| Budget Amount *help |
¥13,700,000 (Direct Cost: ¥13,700,000)
Fiscal Year 2001: ¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 2000: ¥6,900,000 (Direct Cost: ¥6,900,000)
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| Research Abstract |
The Rho/Rho-kinase signaling pathway plays an essential role in neurite retraction and cell rounding in response to G12/13-coupled receptor activation in neuronal cells. The Rho guanine nucleotide exchange factor (RhoGEF) involved in these processes has not been identified. To monitor the activation state of Rho-kinase, we developed a vimentin head/Rho-kinase chimera which is intramolecularly phosphorylated in a Rho-dependent manner at Ser71 of the fused vimentin head. Using this system, we identified a clone termed KIAA0380, which contains the Ga12/13-binding domain as well as a tandem of the DH/PH domain, as an activator of Rho/Rho-kinase signaling. Since KIAA0380 is composed of various functional domains that are commonly found in signaling molecules, we prepared several mutants and analyzed the physiological significance of the functional domains. Interestingly, in addition to DH/PH domain, a proline-rich motif which is C-terminally adjacent to the DH/PH domain is also essential for plasma membrane location of KIAA0380, cortical actin reorganization and cell rounding. In Neuro2a cells, the N-terminus fragment of KIAA0380 inhibited LPA-mediated neurite retraction. Immunological analysis revealed that KIAA0380 is well expressed in Neuro2a cells, but another Ga12/13 binding p115 RhoGEF, was not detected. Taken together, these findings suggest that in neuronal cells, KIAA0380 functions as a RhoGEF at the cell periphery and regulates G12/13-coupled receptor-mediated Rho activation, an event essential for neurite retraction and growth cone collapse.
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