10 (depends on 9)

The isolated polypeptide of claim 9, wherein said GRB2-associating fragment binds a GRB2-SH3 domain.

### Claim 11 (depends on 9)

The isolated polypeptide of claim 9, wherein said polypeptide is a GRB2-associating fragment of the amino acid sequence shown in FIG. 10 (SEQ ID NO:2).

### Claim 12 (depends on 9)

The isolated polypeptide of claim 9, further comprising a heterologous protein fused to said GRB2-associating fragment.

### Claim 13 (depends on 9)

A pharmaceutical composition comprising an effective amount of the polypeptide of claim 9 in combination with a pharmaceutically acceptable carrier.

### Claim 14 (depends on 9)

A kit for determining whether a test compound is an agonist or antagonist of GA5Ptase activity, comprising:
a polypeptide of claim 9;
a GA5Ptase substrate; and
instructions for assaying for the presence of said substrate and a product of GA5Ptase.