Abstract
The Rnd family of proteins, Rnd1, Rnd2 and Rnd3, are atypical Rho family GTPases, which bind to but do not hydrolyse GTP. They interact with plexins, which are receptors for semaphorins, and are hypothesised to regulate plexin signalling. We recently showed that each Rnd protein has a distinct profile of interaction with three plexins, Plexin-B1, Plexin-B2 and Plexin-B3, in mammalian cells, although it is unclear which region(s) of these plexins contribute to this specificity. Here we characterise the binary interactions of the Rnd proteins with the Rho-binding domain (RBD) of Plexin-B1 and Plexin-B2 using biophysical approaches. Isothermal titration calorimetry (ITC) experiments for each of the Rnd proteins with Plexin-B1-RBD and Plexin-B2-RBD showed similar association constants for all six interactions, although Rnd1 displayed a small preference for Plexin-B1-RBD and Rnd3 for Plexin-B2-RBD. Furthermore, mutagenic analysis of Rnd3 suggested similarities in its interaction with both Plexin-B1-RBD and Plexin-B2-RBD. These results suggest that Rnd proteins do not have a clear-cut specificity for different Plexin-B-RBDs, possibly implying the contribution of additional regions of Plexin-B proteins in conferring functional substrate selection.
Highlights
Plexins are a family of transmembrane cell surface receptors [1] which are divided into four families based on structural criteria, Plexins A, B, C and D [2,3]
The association of Rnd2 with Plexin-B1-Rho-binding domain (RBD) could not be fitted to a single binding isotherm under these experimental conditions mainly because the Isothermal titration calorimetry (ITC) data were affected by significant heat contributions from non-specific binding events (S1 Fig) [31,32]
Our ITC measurements using 150 mM NaCl demonstrated a direct interaction between Plexin-B1 and all three Rnd proteins, revealing a monophasic 1:1 binding isotherm and a binding affinity in the low-micromolar range with dissociation constants (Kd) of 2.4, 7.6 and 6.6 μM for Rnd1, Rnd2 and Rnd3 respectively
Summary
Plexins are a family of transmembrane cell surface receptors [1] which are divided into four families based on structural criteria, Plexins A, B, C and D [2,3]. The cytoplasmic region of plexins is highly conserved, consisting mainly of a bipartite segment with high homology to the GTPase-activating domain (GAP domain) found in proteins that act as GAPs for Ras family proteins, such as p120RasGAP [4]. The two regions in Plexins that together form the RasGAP domain are separated by approximately 200 residues, and a ~120 residue region within this forms an independent folding unit that binds to several Rho family GTPases, termed the Rho-binding domain (RBD) [5,6]. Of the Plexin-B family, Plexin-B1 is the best studied, primarily transducing signals from the semaphorin Sema4D and with several roles in development and disease [7,8,9].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
