Heterodimerization of Neuropilin-1 Transmembrane Helices and Plexin Signaling

Abstract

Heterodimerization of transmembrane helices of cell surface receptors is emerging as an important theme in cellular signaling. The most common motif for dimerization of alpha-helical transmembrane proteins is the GXXG motif. Neuropilin-1 (Nrp1) and Plexin D1 (Pld1) are both single-pass transmembrane receptors expressed in all vertebrate systems. The ligands for Nrp1 and Pld1 are a class of guidance molecules called Semaphorin 3 (Sema3). Signaling by all Sema3 ligands (except Sema3E) through Pld1 requires Nrp1. The Sema3/Nrp1/Pld1 signaling axis plays a role in angiogenesis, immune responses, cancer metastasis as well as invasiveness. Despite the established roles in normal physiology and pathology, the molecular details of signaling pathways involved are not well documented. There are no structural/mechanistic level details; it is still unknown which domain/domains of Nrp1 and Pld1 interact. Here we report heterodimerization of the transmembrane (TM) helices of Nrp1 and Pld1. Nrp1 has two GXXG motifs and it is established that the dimerization of the Nrp1 TM helices is important for signaling. Using NMR and all-atom molecular dynamics simulations we show that Nrp1 and Pld1 forms a parallel transmembrane helix dimer. Further, we compare this heterodimer with Nrp1 homodimer and show that the homodimer utilizes the C-terminal GXXG motif of Nrp1 for dimerization. Whereas, the Nrp1/Pld1 heterodimer uses the N-terminal GXXG motif. The ability to use alternative surfaces for dimerization likely has implications for the higher-order assembly of Nrp1/Pld1 in the membrane. Further, using functional assays we show that the heterodimerization of Nrp1 TM helices is essential for Sema3/Nrp1/Pld1 signaling. This to our knowledge is the first report of a functional role for transmembrane helix dimerization in Plexin signaling.