TI - Sema4D induces Met activation in immortalized GnRH-1 cells . AB - The manipulation of the GnRH-1 migratory system and functional studies on these neurons have been challenging because of their limited number ( 800 in mice and 1,000-2,000 in primates ) and widely dispersed distribution in the olfactory system . The generation of immortalized GnRH-1 neurons has permitted the study of more immature , migratory neurons . In particular , GN11 cells display a remarkable motility in vitro , and they have been largely used to investigate the molecular mechanisms controlling the directional migration of GnRH-1 neurons . We previously demonstrated that migratory GnRH-1 cells ( both primary GnRH-1 neurons and GN11 cells ) express Met and are functionally regulated by its ligand HGF . To exploit these cells for functional studies in response to Sema4D , we first verified that GN11 cells retained coexpression of Met and PlexinB1 receptors , as seen for their counterparts in vivo . Double-labeling experiments followed by confocal microscopy analysis showed that Met and PlexinB1 receptors are codistributed throughout the cell surface of GN11 cells ( Fig 5 a ) and often colocalize in dot-like clusters , as shown by single confocal plane images ( Fig 5 a , merge ) . Western blot analysis confirmed Met and PlexinB1 coexpression in GN11 cells ( Fig 5 b ) , and coimmunoprecipitation studies demonstrated PlexinB1-Met association in a molecular complex that increased upon Sema4D stimulation ( Fig 5 c ) . Moreover , when GN11 cells were treated for 30 min with either HGF ( 05 nM ) or soluble Sema4D ( 05 , 125 , 5 , and 25 nM ) , the Met receptor became tyrosine PHOSphorylated , which indicates activation of its signal transduction ( Fig 5 d ) . Interestingly , as known for other receptors , we observed a bell-shaped curve of tyrosine kinase activation in response to increasing concentrations of Sema4D ( Fig 5 d ) .