Molecule associating with intracellular C-terminal domain of receptor
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. A method of identifying an inhibitor of an agonist to receptor(s) CCR2 and/or CCR5 comprising:
forcibly expressing a marker-labeled FROUNT protein having an amino acid sequence represented by SEQ ID NO:2 selected 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
judging whether or not the candidate has an inhibitory effect on the agonist.
2. The identification method as claimed in claim 1 wherein the marker-labeled FROUNT protein is a FROUNT protein fused with a visible color fluorescent protein.
3. The identification method as claimed in claim 2 wherein the visible color fluorescent protein is a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein or a yellow fluorescent protein.
4. The method of identifying an inhibitor as claimed in claim 1 wherein 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.