Dissecting the Function of FARP1 and Rho GTPases in Semaphorin-Plexin Signaling: Structural Perspective
MetadataShow full item record
The Semaphorin-Plexin signaling is important for regulating axon guidance. Binding of Semaphorin to the Plexin receptor induces the dimerization of Plexin and stimulates its cytoplasmic GAP activity towards Rap, resulting in cytoskeleton re-organization. The growth cone of an axon then turns around, which ultimately leads to the repulsive guidance of the axon. Structural studies by our laboratory and others have revealed how the RapGAP activity of Plexin cytoplasmic region is stimulated by dimerization, how the Semaphorin ligand interacts with Plexin ectodomain, and how extracellular domains prevent Plexin from premature activation. Several downstream effectors interacting with Plexin cytoplasmic domain were found; however, the molecular mechanisms by which these effectors regulate Plexin signaling remain largely unknown. Our laboratory was interested in one of the effectors named FARP. Previous studies in our laboratory determined the crystal structures of the functional units of FARP1 and FARP2 but failed to detect any GEF activity in vitro. A recent screening with yeast two-hybrid identified a RhoGTPase, Rif, as a novel interacting protein for FARP1. To investigate how Rif/FARP1 interaction is involved in Plexin signaling, I determined the crystal structure of Rif-bound FARP1. This complex structure explains the functional roles that FARP1 and Rif might exert in dendritic spine formation and neurite outgrowth. Also, the N-terminal FERM domain of FARPs is known to interact with the cytoplasmic domain of Plexin. To interrogate how FERM interacts with Plexin and how this interaction might regulate Plexin signaling, I characterized the interaction of FERM with Plexin both biophysically and biochemically. The last question that I attempted to address in this dissertation is how a RhoGTPase, RhoD, inhibits activation of Plexin. RhoD was known for its remarkable inhibitory effect on Plexin signaling presumably through binding to the defined RhoGTPase binding domain of Plexin. The structure of Rac1 or Rnd1 in complex with Plexin did not explain how this binding regulates Plexin activity. I solved the crystal structure of the RhoD/Plexin complex. Modeling of this structure with that of Plexin active dimer in the context of the plasma membrane reveals a mechanism by which RhoD inhibits Plexin activation. Structure studies in this dissertation added another layer of comprehension on how the Semaphorin-Plexin signaling is regulated.