Trafficking dynamics of VEGFR1, VEGFR2, and NRP1 in human endothelial cells.

The vascular endothelial growth factor (VEGF) family of cytokines are key drivers of blood vessel growth and remodeling. These ligands act via multiple VEGF receptors (VEGFR) and co-receptors such as Neuropilin (NRP) expressed on endothelial cells. These membrane-associated receptors are not solely expressed on the cell surface, they move between the surface and intracellular locations, where they can function differently. The location of the receptor alters its ability to 'see' (access and bind to) its ligands, which regulates receptor activation; location also alters receptor exposure to subcellularly localized phosphatases, which regulates its deactivation. Thus, receptors in different subcellular locations initiate different signaling, both in terms of quantity and quality. Similarly, the local levels of co-expression of other receptors alters competition for ligands. Subcellular localization is controlled by intracellular trafficking processes, which thus control VEGFR activity; therefore, to understand VEGFR activity, we must understand receptor trafficking. Here, for the first time, we simultaneously quantify the trafficking of VEGFR1, VEGFR2, and NRP1 on the same cells-specifically human umbilical vein endothelial cells (HUVECs). We build a computational model describing the expression, interaction, and trafficking of these receptors, and use it to simulate cell culture experiments. We use new quantitative experimental data to parameterize the model, which then provides mechanistic insight into the trafficking and localization of this receptor network. We show that VEGFR2 and NRP1 trafficking is not the same on HUVECs as on non-human ECs; and we show that VEGFR1 trafficking is not the same as VEGFR2 trafficking, but rather is faster in both internalization and recycling. As a consequence, the VEGF receptors are not evenly distributed between the cell surface and intracellular locations, with a very low percentage of VEGFR1 being on the cell surface, and high levels of NRP1 on the cell surface. Our findings have implications both for the sensing of extracellular ligands and for the composition of signaling complexes at the cell surface versus inside the cell.

Tumor and endothelial cells collaborate via transcellular receptor complexes.

Many cellular signaling pathways are initiated by cell-surface ligand-sensing complexes that incorporate not just one but multiple receptors. Most studies focus on receptors coexpressed on a single cell (cis interactions), but complexes containing receptors on adjacent cells (trans interactions) are also possible. Recent work by Morin et al published in this journal provides critical evidence for such trans interactions between Neuropilin-1 (NRP1) expressed on human tumor cells and vascular endothelial growth factor receptor 2 (VEGFR2) expressed on adjacent endothelial cells, with the ligand VEGFA binding and bridging the two receptors. They show that the formation of these complexes is correlated with reduced tumor proliferation and increased patient survival. They also observe trans NRP1-VEGFA-VEGFR2 repressing angiogenesis and cis NRP1-VEGFA-VEGFR2 increasing angiogenesis in selected cancers. The distinct molecular signature of each tumor and each patient will determine which type of complexes dominate and will influence prognosis and treatment. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

VEGF-A121a binding to Neuropilins - A concept revisited.

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 tissue 1 , 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.

Parallels and Distinctions in FGFR, VEGFR, and EGFR Mechanisms of Transmembrane Signaling.

Receptor tyrosine kinase (RTK) signal transduction is essential in human skeletal, nervous, and vascular development, in homeostasis, and in disease. RTKs are activated by dimerization in the plasma membrane. The mechanisms of receptor dimerization and activation are multifaceted and complex, and unraveling them remains challenging. Most studies of RTKs have been devoted to crystallographic analysis of their isolated extracellular domain and biochemical analysis of the catalytic domain. However, the past few years have seen direct biophysical studies of (intact) RTK dimerization in native membranes lead to significant progress in our fundamental understanding of the mechanisms of their signal transduction across the plasma membrane. This perspective focuses on recent insights into the mechanisms of fibroblast growth factor receptor and vascular endothelial growth factor receptor transmembrane signaling, derived from studies of wild-type and mutant RTKs in a number of environments, including plasma membrane-derived vesicles. These insights reveal distinct steps in and factors of RTK signaling across the plasma membrane that can guide the drug discovery process for RTK targeting therapeutics.

Pathogenic Cysteine Removal Mutations in FGFR Extracellular Domains Stabilize Receptor Dimers and Perturb the TM Dimer Structure.

Missense mutations that introduce or remove cysteine residues in receptor tyrosine kinases are believed to cause pathologies by stabilizing the active receptor tyrosine kinase dimers. However, the magnitude of this stabilizing effect has not been measured for full-length receptors. Here, we characterize the dimer stabilities of three full-length fibroblast growth factor receptor (FGFR) mutants harboring pathogenic cysteine substitutions: the C178S FGFR1 mutant, the C342R FGFR2 mutant, and the C228R FGFR3 mutant. We find that the three mutations stabilize the FGFR dimers. We further see that the mutations alter the configuration of the FGFR transmembrane dimers. Thus, both aberrant dimerization and perturbed dimer structure likely contribute to the pathological phenotypes arising due to these mutations.

VEGFR-2 conformational switch in response to ligand binding.

VEGFR-2 is the primary regulator of angiogenesis, the development of new blood vessels from pre-existing ones. VEGFR-2 has been hypothesized to be monomeric in the absence of bound ligand, and to undergo dimerization and activation only upon ligand binding. Using quantitative FRET and biochemical analysis, we show that VEGFR-2 forms dimers also in the absence of ligand when expressed at physiological levels, and that these dimers are phosphorylated. Ligand binding leads to a change in the TM domain conformation, resulting in increased kinase domain phosphorylation. Inter-receptor contacts within the extracellular and TM domains are critical for the establishment of the unliganded dimer structure, and for the transition to the ligand-bound active conformation. We further show that the pathogenic C482R VEGFR-2 mutant, linked to infantile hemangioma, promotes ligand-independent signaling by mimicking the structure of the ligand-bound wild-type VEGFR-2 dimer.

