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Writing recommendation letters on behalf of students and other early-career researchers is an important mentoring task within academia. An effective recommendation letter describes key candidate qualities such as academic achievements, extracurricular activities, outstanding personality traits, participation in and dedication to a particular discipline, and the mentor’s confidence in the candidate’s abilities. In this Words of Advice, we provide guidance to researchers on composing constructive and supportive recommendation letters, including tips for structuring and providing specific and effective examples, while maintaining a balance in language and avoiding potential biases.

The SARS‐CoV‐2 pandemic has significantly impacted global health. Research on viral mechanisms, highly effective vaccines, and other therapies is in progress. Neuropilins have recently been identified as host cell receptors enabling viral fusion. Here, we provide context to neuropilin's tissue‐specific role in infection and the potential impact of NRP‐based therapeutics. We conclude that the central roles of neuropilins in vascular, neural, and other pathways may render it a less suitable target for treating SARS‐CoV‐2 than agents that target its binding partner, the viral spike protein., The SARS‐CoV‐2 pandemic has significantly impacted global health. Research on viral mechanisms, highly effective vaccines, and other therapies is in progress. Neuropilins have recently been identified as host cell receptors enabling viral fusion. We provide context to the tissue‐specific role of neuropilins in infection and the potential impact of NRP‐based therapeutics. We conclude that the central roles of neuropilins in vascular, neural, and other pathways may render it a less suitable target for treating SARS‐CoV‐2 than agents that target its binding partner, the viral spike protein.

Open and reproducible research practices increase the reusability and impact of scientific research. The reproducibility of research results is influenced by many factors, most of which can be addressed by improved education and training. Here we describe how workshops developed by the Reproducibility for Everyone (R4E) initiative can be customized to provide researchers at all career stages and across most disciplines with education and training in reproducible research practices. The R4E initiative, which is led by volunteers, has reached more than 3000 researchers worldwide to date, and all workshop materials, including accompanying resources, are available under a CC-BY 4.0 license at https://www.repro4everyone.org/.

Mentorship is experience and/or knowledge-based guidance. Mentors support, sponsor and advocate for mentees. Having one or more mentors when you seek advice can significantly influence and improve your research endeavours, well-being and career development. Positive mentee-mentor relationships are vital for maintaining work-life balance and success in careers. Early-career researchers (ECRs), in particular, can benefit from mentorship to navigate challenges in academic and nonacademic life and careers. Yet, strategies for selecting mentors and maintaining interactions with them are often underdiscussed within research environments. In this Words of Advice, we provide recommendations for ECRs to seek and manage mentorship interactions. Our article draws from our experiences as ECRs and published work, to provide suggestions for mentees to proactively promote beneficial mentorship interactions. The recommended practices highlight the importance of identifying mentorship needs, planning and selecting multiple and diverse mentors, setting goals, and maintaining constructive, and mutually beneficial working relationships with mentors.

Scientists routinely use images to display data. Readers often examine figures first; therefore, it is important that figures are accessible to a broad audience. Many resources discuss fraudulent image manipulation and technical specifications for image acquisition; however, data on the legibility and interpretability of images are scarce. We systematically examined these factors in non-blot images published in the top 15 journals in 3 fields; plant sciences, cell biology, and physiology (n = 580 papers). Common problems included missing scale bars, misplaced or poorly marked insets, images or labels that were not accessible to colorblind readers, and insufficient explanations of colors, labels, annotations, or the species and tissue or object depicted in the image. Papers that met all good practice criteria examined for all image-based figures were uncommon (physiology 16%, cell biology 12%, plant sciences 2%). We present detailed descriptions and visual examples to help scientists avoid common pitfalls when publishing images. Our recommendations address image magnification, scale information, insets, annotation, and color and may encourage discussion about quality standards for bioimage publishing.

Scientific conferences have an important role in the exchange of ideas and knowledge within the scientific community. Conferences also provide early-career researchers with opportunities to make themselves known within their field of research. Although the COVID-19 pandemic has brought traditional in-person conferences to a halt for the foreseeable future, the growth of virtual conferences has highlighted many of the disadvantages associated with the in-person format and demonstrated the advantages of moving these events online. Here, based on data from in-person and virtual conferences in a range of subjects, we describe how virtual conferences are more inclusive, more affordable, less time-consuming and more accessible worldwide, especially for early-career researchers. Making conferences more open and inclusive will provide both immediate and long-term benefits to the scientific community.

The need to protect public health during the current COVID-19 pandemic has necessitated conference cancellations on an unprecedented scale. As the scientific community adapts to new working conditions, it is important to recognize that some of our actions may disproportionately affect early-career researchers and scientists from countries with limited research funding. We encourage all conference organizers, funders and institutions who are able to do so to consider how they can mitigate the unintended consequences of conference and travel cancellations and we provide seven recommendations for how this could be achieved. The proposed solutions may also offer long-term benefits for those who normally cannot attend conferences, and thus lead to a more equitable future for generations of researchers.

