A Dual Binding Mode for RhoGTPases in Plexin Signalling

Abstract

Plexins are cell surface receptors for the semaphorin family of cell guidance cues. The cytoplasmic region comprises a Ras GTPase-activating protein (GAP) domain and a RhoGTPase binding domain. Concomitant binding of extracellular semaphorin and intracellular RhoGTPase triggers GAP activity and signal transduction. The mechanism of this intricate regulation remains elusive. We present two crystal structures of the human Plexin-B1 cytoplasmic region in complex with a constitutively active RhoGTPase, Rac1. The structure of truncated Plexin-B1-Rac1 complex provides no mechanism for coupling RhoGTPase and Ras binding sites. On inclusion of the juxtamembrane helix, a trimeric structure of Plexin-B1-Rac1 complexes is stabilised by a second, novel, RhoGTPase binding site adjacent to the Ras site. Site-directed mutagenesis combined with cellular and biophysical assays demonstrate that this new binding site is essential for signalling. Our findings are consistent with a model in which extracellular and intracellular plexin clustering events combine into a single signalling output. Axon guidance is fundamental to the development of the central nervous system. The growing axon is guided to its correct location by a plethora of extracellular signals. One of the most important extracellular signals is semaphorin, which binds to plexin receptors on the axon. Usually, this kind of extracellular ligand binding is sufficient to transmit the extracellular signal to the intracellular space to trigger changes in the cell, like axon growth. However, activation of plexin receptors requires a “dual” ligand binding: semaphorin on the extracellular side, and a RhoGTPase on the intracellular side. Signal transduction can only occur if both ligands are present. How this intricate regulation mechanism is organized and how concomitant ligand binding can be integrated into a single signalling output within the cell has remained largely unclear. Here, we present crystal structures of one plexin receptor, Plexin-B1, in complex with an intracellular RhoGTPase ligand (Rac1) and show that binding of Rac1 brings together three Plexin-B1 molecules. In this trimeric arrangement each plexin molecule interacts with two Rac1 ligand molecules. This leads to a previously unidentified plexin-Rac1 ligand interface that is crucial for its function. Further biophysical and cellular analysis in combination with previous findings on the extracellular plexin-semaphorin complex allow us to propose a model for how ligand-induced clustering events on the extra- as well as intracellular side are combined to trigger signal transduction.