Cysteines in the neuropilin‐2 MAM domain modulate receptor homooligomerization and signal transduction

Barton, Rachael; Driscoll, Alyssa; Flores, Samuel Coulbourn; Mudbhari, Durlav; Collins, Theresa; Iovine, M. Kathryn; Berger, Bryan W.

doi:10.1002/bip.22619

Cited by 4 publications

(2 citation statements)

References 44 publications

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“…27 This shows that well-characterized VEGF-A121a, possessing the correct sequence (including exon 8a), has a three dimensional structure that enables its binding to NRP1 with binding affinity comparable to VEGF-A165a (K d of 220 nM compared to K d of 110 nM). Note that SPR measurements represent the affinities for the interaction of a ligand with monomeric truncated NRP1, comprising only the a1/12-b1/b2 domains and lacking the MAM domain known to induce NRP oligomerization, 28 the transmembrane domain known to homodimerize 29 and the NRP1 GAG modification 30 , which may explain why these SPR-measured affinities are lower than those measured for on-cell VEGF-NRP interactions. 31 …”

Section: Evidence Supporting Direct Vegf-a121a Binding To Neuropilinsmentioning

confidence: 99%

VEGF-A121a binding to Neuropilins – A concept revisited

Sarabipour

Gabhann

2017

Cell Adhesion & Migration

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All known splice isoforms of vascular endothelial growth factor A (VEGF-A) can bind to the receptor tyrosine kinases VEGFR-1 and VEGFR-2. We focus here on VEGF-A121a and VEGF-A165a, two of the most abundant VEGF-A splice isoforms in human tissue1, and their ability to bind the Neuropilin co-receptors NRP1 and NRP2. The Neuropilins are key vascular, immune, and nervous system receptors on endothelial cells, neuronal axons, and regulatory T cells respectively. They serve as co-receptors for the Plexins in Semaphorin binding on neuronal and vascular endothelial cells, and for the VEGFRs in VEGF binding on vascular and lymphatic endothelial cells, and thus regulate the initiation and coordination of cell signaling by Semaphorins and VEGFs.2 There is conflicting evidence in the literature as to whether only heparin-binding VEGF-A isoforms – that is, isoforms with domains encoded by exons 6 and/or 7 plus 8a – bind to Neuropilins on endothelial cells. While it is clear that VEGF-A165a binds to both NRP1 and NRP2, published studies do not all agree on the ability of VEGF-A121a to bind NRPs. Here, we review and attempt to reconcile evidence for and against VEGF-A121a binding to Neuropilins. This evidence suggests that, in vitro, VEGF-A121a can bind to both NRP1 and NRP2 via domains encoded by exons 5 and 8a; in the case of NRP1, VEGF-A121a binds with lower affinity than VEGF-A165a. In in vitro cell culture experiments, both NRP1 and NRP2 can enhance VEGF-A121a-induced phosphorylation of VEGFR2 and downstream signaling including proliferation. However, unlike VEGFA-165a, experiments have shown that VEGF-A121a does not ‘bridge’ VEGFR2 and NRP1, i.e. it does not bind both receptors simultaneously at their extracellular domain. Thus, the mechanism by which Neuropilins potentiate VEGF-A121a-mediated VEGFR2 signaling may be different from that for VEGF-A165a. We suggest such an alternate mechanism: interactions between NRP1 and VEGFR2 transmembrane (TM) and intracellular (IC) domains.

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Section: Evidence Supporting Direct Vegf-a121a Binding To Neuropilinsmentioning

confidence: 99%

VEGF-A121a binding to Neuropilins – A concept revisited

Sarabipour

Gabhann

2017

Cell Adhesion & Migration

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“…In contrast to the meprin MAM domain (Arolas et al., 2012), which contains five cysteine residues in the MAM domain of NRP1, no intermolecular disulfide bond can be formed as the four cysteine side chains present form intramolecular disulfide bonds, thus eliminating cysteine-dependent oligomerization as a mechanism of NRP1 self-association. However, it has recently been postulated that the integrity of the intramolecular disulfide bonds might regulate NRP oligomerization (Barton et al., 2015). The crystal structure presented here indicates that disruption of the disulfide bonds would affect the stability of the MAM domain and consequently the full ectopic domain.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of the Neuropilin-1 MAM Domain: Completing the Neuropilin-1 Ectodomain Picture

Yelland

Djordjević

2016

Structure

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SummaryNeuropilins (NRPs) are single-pass transmembrane receptors involved in several signaling pathways that regulate key physiological processes such as vascular morphogenesis and axon guidance. The MAM domain of NRP, which has previously been implicated in receptor multimerization, was the only portion of the ectopic domain of the NRPs for which the structure, until now, has been elusive. Using site-directed mutagenesis in the linker region preceding the MAM domain we generated a protein construct amenable to crystallization. Here we present the crystal structure of the MAM domain of human NRP1 at 2.24 Å resolution. The protein exhibits a jellyroll topology, with Ca2+ ions bound at the inter-strand space enhancing the thermostability of the domain. We show that the MAM domain of NRP1 is monomeric in solution and insufficient to drive receptor dimerization, which leads us to propose a different role for this domain in the context of NRP membrane assembly and signaling.

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