Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-03-15
2001-07-24
Eyler, Yvonne (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387100, C530S387300, C530S388100
Reexamination Certificate
active
06265550
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of a human insulin receptor tyrosine kinase substrate and to the use of these sequences in the diagnosis, prevention, and treatment of cancer, inflammation, and disorders associated with insulin response.
BACKGROUND OF THE INVENTION
Insulin controls blood glucose levels by stimulating glucose influx and metabolism in muscle and adipocytes and by inhibiting gluconeogenesis in the liver. Insulin also modifies the expression or the activity of a variety of enzymes and transport systems in nearly all cells.
Insulin action is mediated through the insulin receptor (IR), a transmembrane glycoprotein with protein tyrosine kinase (PTK) activity. Insulin binding triggers receptor autophosphorylation which activates PTK activity. The cellular response to insulin is mediated through tyrosine phosphorylation of cytosolic polypeptide substrates which act as second messengers in IR signal transduction. Once phosphorylated, the substrates bind to and activate various signal transduction proteins. The signal transduction proteins contain Src-homology-2 (SH2)-domains which bind phosphotyrosine-containing peptide motifs.
Several IR-PTK substrates have been described. The most extensively characterized substrate is the 185-kdal insulin receptor substrate-1 (IRS-1). IRS-1 is found in a variety of insulin responsive cells and tissues. It exhibits no intrinsic enzyme activity but, once phosphorylated, binds to and activates SH2-containing signal transduction proteins including phosphatidylinositol (PI) 3′-kinase and GRB-2, a regulator of the Ras pathway (White, M. F. et al. (1994) J. Biol. Chem. 269:1-4). Mutations in the IRS-1 gene impairs insulin-stimulated signaling and may contribute to insulin resistance in normal and diabetic populations (Almind, K. et al. (1996) J. Clin. Invest. 97:2569-2575).
Two 60-kdal protein substrates of the IR-PTK have been identified. One associates with the GTPase activator of Ras (termed GAP) and the other associates with PI 3′-kinase (Yeh, T. et al. (1996) J. Biol. Chem 271:2921-2928). Two additional substrates for IR-PTK with molecular masses of 53 and 58 kdal were recently identified in rodents. These proteins, p53 and p58, are closely related and may arise from alternative splicing of mRNA or differential post-translational modifications. P53 and p58 do not associate with GAP or PI 3′-kinase and are immunologically distinct from the 60-kDa GAP-associated protein and the 60-kDa PI 3′-kinase-associated protein (Yeh, et al., supra).
Post-receptor defects in the insulin signaling pathway are a common feature of type 2 (non-insulin-dependent) diabetes mellitus (Stoffel M. et al. (1993) Diabetologia 36: 335-337). Other disorders or conditions associated with disturbances in insulin response include hyperglycemia, myotonic muscular dystrophy, acanthosis nigricans, retinopathy, nephropathy, atherosclerotic coronary and peripheral arterial disease, and peripheral and autonomic neuropathies.
The discovery of a new human insulin receptor tyrosine kinase substrate and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention and treatment of cancer, inflammation, and disorders associated with insulin response.
SUMMARY OF THE INVENTION
The invention features a substantially purified polypeptide, human insulin receptor tyrosine kinase substrate (IRS-p53h), having the amino acid sequence shown in SEQ ID NO:1, or fragments thereof.
The invention further provides an isolated and substantially purified polynucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or fragments thereof and a composition comprising said polynucleotide sequence. The invention also provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of said polynucleotide sequence. The invention further provides a polynucleotide sequence comprising the complement of the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or variants of said polynucleotide sequence.
The invention also provides an isolated and purified sequence comprising SEQ ID NO.2 or variants thereof. In addition, the invention provides a polynucleotide sequence which hybridizes under stringent conditions to the polynucleotide sequence of SEQ ID NO:2.
In another aspect the invention provides a composition comprising an isolated and purified polynucleotide sequence comprising the complement of SEQ ID NO:2, or fragments or variants thereof. The invention also provides a polynucleotide sequence comprising the complement of SEQ ID NO:2.
The present invention further provides an expression vector containing at least a fragment of any of the claimed polynucleotide sequences. In yet another aspect, the expression vector containing the polynucleotide sequence is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof, the method comprising the steps of: a) culturing the host cell containing an expression vector containing at least a fragment of the polynucleotide sequence encoding IRS-p53h under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified IRS-p53h having the amino acid sequence of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention also provides a purified antagonist which decreases the activity of a polypeptide of SEQ ID NO:1. In one aspect the invention provides a purified antibody which binds to a polypeptide comprising at least a fragment of the amino acid sequence of SEQ ID NO:1.
Still further, the invention provides a purified agonist which modulates the activity of the polypeptide of SEQ ID NO:1.
The invention also provides a method for treating or preventing a disorder associated with insulin response comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising purified IRS-p53h.
The invention also provides a method for treating or preventing cancer comprising administering to a subject in need of such treatment an effective amount of an antagonist to IRS-p53h.
The invention also provides a method for treating or preventing inflammation comprising administering to a subject in need of such treatment an effective amount of an antagonist to IRS-p53h.
The invention also provides a method for detecting a polynucleotide which encodes IRS-p53h in a biological sample comprising the steps of: a) hybridizing a polynucleotide sequence complementary to IRS-p53h (SEQ ID NO:1) to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding IRS-p53h in the biological sample. In a preferred embodiment, prior to hybridization, the nucleic acid material of the biological sample is amplified by the polymerase chain reaction.
REFERENCES:
Morrison, in Advances in Immunology 44:65-92, Academic Press, Inc., 1989.*
White, M.F. et al., “The Insulin Signaling System”,J. Biol. Chem.,269: 1-4 (1994).
Almind, K. et al., “A Common Amino Acid Polymorphism in Insulin Receptor Substrate-1 Causes Impaired Insulin Signaling”,J. Clin. Invest.,97: 2569-2575 (1996).
Yeh, T. et al., “Characterization and Cloning of a 58/53-kDa Substrate of the Insulin Receptor Tyrosine Kinase”,J. Biol. Chem.,271: 2921-2928 (1996) (GI 1203820).
Stoffel, M. et al., “Human insulin receptor substrate-1 gene (IRS1): chromosomal localization to 2q35-q36.1 and identification of a simple tandem repeat DNA polymorphism”,Diabetologia,36: 335-337 (1993).
Yeh, T.C. et al., (Direct Submission), GenBank Sequence Dat
Hillman Jennifer L.
Lal Preeti
Shah Purvi
Eyler Yvonne
Incyte Genomics Inc.
Incyte Genomics, Inc.
Lazar-Wesley Eliane
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