Plexins are the cell surface receptors of semaphorins. Plexin-mediated semaphorin signaling is essential for processes such as the development of the nervous system and the cardiovascular system and regulation of immune responses and bone homeostasis. Malfunction of plexins has been associated with neurological disorder and cancer. Understanding how plexins function will pave the way for developing targeted therapeutics for fighting the associated diseases and improving neuronal regeneration after injury. The plexin intracellular region contains a GTPase Activating Protein (GAP) domain that is essential for function. In the previous period, we have identified the small GTPase Rap as the authentic substrate for the plexin GAP domain, and have determined the structural basis for how the GAP domain is activated by semaphorin-induced dimerization and how it inactivates Rap through a non-canonical catalytic mechanism. Objectives. To study additional layers of regulation mechanisms of plexins, and mutual regulation between plexins and several of their key binding partners. Research Design. Based on a new crystal structure of ours, we will first analyze the role of the inhibitory dimer in plexin regulation, a long-standing question in the fiel. Plexin signaling requires not only its RapGAP activity, but also its ability to assemble and contro the activity of a multi-protein signaling complex at the plasma membrane. Many proteins interact with plexins, but the structural basis of their actions is largely unknown. We will focus on some of the essential binding partners, address the questions how they bind plexin and exert mutual regulation with plexins. In Aim 1 we will test an inhibitory dimer model in plexin regulation. We have determined two crystal structures of PlexinA4, which adopts a new conformation and forms a compact dimer with the GAP active site buried in the dimer interface. We propose that this dimer and the apo dimer structure of the plexin extracellular region reported previously together mediate the autoinhibited dimeric state of full-length plexin on the cell surface. Structure-based mutational analyses will be performed to test this hypothesis. In Aim 2 we will study the basis for the opposite effects of RND1/Rac and RhoD on plexin signaling. The RhoGTPases Rac1 and RND1 interact with plexin and facilitate its binding and activation by semaphorin. In contrast, RhoD inhibits plexin signaling, although it binds plexin in the same mode with similar affinity. Our structure analyses led to a hypothesis that explains this paradox, which will be tested in this aim. In Aim 3 we will analyze the interaction and regulation of FARPs by plexin. FARP1 and FARP2 are two related guanine nucleotide exchange factors (GEFs) that have been shown to interact directly with plexin and make essential contributing to its signaling. We will pursue a structure of the plexin/FARP complex to elucidate the basis for their interaction, and analyze how this interaction helps release the autoinhibition of FARPs.