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.

Epithelial cadherin (Ecadherin) is responsible for the intercellular cohesion of epithelial tissues. It forms lateral clusters within adherens cell-cell junctions, but its association state outside these clusters is unknown. Here, we use a quantitative Forster resonance energy transfer (FRET) approach to show that Ecadherin forms constitutive dimers and that these dimers exist independently of the actin cytoskeleton or cytoplasmic proteins. The dimers are stabilized by intermolecular contacts that occur along the entire length of Ecadherin, with the intracellular domains having a surprisingly strong favorable contribution. We further show that Ecadherin mutations and calcium depletion induce structural alterations that propagate from the N terminus all the way to the C terminus, without destabilizing the dimeric state. These findings provide context for the interpretation of Ecadherin adhesion experiments. They also suggest that early events of adherens junction assembly involve interactions between from preformed Ecadherin dimers.

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.

Many postdoctoral researchers apply for faculty positions knowing relatively little about the hiring process or what is needed to secure a job offer. To address this lack of knowledge about the hiring process we conducted a survey of applicants for faculty positions: the survey ran between May 2018 and May 2019, and received 317 responses. We analyzed the responses to explore the interplay between various scholarly metrics and hiring outcomes. We concluded that, above a certain threshold, the benchmarks traditionally used to measure research success – including funding, number of publications or journals published in – were unable to completely differentiate applicants with and without job offers. Respondents also reported that the hiring process was unnecessarily stressful, time-consuming, and lacking in feedback, irrespective of outcome. Our findings suggest that there is considerable scope to improve the transparency of the hiring process.

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.

Receptor Tyrosine Kinases (RTKs) conduct biochemical signals upon dimerization in the membrane plane. While RTKs are generally known to be activated in response to ligand binding, many of these receptors are capable of forming unliganded dimers that are likely important intermediates in the signaling process. All 58 RTKs consist of an extracellular domain, a transmembrane (TM) domain, and an intracellular domain which includes a juxtamembrane (JM) sequence and a kinase domain. Here we investigate directly the effect of the JM domain on unliganded dimer stability of FGFR3, a receptor that is critically important for skeletal development. The data suggest that FGFR3 unliganded dimers are stabilized by receptor-receptor contacts that involve the JM domains. The contribution is significant, as it is similar in magnitude to the stabilizing contribution of a pathogenic mutation and the repulsive contribution of the extracellular domain. Furthermore, we show that the effects of the JM domain and a TM pathogenic mutation on unliganded FGFR3 dimer stability are additive. We observe that the JM-mediated dimer stabilization occurs when the JM domain is linked to FGFR3 TM domain and not simply anchored to the plasma membrane. These results point to a coordinated stabilization of the unliganded dimeric state of FGFR3 by its JM and TM domains via a mechanism that is distinctly different from the case of another well studied receptor, EGFR.

Forster 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 experi- ments 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 Au. 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 tetra- meric membrane proteins, as well as membrane proteins existing in monomer-dimer equilibria. We also perform rigorous exper- imental 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 mea- surements 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.

Peer-reviewed journal publication is the main means for academic researchers in the life sciences to create a permanent public record of their work. These publications are also the de facto currency for career progress, with a strong link between journal brand recognition and perceived value. The current peer-review process can lead to long delays between submission and publication, with cycles of rejection, revision, and resubmission causing redundant peer review. This situation creates unique challenges for early career researchers (ECRs), who rely heavily on timely publication of their work to gain recognition for their efforts. Today, ECRs face a changing academic landscape, including the increased interdisciplinarity of life sciences research, expansion of the researcher population, and consequent shifts in employer and funding demands. The publication of preprints, publicly available scientific manuscripts posted on dedicated preprint servers prior to journal-managed peer review, can play a key role in addressing these ECR challenges. Preprinting benefits include rapid dissemination of academic work, open access, establishing priority or concurrence, receiving feedback, and facilitating collaborations. Although there is a growing appreciation for and adoption of preprints, a minority of all articles in life sciences and medicine are preprinted. The current low rate of preprint submissions in life sciences and ECR concerns regarding preprinting need to be addressed. We provide a perspective from an interdisciplinary group of ECRs on the value of preprints and advocate their wide adoption to advance knowledge and facilitate career development., This Perspective article from an interdisciplinary group of early career researchers explores the value of preprints, advocates the wider adoption of preprints to advance knowledge and facilitate career development, and tackles concerns and opportunities regarding their use.

Isolated receptor tyrosine kinase transmembrane (TM) domains have been shown to form sequence-specific dimers in membranes. Yet, it is not clear whether studies of isolated TM domains yield knowledge that is relevant to full-length receptors or whether the large glycosylated extracellular domains alter the interactions between the TM helices. Here, we address this question by quantifying the effect of the pathogenic A391E TM domain mutation on the stability of the fibroblast growth factor receptor 3 dimer in the presence of the extracellular domain and comparing these results to the case of the isolated TM fibroblast growth factor receptor 3 domains. We perform the measurements in plasma membrane-derived vesicles using a Forster-resonance-energy-transfer-based method. The effect of the mutation on dimer stability in both cases is the same (∼−1.5 kcal/mol), suggesting that the interactions observed in simple TM-peptide model systems are relevant in a biological context.

