Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-06-27
2002-06-04
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023500, C435S325000, C435S471000, C435S252300, C435S320100
Reexamination Certificate
active
06399326
ABSTRACT:
BACKGROUND OF THE INVENTION
Protein tyrosine phosphorylation has been extensively characterized as a major mechanism of transducing signals within cells. The balance of tyrosine phosphorylation is maintained and modulated by two opposing sets of enzymes, the protein tyrosine kinases (PTKs) and the protein tyrosine phosphatases (PTPs). During embryonic development, several protein tyrosine kinases are known to have powerful and specific roles (Cantley et al., (1991)
Cell
64:281-302; Fantl et al., (1993)
Annu. Rev. Biochem
. 62:453-481; Imamoto et al., (1994)
Curr. Opin. Gen. Dev
. 4:40-46; van der Geer et al., (1994)
Annu. Rev. Cell Biol
. 10:251-337). Though the PTPs are less well characterized, there is genetic evidence to indicate important functions in specific tissues during development. The Drosophila gene corkscrew, for example, encoding an intracellular PTP, is required for the development of the head and tail of the embryo (Perkins et al., (1992)
Cell
70:225-236). Mice homozygous for the moth-eaten (me) allele which encodes a mutated version of the intracellular PTP, hematopoietic cell phosphatase (HCP, also known as SH-PTP1 and PTP1C), have a variety of defects in the immune system (Shultz et al., (1993)
Cell
73:1445-1454; Tsui et al., (1993)
Nat. Gen
. 4:124-129). Important roles for other PTPs are also indicated by biochemical studies. For instance, an intracellular PTP, FAP-1 (also known as PTP-BAS) has been found to be involved in the signal transduction pathway of apoptosis (Sato et al., (1995)
Science
268:411-415).
In addition to the phosphatase catalytic domain, many PTPs contain a transmembrane and extracellular domain (Cohen and Cohen, (1989)
J. Biol. Chem
. 264:21345-21438; Hunter, (1989)
Cell
58:1013-1016; Walton and Dixon, (1993)
Annu. Rev. Biochem
. 62:101-120; Brady-Kalnay and Tonks, (1995)
Curr. Opin. Cell Biol
. 7:650-657). Like the transmembrane PTKs, the transmembrane PTPs could be receptors with the potential to regulate the phosphorylation state of downstream targets in response to binding of extracellular ligands. However, there has heretofore been little evidence on ligands, or on the potential for ligand-induced signaling. Two transmembrane PTPs, PTPm and PTPk, have been demonstrated to exhibit homophilic binding which can cause cell-cell adhesion (Brady-Kalnay et al., (1993)
Curr. Opin. Cell Biol
. 7:650-657; Gebbink et al., (1993)
J. Biol. Chem
. 268:16101-16104; Sap et al., (1994)
Mol. Cell. Biol
. 14:1-9). Another transmembrane PTP, RPTPb, was found to correspond to phosphacan, a proteoglycan that can interact with the adhesion molecules N-CAM and Ng-CAM and the extracellular matrix protein tenascin (Milev et al., (1991)
J. Cell Biol
. 127:1703-1715; Barnea et al., (1994)
J. Biol. Chem
. 269:14349-14352; Grumet et al., (1994)
J. Biol. Chem
. 269:12142-12146), and was also identified as a ligand of the neuronal cell surface molecule contactin (Peles et al., (1995)
Cell
82:251-260).
In vertebrates and invertebrates, several receptor-type PTPs have been identified with restricted expression patterns in the developing nervous system. In Drosophila, DPTP99A, DPTP10D, and DLAR are transmembrane PTPs with neuron-specific expression, and immunolocalization of these molecules on axons has led to proposals that they may be involved in axon outgrowth and guidance (Tian et al., (1991)
Cell
67:687-700; Yang et al., (1991)
Cell
67:661-673). In vertebrates, RPTPb shows expression restricted to the developing nervous system (Carnoll et al., (1993)
Brain Res. Dev. Brain Res
. 75:293-298; Levy et al., (1993)
J. Biol. Chem
. 268:10573-10581). LAR, RPTPs ,and CRYPa were also found expressed in embryonic neuronal tissues (Yan et al., (1993)
J. Biol. Chem
. 268:24880-24886; Stoker (1994)
Mech. Dev
. 46:201-217; Wang et al., (1995)
J. Neurosci. Res
. 41:297-310). In addition, PTPa was found to be induced during neuronal differentiation of P 19 cells (den Hertog et al., (1993)
EMBO J
. 12:3789-3798). Similarly, the expression of two other PTPs, PC12-PTP1 and LAR, was induced in differentiating PC12 cells (Sharama and Lombroso, (1995)
J. Biol. Chem
. 270:49-53; Zhang and Longo, (1995)
J. Cell. Biol
. 128:415-431).
