Transmembrane domain interactions control biological functions of neuropilin-1

Abstract

Neuropilin-1 (NRP1) is a transmembrane receptor playing a pivotal role in the control of semaphorins and VEGF signaling pathways. The exact mechanism controlling semaphorin receptor complex formation is unknown. A structural analysis and modeling of NRP1 revealed a putative dimerization GxxxG motif potentially important for NRP1 dimerization and oligomerization. Our data show that this motif mediates the dimerization of the transmembrane domain of NRP1 as demonstrated by a dimerization assay (ToxLuc assay) performed in natural membrane and FRET analysis. A synthetic peptide derived from the transmembrane segment of NRP1 abolished the inhibitory effect of Sema3A. This effect depends on the capacity of the peptide to interfere with NRP1 dimerization and the formation of oligomeric complexes. Mutation of the GxxxG dimerization motif in the transmembrane domain of NRP1 confirmed its biological importance for Sema3A signaling. Overall, our results shed light on an essential step required for semaphorin signaling and provide novel evidence for the crucial role of transmembrane domain of bitopic protein containing GxxxG motif in the formation of receptor complexes that are a prerequisite for cell signaling.

Figure 1.

Predicted structural features of the NRP1 TM domain. (A) Helical-wheel representation of the dimer interface in the NRP1 TM domain sequence from Ile⁸⁵⁹ to Y⁸⁸⁰. The three glycines residues in the putative interface are indicated in bold characters and labeled with their residue number besides their one-letter amino acid codes. (B–D) A possible structure of the NRP1 TM domain homo-dimer. This TM domain dimer model was made by aligning the double GxxxG motif of the NRP1 TM sequence with the GxxxGxxxT dimerization motif of glycophorin A (PDB code 1AFO; MacKenzie and Engelman, 1998), manually adjusting helices distance and angles, and by minimizing the energy of the dimeric structure to remove clashes using the Swiss-PDB viewer program (Guex and Peitsch, 1997). (B) A view down the dimer axis (C-terminus on top) shows the close packing of the glycine residues. (C) A view of the monomer shows the alignment of the three glycine residues (in red) on the same face of the helix. (D) A view along the dimer interface highlights the three glycines (in red) and the adjacent residues. These representations were made with the PyMol program (DeLano, 2002; http://www.pymol.org).

Figure 2.

Transmembrane domain dimerization capacity. (A) Luciferase luminescence induced by several constructs. DH5α, bacteria without construct; pcc, empty plasmid; GPA, glycophorin A; NRP1, neuropilin 1. Student's t test analysis (*p < 0.001, ns, nonsignificant). (B) Homo- and heterodimer formation in detergent micelles. Initial samples were prepared with an equimolar mixture of donor (pyrene) and acceptor (coumarin)-labeled peptides at a total concentration of 2 μM. Total peptide concentration was varied by a serial dilution in detergent buffer. Excitation spectra were collected, and pyrene (sensitized emission) and coumarin (direct emission) contribution to emission at 500 nm were measured and converted into the FRET ratio, which is proportional to the dimer fraction (see Materials and Methods for details). The dimer fraction was plotted as a function of total peptide concentration in semilogarithmic form. Homodimerization between pTM-NRP1 (●), pTM-NRP1^mut (○), pTM-GpA (△), and heterodimerization between pTM-NRP1 and pTM-NRP1^mut (peptides, ■), pTM-NRP1 and pTM-GPA (▲) as well as pTM-NRP1^mut and pTM-GpA (◇) are depicted. Results represent mean ± SEM of 2–4 independent experiments. The only significant dimerization occurred between pTM-NRP1 peptides and between the positive control pTM-GPA peptides.

Figure 3.

Functional study on embryonic cortical neurons. Explants of E15 neocortex were grown for 48 h to obtain a sufficient axonal growth to perform a collapse assay. Sema3A or combination of Sema3A and peptides were added to the culture medium for 2 h. For each explant, total number of growth cones and collapsed growth cones were counted. The percentage of growth cone collapse was calculated for each condition tested and statistical analyses were made by using χ² test analysis (*p < 0.001). (A) Effects of increasing concentrations of pTM-NRP1. (B) Effects of the mutated peptide at 10⁻⁸ M. (C) pTM-NRP1 blocking properties of Sema3A inhibitory effects were also tested at 10⁻⁸ M in DMSO as an alternative detergent vehicle. (D) Effects of pTM-ErbB2 (10⁻⁸ M), a NRP1-unrelated peptide containing a GxxxG motif. A minimum of 500 growth cones were analyzed in each conditions in at least three independent experiments.

Figure 4.

Functional study on COS cells. COS cells coexpressing NRP1 or NRP1^mut and PlexA1 were incubated with control medium or Sema3A containing medium for 4 h. pTM-NRP1 and pTM-NRP1^mut were added at a concentration of 1 nM 1 h before treatments with Sema3A. In a collapse assay, percentage of cell collapse was determined for NRP1/PlexA1-expressing COS cells and for NRP1^mut/PlexA1-expressing cells. For each condition, 400 cells were counted and categorized as normal (spread cells) or collapsed cells (round cells). (χ² test analysis; *p < 0.001, ns, nonsignificant).

Figure 5.

Binding experiments on COS cells. (A) NRP1 expressing COS cells (COS-NRP1) were incubated with pTM-NRP1 or pTM-NRP1^mut and then for 2 h at 4°C with conditioned medium containing AP-Sema3A. Bound AP-Sema3A was detected by addition of an AP luminescent substrate (Lumi-phos, Lumigen). (B) Binding experiments were performed on NRP1-expressing COS cells (COS-NRP1) and mutated NRP1-expressing COS cells (COS-NRP1^mut). (C) Similarly, the expression of the mutated NRP1 caused a significant reduction of VEGF binding. (Student's t test analysis; *p < 0.05).

Figure 6.

Analysis of sedimentation constants by centrifugation in sucrose gradients. Confluent COS-1 cells expressing NRP1 or mutated NRP1 and PlexA1 were incubated with TM peptide for 1 h at 37°C. The culture medium was replaced by conditioned medium containing AP-coupled Sema3A. Cellular lysates were submitted to sucrose gradient centrifugation. Fractions were collected from the bottom and subjected to SDS-PAGE. For sedimentation constant(s) evaluation, the gradient was calibrated with thyroglobulin (19.3S), ferritin (17.7S), catalase (11S), lactate dehydrogenase (7S), and albumin (4.3S).