Interactions Between the Transmembrane Domains of Plexin, Semaphorin, and Neuropilin

Plexins and semaphorins are a large family of secreted and membrane bound signaling proteins, all with a conserved Sema domain. Broad expression across tissue types is thought to aid in organization of tissues, and neuoral and vasculature guidance. The plexins are the receptor moiety and act through intracellular domains that contain a region with homology to GTPase activating proteins. Canonical signaling occurs through trans interactions via the Sema domains of semaphorin ligand and plexin receptor or in some cases by the use of neuropilin co-receptors. Recent developments show cis interactions may exist between certain family members, where the ligand semaphorin expressed in the same cell as the receptor causes changes in trans signaling. Some of these associations have been attributed to the transmembrane (TM) domains. Due to the sequence characteristics of the TM domains of these families, our study aims to experimentally test the homo- and hetero-cis interactions of these TM domains in cell membranes. Here we use pulsed interleaved fluorescence cross correlation spectroscopy to resolve the contribution of the TM domain to plexin-semaphorin-neuropilin oligomerization in live cells. We designed constructs consisting of the TM domain and short regions of the extracellular and intracellular domains attached to a fluorescent protein. Using these we found a diverse set of correlations between TM domains of the protein family, with most interactions in agreement with the computational models. We also began to determine how the TM correlates and oligomerizes in context with the full length proteins. These results suggest that the TM domain can used as a potential therapeutic target and could aid in the creation of TM-specific inhibitor peptides for plexin-semaphorin-neuropilin signaling related diseases.