Compositions of alpha platelet derived growth factor receptor nucleic acid and protein and method of making
Inventors
Matsui, Toshimitsu • Aaronson, Stuart A. • Pierce, Jacalyn H.
Assignees
US Department of Health and Human Services
Publication Number
US-7427671-B1
Publication Date
2008-09-23
Expiration Date
2025-09-23
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Abstract
A DNA sequence which encodes a human type α platelet derived growth factor receptor protein which preferentially binds to the AA homodimer and AB heterodimer forms of platelet derived growth factor and also binds the BB homodimer at high affinity, is described. Substantially pure human a platelet derived growth factor receptor protein and methods for recombinantly producing human α platelet derived growth factor receptor protein are also described.
Core Innovation
The invention relates to the identification, isolation, and cloning of a novel human gene encoding a type α platelet derived growth factor receptor (α PDGF-R) protein. This receptor protein preferentially binds to the AA homodimer and AB heterodimer forms of platelet derived growth factor and also binds the BB homodimer at high affinity. The invention includes DNA segments encoding the receptor, recombinant DNA molecules comprising such DNA, methods for expressing these DNA in host cells to produce the receptor protein, and the use of such proteins for functional studies and bioassays.
The problem addressed is uncertainty in the ability of known PDGF receptors to respond differently to PDGF isoforms, particularly the main AB heterodimer form. Prior studies showed conflicting findings regarding binding and functional responses to PDGF isoforms, possibly due to processing differences of receptor gene products or accessory proteins. There was a need for improved methods and compositions to analyze connective tissue growth regulation mechanisms, novel diagnostics, and therapies targeting PDGF receptors involved in physiological and pathological processes such as wound healing, neoplastic growth, arteriosclerosis, arthritis, and fibrotic diseases.
The invention overcomes these issues by providing the DNA encoding a novel PDGF receptor distinct from the known PDGF-R gene, designated as type α PDGF receptor. This receptor is coexpressed with the known type β receptor but exhibits distinct binding preferences, especially for the main human platelet PDGF isoform (AB). The invention also includes methods to express this receptor in host cells, bioassays to detect its expression, antibodies specific to the type α receptor, and recombinant cell lines for studying receptor functions and developing therapeutics.
Claims Coverage
The patent claims cover 15 claims focusing on the novel human type α platelet derived growth factor receptor protein and nucleic acids encoding it, along with recombinant expression systems and methods of producing the protein. There are several inventive features that encompass pure protein forms, cDNA sequences, recombinant DNA constructs, transformed cell cultures, and protein production methods.
Substantially pure human α-platelet derived growth factor receptor protein
Claims a substantially pure human α PDGFR protein having the amino acid sequence of amino acids 1-1089 or 24-1089 of the sequence shown in FIG. 3, including forms with the signal peptide intact or cleaved.
cDNA encoding human α PDGFR protein
Claims cDNA sequences encoding the α PDGFR protein, including several specified variants corresponding to sequences shown in FIG. 3 such as the entire T11 cDNA, TR4 cDNA with or without the signal peptide coding sequence, and the pHF1 cDNA clone.
Recombinant DNA vectors comprising α PDGFR cDNA
Claims recombinant DNA vectors carrying the cDNAs encoding the α PDGFR as defined in the preceding claims, enabling expression in host cells.
Transformed in vitro cell cultures expressing α PDGFR protein
Claims in vitro cultured cells transformed with the recombinant DNA vectors carrying α PDGFR cDNA that produce the α PDGFR protein.
Method of producing human α PDGFR protein
Claims a method of producing human α PDGFR protein by culturing transformed cells expressing the α PDGFR and isolating the protein from those cells.
The claims encompass purified α PDGFR protein forms, specific cDNA sequences encoding this receptor, recombinant DNA constructs for expression, transformed cell cultures producing the receptor, and methods for producing the α PDGFR protein, collectively enabling recombinant production and characterization of this distinct human PDGF receptor.
Stated Advantages
The type α PDGF receptor is the main effector for the most abundant human PDGF form found in platelets, enabling targeted diagnostic and therapeutic approaches.
DNA probes and antibodies specific to the type α receptor allow detection and differentiation from the type β receptor, aiding in diagnosis of tumors and pathological conditions.
Expression of the α PDGF receptor in host cells permits production of pure receptor protein for biochemical and functional studies.
The invention provides improved understanding of PDGF receptor roles in normal growth control, wound healing, and diseases such as cancer, arteriosclerosis, arthritis, and fibrosis.
Availability of stable expression systems for each receptor type enables elucidation of receptor-specific binding and signaling leading to development of receptor analogs as agonists or antagonists for therapy.
Documented Applications
Use of α PDGF receptor proteins for functional studies including qualitative and quantitative receptor binding assays.
Recombinant production of α PDGF receptor proteins in mammalian cells such as COS-1 cells.
Development of bioassays employing DNA probes to detect expression levels of α PDGF receptor mRNAs distinct from β PDGF receptor transcripts.
Use of specific antibodies for detection, purification, and potential therapeutic targeting of α PDGF receptor expressing tissues and tumors.
Reconstitution of PDGF receptor function in null cells to study PDGF-mediated mitogenic signaling, chemotaxis, phosphoinositide turnover, and calcium mobilization.
Application in diagnostics and treatments for diseases involving abnormal connective tissue growth regulation including cancers, arteriosclerosis, arthritis, and fibrotic disorders.
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