Molecules associating to c-terminal domain in receptor cell

Abstract

Concerning intracellular signal transduction mechanism, there has been drawn a novel hypothesis that, even in the case where phosphorylation does not occur in the intracellular C-terminal domain of a receptor, an unknown molecule associates with the Pro-C terminal domain of a G protein-coupled receptor for each chemokine and thus leukocyte chemotaxis depending on the receptor is controlled. To examine this hypothesis and clarify therapeutic targets in inflammatory diseases as well as other various diseases, attempts are made to search for a CCR2-binding protein. As a result, a novel cytoplasmic protein associating directly and specifically with the Pro-12-C-terminal domain of CCR2 is found out and it is clarified that this protein forms clusters with CCR2 after stimulation with CCL2. Thus, it is confirmed that there is a novel signal transduction system in the G protein relating signal transduction in the CCL2-CCR2 pathway. It is also found out that this novel protein associates with the intracellular C-terminal domain of a receptor CCR5 too.

1 . An isolated DNA encoding the amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38.  
     
     
         2 . An isolated DNA represented by any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 or 37.  
     
     
         3 . An isolated DNA of the sequence having at least 90% identity to the DNA as claimed in  claim 1  or  2  and encoding a polypeptide having a function of the FROUNT protein.  
     
     
         4 . A FROUNT protein having an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10. 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38.  
     
     
         5 . A polypeptide having an amino acid sequence having at least 90% identity to the amino acid sequence as claimed in  claim 4  and having the function of the FROUNT protein.  
     
     
         6 . An antisense DNA or an antisense RNA inhibiting the expression of a FROUNT protein having an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38.  
     
     
         7 . An antisense DNA or an antisense RNA directed against the full length or a part of the DNA as claimed in any of  claims 1  to  3 .  
     
     
         8 . An antisense RNA having the full length or a part of the sequence represented by SEQ ID NO:39.  
     
     
         9 . An antisense RNA having at least 90% identity to the sequence of the RNA as claimed in  claim 8  and inhibiting the expression of a FROUNT protein.  
     
     
         10 . DNA for producing the RNA as claimed in  claim 8  or  9  which consists of the DNA sequence represented by SEQ ID NO:40 or the full length or a part of a sequence having at least 90% identity to this sequence.  
     
     
         11 . A ribozyme against an RNA corresponding to a DNA encoding the amino acid sequence represented by SEQ ID NO:2 or the DNA sequence represented by SEQ ID NO:1.  
     
     
         12 . A plasmid containing the DNA as claimed in any of  claims 1  to  3 .  
     
     
         13 . A liposome preparation containing the DNA as claimed in any of  claims 1  to  3 .  
     
     
         14 . A plasmid containing the DNA as claimed in  claim 7  or  10 .  
     
     
         15 . A liposome preparation containing the DNA or RNA as claimed in any of  claims 7  to  10 .  
     
     
         16 . A liposome preparation containing the ribozyme as claimed in  claim 11 .  
     
     
         17 . An isolated antibody binding specifically to the polypeptide as claimed in  claim 4  or  5 .  
     
     
         18 . A composition for treating chronic inflammatory disease or autoimmune diseases or for treating or preventing infectious diseases which contains as the active ingredient the plasmid or the liposome preparation as claimed in any of  claims 14  to  16 .  
     
     
         19 . A composition for treating atherosclerosis, chronic glomerulonephritis or multiple sclerosis, an immunomodulator or an antiallergic agent which contains as the active ingredient the plasmid or the liposome preparation as claimed in any of  claims 14  to  16 .  
     
     
         20 . A pharmaceutical composition which contains as the active ingredient the DNA as claimed in any of  claims 1  to  3 .  
     
     
         21 . An immunoenhancer, a self-defensive reaction promoter or a composition for treating or preventing infectious diseases which contains as the active ingredient the DNA as claimed in any of  claims 1  to  3 .  
     
     
         22 . An immunoenhancer, a self-defensive reaction promoter or a composition for treating or preventing infectious diseases which contains as the active ingredient the plasmid or the liposome preparation as claimed in  claim 12  or  13 .  
     
     
         23 . A method of examining the presence or absence of an abnormality in the CCL2-CCR2 pathway or the CCL3, 4 or 5-CCR5 pathway characterized by comprising comparing the full length or a part of the DNA sequence as claimed in any of  claims 1  to  3  with a DNA sequence collected from a specimen and thus judging whether or not the DNA collected from the specimen has an abnormality.  
     
     
         24 . A probe for examining the presence or absence of an abnormality in the CCL2-CCR2 pathway or the CCL3, 4 or 5-CCR5 pathway which consists of the full length or a part of a sequence complementary to the DNA as claimed in any of  claims 1  to  3 .  
     
