Understanding the Role of Transmembrane and Juxtamembrane Domains in Plexin and Neuropilin Signaling

Neuropilins (nrps) and plexins (plxns) are transmembrane (TM) proteins that form co-receptor complexes to guide neuronal, vascular, lymphatic, and bone development as well as cancer metastasis. While it is understood that nrp serves as the extracellular ligand-binding receptor and plxn as the signal-transducing portion of the complex, little is understood about the mechanism of activation of the signal transduction cascade beyond ligand binding. Understanding the mechanisms of plxn and nrp activation may provide insight necessary for rational design of novel cancer therapeutics.Co-receptor clustering is believed to induce activation. Previous studies suggest deletion of the plxn extracellular domain leads to a constitutively active plxn, but lack of membrane-anchorage of the cytosolic domain yields inactivity, implying a role for the plxn TM and juxtamembrane (JM) domains in clustering and subsequent activation. We demonstrate that a heptad repeat in the cytosolic JM domain modulates Danio rerio PlxnA3 homodimerization of the TM + JM domains in a bacterial membrane via the AraTM homodimer assay and of the TM + JM domains with a full extracellular domain intact via a bioluminescence resonance energy transfer (BRET2) assay. A specific mutation (M1281L) that enhances homodimerization in the BRET2 assay in the presence of a Nrp2a co-receptor and semaphorin-3F ligand also fails to rescue motor neuron patterning in PlxnA3-knockout zebrafish embryos, in contrast to the wild-type protein. We also demonstrate via these same techniques that a glycine-rich segment of the PlxnA3 TM domain modulates receptor homodimerization, competing with the dimerization motif of the JM domain. Specifically, mutations to small-x3-small motifs in the PlxnA3 TM domain enhance dimerization of the TM + JM domains in the AraTM assay. Mutations to both the TM and JM dimerization motifs demonstrate, in the context of the TM + JM system, the heptad repeat in the JM dominates TM + JM dimerization. Mutations to the small-x3-small TM dimerization motifs exhibit reduced functionality in the zebrafish embryo axonal guidance assay. Collectively, these results demonstrate that enhanced PlxnA3 dimerization does not correlate with enhanced function. The TM-driven dimerization serves to weaken the JM dimer, likely allowing switchability between co-receptors as well as active and inactive states.The nrp MAM domain is also believed to contribute to the observed clustering phenomenon with the intact, full-length plxn receptor. We show that cysteines in the Danio rerio Nrp2a MAM domain, in particular residue C711, modulate Nrp2 homodimerization, as determined via the BRET2 assay. Mutation of residue C711 also disrupts ligand binding. While zebrafish embryos injected with wild-type nrp2a RNA exhibit ectopic vascular branching, significantly fewer embryos injected with nrp2a RNA with the C711S mutation exhibit this overexpression phenotype.Collectively, this work provides insight into the dimerization mechanisms important for nrp and plxn activity. The structure-function correlations determined may assist in rational design of targeted therapeutics to alter nrp and plxn activity.