Effect of the achondroplasia mutation on FGFR3 dimerization and FGFR3 structural response to fgf1 and fgf2: A quantitative FRET study in osmotically derived plasma membrane vesicles.

The G380R mutation in the transmembrane domain of FGFR3 is a germline mutation responsible for most cases of Achondroplasia, a common form of human dwarfism. Here we use quantitative FÓ§ster Resonance Energy Transfer (FRET) and osmotically derived plasma membrane vesicles to study the effect of the achondroplasia mutation on the early stages of FGFR3 signaling in response to the ligands fgf1 and fgf2. Using a methodology that allows us to capture structural changes on the cytoplasmic side of the membrane in response to ligand binding to the extracellular domain of FGFR3, we observe no measurable effects of the G380R mutation on FGFR3 ligand-bound dimer configurations. Instead, the most notable effect of the achondroplasia mutation is increased propensity for FGFR3 dimerization in the absence of ligand. This work reveals new information about the molecular events that underlie the achondroplasia phenotype, and highlights differences in FGFR3 activation due to different single amino-acid pathogenic mutations.

Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic single amino acid mutations that cause skeletal and cranial dysplasias, as well as cancer, we also study the effects of these mutations on dimerization. First, we show that the A391E mutation, linked to Crouzon syndrome with acanthosis nigricans and to bladder cancer, significantly enhances FGFR3 dimerization in the absence of ligand and thus induces aberrant receptor interactions. Second, we present results about the effect of three cysteine mutations that cause thanatophoric dysplasia, a lethal phenotype. Such cysteine mutations have been hypothesized previously to cause constitutive dimerization, but we find instead that they have a surprisingly modest effect on dimerization. Most of the studied pathogenic mutations also altered FGFR3 dimer structure, suggesting that both increases in dimerization propensities and changes in dimer structure contribute to the pathological phenotypes. The results acquired with the QI-FRET method further our understanding of the interactions between FGFR3 molecules and RTK molecules in general. Since RTK dimerization regulates RTK signaling, our findings advance our knowledge of RTK activity in health and disease. The utility of the QI-FRET method is not restricted to RTKs, and we thus hope that in the future the QI-FRET method will be applied to other classes of membrane proteins, such as channels and G protein-coupled receptors.

Analytical characterization of plasma membrane-derived vesicles produced via osmotic and chemical vesiculation.

Plasma membrane-derived vesicles are being used in biophysical and biochemical research as a simple, yet native-like model of the cellular membrane. Here we report on the characterization of vesicles produced via two different vesiculation methods from CHO and A431 cell lines. The first method is a recently developed method which utilizes chloride salts to induce osmotic vesiculation. The second is a well established chemical vesiculation method which uses DTT and formaldehyde. We show that both vesiculation methods produce vesicles which contain the lipid species previously reported in the plasma membrane of these cell lines. The two methods lead to small but statistically significant differences in two lipid species only; phosphatidylcholine (PC) and plasmalogen phosphatidylethanolamine (PEp). However, highly significant differences were observed in the degree of incorporation of a membrane receptor and in the degree of retention of soluble cytosolic proteins within the vesicles.

How IGF-1 activates its receptor.

The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation.

The FRET signatures of noninteracting proteins in membranes: simulations and experiments.

Förster resonance energy transfer (FRET) experiments are often used to study interactions between integral membrane proteins in cellular membranes. However, in addition to the FRET of sequence-specific interactions, these experiments invariably record a contribution due to proximity FRET, which occurs when a donor and an acceptor approach each other by chance within distances of ∼100 Å. This effect does not reflect specific interactions in the membrane and is frequently unappreciated, despite the fact that its magnitude can be significant. Here we develop a computational description of proximity FRET, simulating the cases of proximity FRET when fluorescent proteins are used to tag monomeric, dimeric, trimeric, and tetrameric membrane proteins, as well as membrane proteins existing in monomer-dimer equilibria. We also perform rigorous experimental measurements of this effect, by identifying membrane receptors that do not associate in mammalian membranes. We measure the FRET efficiencies between yellow fluorescent protein and mCherry-tagged versions of these receptors in plasma-membrane-derived vesicles as a function of receptor concentration. Finally, we demonstrate that the experimental measurements are well described by our predictions. The work presented here brings additional rigor to FRET-based studies of membrane protein interactions, and should have broad utility in membrane biophysics research.

Glycophorin A transmembrane domain dimerization in plasma membrane vesicles derived from CHO, HEK 293T, and A431 cells.

Membrane protein interactions, which underlie biological function, take place in the complex cellular membrane environment. Plasma membrane derived vesicles are a model system which allows the interactions between membrane proteins to be studied without the need for their extraction, purification, and reconstitution into lipid bilayers. Plasma membrane vesicles can be produced from different cell lines and by different methods, providing a rich variety of native-like model systems. With these choices, however, questions arise as to how the different types of vesicle preparations affect the interactions between membrane proteins. Here we address this question using the glycophorin A transmembrane domain (GpA) as a model system. We compare the dimerization of GpA in six different vesicle preparations derived from Chinese hamster ovary (CHO), Human Embryonic Kidney 293T (HEK 293T) and A431 cells. We accomplish this with the use of a FRET-based method which yields the FRET efficiency, the donor concentration, and the acceptor concentration in each vesicle. We show that the vesicle preparation protocol has no statistically significant effect on GpA dimerization. Based on these results, we propose that any of the six plasma membrane preparations investigated here can be used as a model system for studies of membrane protein interactions.