Unlike the nervous system, there is little information on molecular mechanisms for cell-cell signaling in pancreatic development, and no cell-cell signaling molecules specific to the pancreatic lineage have yet been identified. The pancreas is of enormous medical importance, because of its role in widespread diseases, notably juvenile diabetes and pancreatic cancer. Formation of the pancreas during development has been well studied at the morphological and cellular level, but little is known about control of the induction, growth or differentiation of the pancreas at the molecular level (Slack, (1995)
Development
121:1569-1580). Previous studies have shown some transcription factors expressed in early developing pancreas. In particular, STF-1 (also known as IPF-1, IDX-1, or PDX: Ohlsson et al., (1993)
EMBO J
. 12:4251-4259; Miller et al., (1994)
EMBO J
. 13:1145-1156; and Guz et al., (1995)
Development
121:11-18), a homeobox gene, is expressed in the pancreatic primordium and adjacent gut endothelium, and has been shown by targeted mutagenesis to be critical for the development of the pancreas (Jonsson et al., (1994)
Development
114:75-87). However, even though extracellular signals to control pancreatic endocrine development could be clinically useful, the mechanisms of extracellular signaling that control pancreas formation and endocrine cell development are still being elucidated.
SUMMARY OF THE INVENTION
The present invention relates to the discovery of a new class of the receptor protein tyrosine phosphatases (PTP), referred to herein as PTP-NP (for neural and pancreatic) receptors.
In general, the invention features isolated PTP-NP polypeptides, preferably substantially pure preparations of the subject PTP-NP polypeptides. The invention also provides recombinantly produced PTP-NP polypeptides. In preferred embodiments the polypeptide has a biological activity including one or more of: the ability to dephosphorylate a phosphotyrosine residue; hydrolyze a phosphatase substrate such as p-nitrophenylphosphate; bind to a ligand expressed on pancreatic &bgr; cells. However, PTP-NP polypeptides which specifically antagonize such activities, such as may be provided by truncation mutants or other dominant negative mutants, are also specifically contemplated.
The PTP-NP proteins of the present invention can be characterized as including one or more of the following domains/motifs: an extracellular domain, having a cys
4
domain, which mediate ligand binding, a transmembrane domain, and an intracellular domain including a phosphatase domain. The protein may also include a secretion signal sequence, and (optionally) glycosylated amino acid residues.
In one embodiment, the polypeptide is identical with or homologous to a PTP-NP protein represented in SEQ ID NO: 2. Related members of the PTP-NP family are also contemplated, for instance, a PTP-NP polypeptide preferably has an amino acid sequence at least 65%, 70%, 75% or 80% homologous to the polypeptide represented by. SEQ ID NO: 2, though polypeptides with higher sequence homologies of, for example, 85, 90% and 95% or are also contemplated. In a preferred embodiment, the PTP-NP polypeptide is encoded by a nucleic acid which hybridizes under stringent conditions with a nucleic acid sequence represented in SEQ ID NO: 1. Homologs of the subject PTP-NP proteins also include versions of the protein which are resistant to post-translation modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with extracellular ligands or with intracellular proteins involved in signal transduction.
The PTP-NP polypeptide can comprise a full length protein, such as represented in SEQ ID NO: 2, or it can comprise a frag
Chiang Ming-Ko
Flanagan John G.
Clauss Isabelle M.
Foley Hoag & Eliot
Kunz Gary L.
Landsman Robert S.
President and Fellows of Harvard College
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