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. 2021 Aug;297(2):100965.
doi: 10.1016/j.jbc.2021.100965. Epub 2021 Jul 13.

Interactions between semaphorins and plexin-neuropilin receptor complexes in the membranes of live cells

Shaun M Christie  1 , Jing Hao  2 , Erin Tracy  1 , Matthias Buck  3 , Jennifer S Yu  4 , Adam W Smith  5
Affiliations

Interactions between semaphorins and plexin-neuropilin receptor complexes in the membranes of live cells

Shaun M Christie et al. J Biol Chem. 2021 Aug.

Abstract

Signaling of semaphorin ligands via their plexin-neuropilin receptors is involved in tissue patterning in the developing embryo. These proteins play roles in cell migration and adhesion but are also important in disease etiology, including in cancer angiogenesis and metastasis. While some structures of the soluble domains of these receptors have been determined, the conformations of the full-length receptor complexes are just beginning to be elucidated, especially within the context of the plasma membrane. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy allows direct insight into the formation of protein-protein interactions in the membranes of live cells. Here, we investigated the homodimerization of neuropilin-1 (Nrp1), plexin A2, plexin A4, and plexin D1 using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy. Consistent with previous studies, we found that Nrp1, plexin A2, and plexin A4 are present as dimers in the absence of exogenous ligand. Plexin D1, on the other hand, was monomeric under similar conditions, which had not been previously reported. We also found that plexin A2 and A4 assemble into a heteromeric complex. Stimulation with semaphorin 3A or semaphorin 3C neither disrupts nor enhances the dimerization of the receptors when expressed alone, suggesting that activation involves a conformational change rather than a shift in the monomer-dimer equilibrium. However, upon stimulation with semaphorin 3C, plexin D1 and Nrp1 form a heteromeric complex. This analysis of interactions provides a complementary approach to the existing structural and biochemical data that will aid in the development of new therapeutic strategies to target these receptors in cancer.

Keywords: cancer; cell signaling; fluorescence correlation spectroscopy; membrane biophysics; membrane protein; protein–protein interaction.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Schematic of hypothesized plexin–neuropilin–class 3 semaphorin signaling. Using previously available structural and biochemical data, plexins (light blue) and neuropilins (purple) likely form inhibitory homodimers. Addition of a soluble and dimeric class 3 semaphorin induces a tripartite complex formation where neuropilins act as a bridge between plexins and class 3 semaphorins. Conformational changes to the intracellular region of plexins then allow for interactions with GTPases, such as Rac1, R-Ras, and Rap1/2, which control downstream cytoskeletal dynamics.
Figure 2
Figure 2
Homotypic interaction of Nrp1, plexin A2, plexin A4, and plexin D1.A, fraction correlated for Nrp1, plexin A2, and plexin A4 fall in the range of homodimers, whereas plexin D1 diffuses as a monomer. Gray numbers above each column represent the number of single cells analyzed. B, the average diffusion coefficients agree with the cross-correlation results, where plexin D1 (monomer) diffuses at a faster rate than the dimers, but the slow diffusion for Nrp1 suggests that multimers may form as well, possibly involving interactions with other endogenous proteins. Nrp1, neuropilin-1.
Figure 3
Figure 3
Heterotypic interaction of Nrp1 with plexin receptors.A, the fraction correlated indicates no interaction between any receptor combinations under nonstimulatory conditions. Gray numbers above each column represent the number of single cells analyzed. Data marked with red + are regarded as outliers and are not included in the analysis. B, comparison of effective diffusion coefficient of Nrp1 when expressed alone (light gray) or coexpressed (dark gray). Comparison with Nrp1-mCh diffusion in the homodimer experiments shows that Nrp1-mCh diffusion is significantly increased when coexpressed with plexin D1 (p < 0.0001) but not with plexin A2 or plexin A4. mCh, mCherry; Nrp1, neuropilin-1.
Figure 4
Figure 4
Heterotypic interactions of plexins.A, fraction correlated for each combination indicates that plexin A2 and plexin A4 form a heterodimer, and neither interacts with plexin D1. Gray numbers above each column represent the number of single cells analyzed. B, diffusion coefficient change for plexin A2 and plexin A4 when expressed alone (light gray) or together (dark gray) likely indicating that neither forms a complex larger than a homodimer or a heterodimer.
Figure 5
Figure 5
Homotypic interaction of Nrp1, plexin A2, plexin A4, and plexin D1 following stimulation with semaphorin 3C and semaphorin 3A. Cells expressing homotypic receptor combinations were incubated with 500 ng/ml of semaphorin 3C or semaphorin 3A 10 min prior to data acquisition. Fraction correlated is unchanged from nonstimulatory conditions. Gray numbers above each column represent the number of single cells analyzed. Nrp1, neuropilin-1.
Figure 6
Figure 6
Heterotypic interaction of Nrp1, plexin A2, plexin A4, and plexin D1 following stimulation with semaphorin 3C.A, fraction correlated for Nrp1 coexpressed with each plexin receptor. Plexin D1 and Nrp1 exhibit extensive dimerization. Gray numbers above each column represent the number of single cells analyzed. B, diffusion change for plexin D1-eGFP when coexpressed with Nrp1. Stimulation with semaphorin 3C significantly decreased (p < 0.01) the average diffusion coefficient indicating increased molecular weight and oligomer state. C, diffusion change for Nrp1-mCh alone or when coexpressed with plexin D1. Again, the average diffusion coefficient is significantly increased from expression alone but not significantly decreased from the unstimulated coexpression. eGFP, enhanced GFP; Nrp1, neuropilin-1.
Figure 7
Figure 7
Heterotypic interaction following semaphorin 3A stimulation.A, fraction correlated after stimulation. No apparent increase is observed like that of plexin D1–Nrp1 following semaphorin 3C stimulation. Gray numbers above each column represent the number of single cells analyzed. B, changes in average fc value following stimulation. Plexin A2–Nrp1 and plexin A4–Nrp1 have significant increases in correlation, whereas the plexin D1–Nrp1 interaction is unchanged. C, diffusion change for plexin A4-eGFP and plexin A2-eGFP when coexpressed with Nrp1 and stimulated with semaphorin 3A. Both plexin A4 and plexin A2 have significantly decreased average diffusion coefficients adding to evidence that a weak/transient interaction is formed. fc, fraction of cross-correlation; Nrp1, neuropilin-1.
Figure 8
Figure 8
Possible stoichiometry of plexin D1–Nrp1–semaphorin 3C complex. Plexin D1 diffuses as a monomer, whereas initially Nrp1 diffuses as a dimer or a multimer. Upon coexpression, Nrp1 likely shifts toward dimers. Because of plexin D1 diffusing as a monomer, it is possible to form a 1:2:2 complex (right) using a plexin D1 monomer to induce signaling rather than a dimer seen for class A plexins. Nrp1, neuropilin-1.
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