Claims
- 1. An amino acid sequence having a PDZ domain and an RGS domain.
- 2. A protein comprising an amino acid sequence substantially identical to that shown in SEQ ID NO: 1.
- 3. A nucleic acid encoding the amino acid sequence according to claim 2.
- 4. A nucleic acid that hybridizes to the nucleic acid according to claim 3.
- 5. A recombinant vector comprising the nucleic acid according to claim 3.
- 6. A recombinant cell containing the vector according to claim 5.
- 7. A protein according to claim 2, wherein the protein is encoded by a gene from a vertebrate.
- 8 A protein according to claim 7, wherein the vertebrate is a mammal.
- 9. A protein encoded by a gene, the protein having an RGS domain and a PDZ domain, the PDZ domain being capable of binding to a portion of a cytoplasmic domain of an ephrin-B2 in a cell.
- 10. A protein according to claim 9, wherein the binding occurs in a two-hybrid system in a yeast cell, wherein the ephrin-B2 cytoplasmic domain is used as the bait of the system.
- 11. A protein according to any of claim 9, wherein the mammalian cDNA library is obtained from a tissue selected from the group consisting of an embryo, a tumor or a leukemia.
- 12. A protein according to claim 11, wherein the tumor is of neural origin.
- 13. A protein according to claim 12, wherein the tumor of neural origin is a neuroblastoma.
- 14. A protein according to claim 2, wherein the protein causes stimulation of ephrin-B1 induced de-adhesion of embryonic test cells at levels of ephrin-B1 that are suboptimal for dissociation.
- 15. A protein according to claim 14, wherein the stimulation is at least 2-fold.
- 16. A protein according to claim 15, wherein the stimulation is at least 4-fold.
- 17. A protein according to claim 16, wherein the stimulation is at least 8-fold.
- 18. A protein according to claim 14, wherein the stimulation is dependent on the presence of an amino acid sequence present in the carboxy terminal RGS domain.
- 19. A protein according to claim 14, wherein the stimulation is reversed in a dose-dependent manner in the presence of the amino terminal PDZ domain and in the absence of the carboxy terminal RGS domain.
- 20. A protein according to claim 15, wherein the embryonic test cells are from an embryo of a cold-blooded vertebrate.
- 21. A protein according to claim 20, wherein the vertebrate is an amphibian.
- 22. A soluble epHB2 receptor capable of binding a cell, such that a pattern of migration of the cell is altered.
- 23. A method of altering sensitivity of a cell to a chemokine, comprising:
transmitting a reverse signal from a recombinant soluble ephB2 receptor to a transmembrane protein in the cell which is a ligand of the ephB2 receptor; binding a cytoplasmic protein, the cytoplasmic protein having an RGS domain and a PDZ domain, to the cytoplasmic domain of the transmembrane protein in the cell; and altering a reaction of a G-protein coupled receptor (GPCR) in the membrane of the cell, such that the cell has altered sensitivity to a chemoattractant chemokine.
- 24. A method according to claim 23, wherein the cell is a granule cell located in an external granule cell layer (EGL) of a developing brain cerebellum.
- 25. A method according to claim 23, wherein the cell is involved in a process selected from the group consisting of cell migration, blood vessel formation, axon pathway selection, and rhombomere compartmentation.
- 26. A method according to claim 23, wherein the transmembrane protein is substantially homologous to an ephrin-B2.
- 27. A method according to claim 23, wherein the chemokine is an SDF-1.
- 28. A method according to claim 23, wherein transmission of the reverse signal requires a presence of the PDZ-RGS3 protein in the cell.
- 29. A method according to claim 27, wherein transmission of the reverse signal causes loss of responsiveness of the cell to the SDF-l chemokine.
- 30. A method according to claim 23, wherein the cell is a leukocyte.
- 31. A method according to claim 30, wherein the method by which the cell has altered sensitivity to the chemokine is a treatment for an inflammatory condition or an autoimmune disease.
- 32. A method according to claim 23, further comprising omitting the recombinant soluble ephB2, and screening for a chemical agent that substitutes functionally for the omitted ephB2.
- 33. A method according to claim 23, further comprising adding a test sample to screen for an agent that alters sensitivity of the cell to the chemokine.
- 34. A method of modulating an intracellular pathway involved in cell migration upon the event of a cell to cell contact, comprising:
initiating ephrin signaling by providing cell to cell contact in a cell having a cytoplasmic protein, the cytoplasmic protein having an amino terminus that interacts with the carboxy terminus of ephrin, and having a carboxy terminus that affects a GTP-linked reaction of a seven transmembrane protein, causing the cell in the presence of sufficient chemokine to otherwise inhibit such migration from an initial anatomical location and towards a target location.