     
         25 . (cancelled)  
     
     
         26 . A method of identifying an inhibitor of an agonist to receptor(s) CCR2 and/or CCR5 characterized by comprising forcibly expressing a marker-labeled FROUNT protein in a cell having the receptor(s) CCR2 and/or CCR5 or expressing the same, treating the cell with an agonist to CCR2 and/or CCR5 and a candidate for the agonist inhibitor, observing whether or not the clusterization of the receptor(s) is induced, and thus judging whether or not the candidate has an inhibitory effect on the agonist.  
     
     
         27 . A method of identifying an agonist inhibitor by using a chimeric receptor cell characterized by comprising preparing a cell having a labeled FROUNT protein and a chimeric receptor by forcibly expressing a chimeric receptor, which is obtained by integrating a DNA sequence encoding the full length or a part of a FROUNT protein-association sequence in the intracellular C-terminal domain of receptor(s) CCR2 and/or CCR5 into the intracellular C-terminal domain of the DNA sequence of a desired receptor, in a cell appropriate for the desired receptor and then forcibly expressing a marker-labeled FROUNT protein in the cell, treating the chimeric receptor cell with an agonist to the receptor and a candidate for an agonist inhibitor, then observing whether or not the clusterization of the receptor is induced and thus judging whether or not the candidate has an inhibitory effect on the agonist.  
     
     
         28 . The identification method as claimed in  claim 27  characterized in that the FROUNT protein-association sequence in the intracellular C-terminal domain of receptor(s) CCR2 and/or CCR5 is the amino acid sequence represented by SEQ ID NO:41.  
     
     
         29 . The identification method as claimed in  claim 26  or  27  characterized in that the marker-labeled FROUNT protein is a FROUNT protein fused with a visible color fluorescent protein.  
     
     
         30 . The identification method as claimed in  claim 29  wherein the visible colorfluorescent protein is a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein or a yellow fluorescent protein.  
     
     
         31 . A DNA encoding the FROUNT protein fused with a visible color fluorescent protein as claimed in  claim 29  or  30 .  
     
     
         32 . A plasmid containing the DNA sequence as claimed in  claim 31 .  
     
     
         33 . A chimeric receptor DNA obtained by integrating a DNA sequence encoding the full length or a part of a FROUNT protein-association sequence in the intracellular C-terminal domain of receptor CCR2 and/or CCR5 into the intracellular C-terminal domain of the DNA sequence of a desired receptor.  
     
     
         34 . The chimeric receptor as claimed in  claim 33  characterized in that the FROUNT protein-association sequence in the intracellular C-terminal domain of receptor(s) CCR2 and/or CCR5 is the amino acid sequence represented by SEQ ID NO:41.  
     
     
         35 . (cancelled)  
     
     
         36 . A cell wherein a marker-labeled FROUNT protein is forcibly expressed and receptor(s) CCR2 and/or CCR5 are further expressed therein.  
     
     
         37 . A cell having a labeled FROUNT protein and a chimeric receptor prepared by forcibly expressing a chimeric receptor, which is obtained by integrating a DNA sequence encoding the full length or a part of a FROUNT protein-association sequence in the intracellular C-terminal domain of receptor(s) CCR2 and/or CCR5 into the intracellular C-terminal domain of the DNA sequence of a desired receptor, in a cell appropriate for the desired receptor and then forcibly expressing a marker-labeled FROUNT protein in the cell.  
     
     
         38 . The cell as claimed in  claim 36  or  37  characterized in that the FROUNT protein-association sequence in the intracellular C-terminal domain of receptor(s) CCR2 and/or CCR5 is the amino acid sequence represented by SEQ ID NO:41.  
     
     
         39 . The cell as claimed in  claim 36  or  37  characterized in that the marker-labeled FROUNT protein is a FROUNT protein fused with a visible color fluorescent protein.  
     
     
         40 . The cell as claimed in  claim 39  wherein the visible color fluorescent protein is a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein or a yellow fluorescent protein.  
     
     
         41 . A method of identifying an intracellular signal transduction pathway inhibitor depending on binding of a FROUNT protein to a receptor which comprises using the binding activity of the FROUNT protein to the receptor as an indication and screening a substance inhibiting the binding activity.  
     
     
         42 . The method of identifying an inhibitor as claimed in  claim 26  or  27  characterized in that the identification is made depending on a color change as an indication by using a cell wherein both of the receptor(s) CCR2 and/or CCR5 and FROUNT protein are labeled with visible color markers being different from each other in color.  
     
     
         43 . A cell wherein both of a receptor(s) CCR2 and/or CCR5 and FROUNT protein which are labeled with visible color markers being different from each other in color, are expressed therein.  
     
     
         44 . A method of judging whether or not a specimen contains a cytotoxic substance which comprises treating the cell as claimed in any of  claim 36  to  40  and  43  with the specimen, then treating it with an agonist to the receptor carried by the cell and observing whether or not clusterization or colocalization is induced.