Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-05-23
2001-02-13
Caputa, Anthony C. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007210, C436S501000, C436S503000
Reexamination Certificate
active
06187548
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a cDNA clone encoding a human calcium sensor protein of parathyroid, placental, and kidney tubule cells.
In WO 88/03271 there is described monoclonal antiparathyroid antibodies identifying a parathyroid cell membrane-bound calcium receptor or sensor, crucially involved in calcium regulation of the parathyroid hormone (PTH) release (1,2). The receptor function is essential for maintenance of normal plasma calcium concentrations, and reduced receptor expression within proliferating parathyroid cells of patients with hyperparathyroidism (HPT) results in calcium insensitivity of the PTH secretion and variably severe hypercalcemia (3-6). Reactivity with the antiparathyroid antibodies was also demonstrated for proximal kidney tubule cells and cytotrophoblast cells of the human placenta, and the cytotrophoblasts were demonstrated to exhibit an almost parathyroid-identical regulation of cytoplasmic calcium [Ca
2+
i] (7,8). The antibody-reactive structure was found to exert calcium sensing function also in the cytotrophoblasts, and as these cells constitute part of the syncytium, the calcium sensor was suggested to be actively involved in the calcium homeostasis of the fetus (7,8). It was proposed that the antibody-reactive structure of the proximal kidney tubule cells exerts a similar calcium sensing function, and that the calcium sensor, thus, plays a more universal role in calcium regulation via different organ systems (1,7,9,10).
On HPT patients with hypercalcemia, surgery is performed to remove one or more of the parathyroid glands. It would be greatly desirable to have alternatives to this surgical procedure as HPT has proven to be a very common disorder and surgery is a relatively costly procedure and sometimes even entails some risks for the patients.
The calcium sensor/receptor has been revealed as a 500 kDa single chain glycoprotein (7). However, the amino acid sequence as well as the corresponding DNA sequences thereof are hitherto unknown.
SUMMARY OF THE INVENTION
Therefore, an object of the present invention was to provide sufficient structural data of the calcium sensor/receptor to enable complete characterization thereof.
In one embodiment, the present invention provides complete amino acid sequence of the human calcium sensor protein of parathyroid, placental and kidney tubule cells.
In another embodiment the invention provides nucleic acid sequence encoding the human calcium sensor and nucleic acid probes for identifying other novel calcium sensor proteins.
Another object is to use said structural data to design novel treatment methods as well as compounds and compositions for treating calcium related disorders.
In other embodiments, the present invention provides identification of peptide regions within the calcium sensor protein cytoplasmic domain which are homologous to SH2 and SH3 binding motifs involved in signal transduction pathways.
Two important human diseases associated with perturbations of the calcium ion homeostasis are hyperthyroidism and osteoporosis. Thus, in one embodiment cells expressing the calcium sensor protein or a fragment thereof or comprising the cDNA encoding the calcium sensor protein of the present invention may be utilized in an assay to identify molecules which block or enhance the activity of the calcium sensor protein, including signal transduction pathways associated with the activity of the sensor. These molecules will be useful in the treatment of mammalian pathological conditions associated with perturbations in the levels of PTH, vitamins D3 production, estrogen, osteoclast activity or osteoblast activity (therefore, bone resorption and/or formation), calcium secretion and calcium ion homeostasis.
The present invention describes the isolation and characterization of cDNA clones encoding the calcium sensor/receptor in human placenta and Northern blots verifying the presence of the corresponding mRNA within the parathyroid and kidney. Close sequence similarity between the calcium sensor and a rat
Heymann nephritis
antigen, gp330 (11, 67), suggests that the common calcium sensor of the placenta, the parathyroid and kidney tubule is related to this antigen, represents the human homologue of gp330, and belongs to a family of large glycoproteins with receptor function and calcium binding ability. Therefore, a further object of this invention is to provide diagnostic assays and therapeutic methods based on human gp330.
The invention provides a method of identifying agonists and/or antagonists of human calcium sensor protein activity comprising contacting potential agonists or antagonists with said calcium sensor protein or a biologically active sequence analog thereof and determining the ability of said potential agonists or antagonists to block or enhance the activity of said calcium sensor protein or biologically active sequence analog thereof.
The present invention further provides a method of identifying agonists and/or antagonists of human calcium sensor protein activity comprising:
a) expressing a cDNA encoding a human calcium sensor protein or a biologically active sequence analog thereof in a host cell;
b) contacting potential agonists or antagonists with said host cell; and
c) determining the ability of said agonists or antagonists to block or enhance the activity of said calcium sensor protein or fragment thereof.
REFERENCES:
patent: 358977 (1990-03-01), None
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Kounnas et al.,JBC, vol. 270, pp. 13070-13075, Jun. 2, 1995.
Juhlin, C. et al., 500-Kilodalton Calcium Sensor Regulating Cytoplasmic Ca2+ in CytotrophoblastCells of Human Placenta, The Journal of Biological Chemistry 265, 8275-8279 (1990).
Saito, A. et al., Complete Cloning and Sequencing of Rat gp330/“Megalin,” a Dinstinctive Member of the Low Density Liproprotein Receptor Gene Family, Proceedings of the National Academy of Science 91, 9725-9729 (1994).
Raychowdhury, R. et al., Autoimmune Target in Heymann Nephritis is a Glycoprotein with Homology to the LDL Receptor, Science 244, 1163-1165 (1989).
Juhlin, C. et al., Monoclonal Antibodies with Exclusive Reactivity Against Parathyroid Cells and Tubule cells of the Kidney, Proceedings of the National Academy of Science 84, 2990-2994 (1987).
Lundgren, S. et al., A Protein Involved in Calcium Sensing of the Human Parathyroid and Placental Cytotrophoblast Cells Belongs to the LDL-Receptor Protein Superfamily, Experimental Cell Research 212, 344-350 (1994).
Brown, E. et al., Molecular Mechanisms Underlysing the Sensing of Extracellular Ca2+ by Parathryroid and Kidney Cells, Europearn Journal of Endocrinology 132, 523-531 (1995).
Moestrup, S., The alpha2-Macroglobulin Receptor and Epithelial Glycoprotein-330: Two Giant Receptors Mediating Endocytosis of Multiple Ligands, Biochimica et Biophysica Acta 1197 197-213 (1994).
Farquhar, M. et al., gp330 and RAP: The Heymann Nephritis Antigenic Complex, Annals New York Academy of Sciences 737, 96-113 (1994).
Kounnas, m. et al., An Overview of the Structure and Function of Glycoprotein 330, a Receptor Related to the alpha2-macroglobulin Receptor, Annals New York Academy of Sciences 737, 114-123 (1994).
Moestrup, S. et al., Binding and Endocytosis of Proteins Mediated by Epithelial gp330, Annals New York Academy of Sciences 737, 124-137 (1994).
Zlokovic et al., Glycoprotein 330/megalin: Probable role in receptor-mediated transport of apolipoprotein J alone and in a complex with Alzheimer disease amyloid &bgr; at the blood-brain and blood-cerebrospinal fluid barriers, Proc. Natl. Acad. Sci., USA 93, 4229-4234 (1996).
Akerstrom Goran
Crumley Gregg R.
Hjälm Göran
Juhlin Claes
Morse Clarence C.
Caputa Anthony C.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Gucker Stephen
Rhone-Poulenc Rorer Pharmaceuticals Inc.
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