- 35. A method according to claim 34, further comprising selecting the cell from a granule cell and a leukocyte, such that the cytoplasmic protein mediates an intracellular pathway, or causing a granule cell to migrate away from the external granule cell layer (EGL), or causing the leukocyte to migrate into an inflamed tissue.
- 36. A method of screening in a test sample for the presence of an agent that alters in vivo functional interactions among the components of an ephrin-B signal pathway involving chemoattraction by a chemokine, comprising:
placing chemokine-sensitive cells having the ephrin-B ligand on a top side of a filter in an upper chamber of a transwell system, wherein the filter has pores of uniform size and separates the upper chamber from a lower chamber, and wherein the lower chamber contains the chemokine; adding a test sample to the lower chamber; and analyzing the lower side of the filter to determine an amount of migration of the cells into the lower chamber.
- 37. A method according to claim 36, having a second transwell such that adding a test sample to the lower chamber of the second transwell is omitted as a control, and further comparing the amount of migration of cells in the presence and absence of the test sample is an indication of the effect of the agent on cell migration.
- 38. A method according to claim 36, wherein the cells are selected from a purified preparation of cerebellar granule cells and a pure cultured leukocyte cell line.
- 39. A method according to claim 38, wherein the chemokine is SDF-1.
- 40. A method according to claim 36, wherein the lower chamber contains the chemokine at a sub-optimal level.
- 41. A method according to claim 40, wherein the agent causes a decrease in cell migration in comparison to the second transwell control.
- 42. A method according to claim 41, wherein the agent is an anti-inflammatory or an anti-autoimmune therapeutic composition.
- 43. A method according to claim 40, wherein the agent causes a increase in cell migration in comparison to the control.
- 44. A method according to claim 43, wherein the agent is a novel chemokine.
- 45. A method according to claim any one of claims 41 or 43, wherein the agent is a low molecular weight synthetic organic chemical.
- 46. A pharmaceutical composition for delivering to a selected site an effective dose of a protein having an RGS domain and a PDZ domain capable of altering the sensitivity of a cell to a chemokine comprising an effective dose of said protein and a suitable carrier, and optionally additional active or inert ingredients such as diluents, stabilizers, and excipients.
- 47. A pharmaceutical composition according to claim 46, further comprising a substance which allows for the slow release of the pharmaceutical composition at the selected site.
- 48. A pharmaceutical composition according to claim 46, wherein the selected site for delivery is a tumor site.
- 49. A pharmaceutical composition according to claim 46, wherein the selected site for delivery is an allergic response site.
- 50. A pharmaceutical composition according to claim 46, wherein the selected site for delivery is an autoimmune response site.
- 51. A pharmaceutical composition for delivering to a selected site an effective dose of a soluble protein having an Eph receptor ectodomain, said soluble protein being capable of altering a cell signaling pathway comprising:
an effective dose of said soluble protein; a suitable carrier; and optionally additional active or inert ingredients such as diluents, stabilizers, and excipients.
- 52. A pharmaceutical composition according to claim 51, wherein the soluble protein is a fusion protein.
- 53. A pharmaceutical composition according to claim 52, wherein the soluble fusion protein comprises GST and the C-terminal 33 amino acids of ephrin B receptors.
- 54. A pharmaceutical composition according to claim 51, further comprising a substance which allows for the slow release of the pharmaceutical composition at the selected site.
- 55. A pharmaceutical composition according to claim 51, wherein the selected site for delivery is a tumor site.
- 56. A pharmaceutical composition according to claim 51, wherein the selected site for delivery is an allergic response site.
- 57. A pharmaceutical composition according to claim 51, wherein the selected site for delivery is an autoimmune response site.
- 58. A viral vector comprising the nucleic acid sequence encoding the protein of claim 2.
- 59. A plasmid comprising the nucleic acid sequence encoding the protein of claim 2.
- 60. A plasmid vector encoding the protein of claim 52.
- 61. A plasmid vector encoding the protein of claim 53.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application gains priority from U.S. Provisional Application No. 60/280,260, filed Mar. 30, 2001, which is hereby incorporated by reference in its entirety herein.
GOVERNMENT FUNDING
[0002] The invention was made in part with government support under grants HD29417 and NS40043 awarded by the National Institutes of Health. The government has certain rights in the invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60280260 |
Mar 2001 |
US |