| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2004: ¥3,000,000 (Direct Cost: ¥3,000,000)
|
|---|
| Research Abstract |
1)Identification of signaling molecules in the Sema6D-Plexin-A1 pathway
To identify the molecules involving in the Sema6D-Plexin-A1 pathway, yeast two-hybrid system will be performed. The binding molecules are screened in the brain cDNA libraries by using the cytoplasmic region of Plexin-A1 as the bait. From 10X10^6 clones, FERM domain containing guanosine-exchange factor for Rac, named FARP2, is identified to bind to the Plexin-A1.
2)Functional analysis of FARP2
Immunoprecipitaion-immunoblot assay shows that FARP2 binds to Plexin-A1 through its FERM domain. This interaction is dependent on the presence of co-receptor Neuropilin-1. When Sema3A, one of the ligand for Plexin-A1-Neuropilin-1 receptor complex, binds to receptor complex, FARP2 is dissociates from Plexin-A1, and the released form activates its Rac GEF activity. The activation of Rac induces the sequential steps of signaling pathway to change the cytoskeletal architecture.
3)In vivo analysis of Sema6D function
(a)generation of Sema6D knockout mice and Plexin-A1 knockout mice
Construction of target vectors : Genomic DNA surrounding Sema6D or Plexin-A1 gene are isolated by the PCR and these fragments are inserted into the pNT1.1 vector. The vectors are electroporated into the mouse ES cells, and the ES cells containing the recombinant vector are selected.
|