The activity of receptor tyrosine kinases (RTKs) is controlled through their lateral association in the plasma membrane. RTKs are believed to form both homodimers and heterodimers, and the different dimers are believed to play unique roles in cell signaling. However, RTK heterodimers remain poorly characterized, as compared with homodimers, because of limitations in current experimental methods. Here, we develop a FRET-based methodology to assess the thermodynamics of hetero-interactions in the plasma membrane. To demonstrate the utility of the methodology, we use it to study the hetero-interactions between three fibroblast growth factor receptors-FGFR1, FGFR2, and FGFR3-in the absence of ligand. Our results show that all possible FGFR heterodimers form, suggesting that the biological roles of FGFR heterodimers may be as significant as the homodimer roles. We further investigate the effect of two pathogenic point mutations in FGFR3 (A391E and G380R) on heterodimerization. We show that each of these mutations stabilize most of the heterodimers, with the largest effects observed for FGFR3 wild-type/mutant heterodimers. We thus demonstrate that the methodology presented here can yield new knowledge about RTK interactions and can further our understanding of signal transduction across the plasma membrane.

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 Fӧster 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.

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A gelatin phantom containing an optically scattering funnel-shaped region of elevated optical density (OD) was used to examine light-scattering-induced artefacts in a cone-beam optical CT scanner used for gel dosimetry. To simulate polymer gel dosimeters, the opacity was introduced by adding a colloidal scatterer to the gelatin. Scatter results in an underestimate of OD (hence dose). In line profiles of OD taken from 3D reconstructions of the funnel, those profiles with a long pathlength through high OD regions exhibited a 'dishing' (or 'cupping') artefact, while those of short pathlength exhibited the opposite effect-'doming'. These phenomena are accounted for by a model that includes the effect of stray, scattered light.

Here we describe an experimental tool, termed Quantitative Imaging Förster Resonance Energy Transfer (QI-FRET), which 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 which 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), an 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.

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. DOI: http://dx.doi.org/10.7554/eLife.03772.001, eLife digest Hormones are chemicals that are produced to carry signals around the body. Mammals, including humans, need hormones called insulin and insulin-like growth factors (or IGF for short) to grow and develop normally. These hormones bind to, and activate, specific proteins—known as receptors—that span from the outside of the cell to the inside through the cell's surface membrane. The insulin and IGF receptors are complex molecules, each composed of two identical protein subunits that are linked together. The hormones bind to the part of the proteins (known as the extracellular domains) that are on the outside surface of cells. When no hormone is bound to the receptor, these two extracellular domains form an inverted ‘V’ shape and the two regions that cross the membrane are held far apart. It was unclear, however, how this shape changes when the hormone binds, and how this change in shape activates the receptor. Kavran et al. have now compared the known three-dimensional structures of the extracellular domains of the insulin receptor, both with and without a molecule of insulin bound to it. This comparison highlighted an interaction between the two receptor subunits that was disrupted when insulin was bound to the receptor. This interaction appeared to stabilize the inverted ‘V’ structure and hold apart the parts of the proteins that span the surface—which in turn separates the regions of the proteins that are inside the cell. Kavran et al. suggest that this separation keeps the insulin receptor in an inactive state. Removing either the whole extracellular domain of the IGF receptor—or a smaller portion that keeps the regions that cross the cell membrane separate—resulted in the receptor being activated, even when insulin-like growth factor was not bound to the receptor. Kavran et al. then confirmed that when a molecule of insulin-like growth factor binds to the extracellular domain of the IGF receptor, it causes a shape change that moves the parts of the receptor that span the cell membrane closer together. This results in the regions of the receptor proteins located inside the cell adding chemical tags, called phosphate groups, to one another—which activates the receptor. When there are problems with how these receptors are activated or inactivated in humans, serious disorders including diabetes and cancer can occur. As such, the findings of Kavran et al. might help future work aimed at regulating the activation of the insulin and IGF receptors to treat these and other diseases. DOI: http://dx.doi.org/10.7554/eLife.03772.002

The A391E mutation in fibroblast growth factor receptor 3 (FGFR3) is the genetic cause for Crouzon syndrome with Acanthosis Nigricans. Here we investigate the effect of this mutation on FGFR3 activation in HEK 293 T cells over a wide range of fibroblast growth factor 1 concentrations using a physical-chemical approach that deconvolutes the effects of the mutation on dimerization, ligand binding, and efficiency of phosphorylation. It is believed that the mutation increases FGFR3 dimerization, and our results verify this. However, our results also demonstrate that the increase in dimerization is not the sole effect of the mutation, as the mutation also facilitates the phosphorylation of critical tyrosines in the activation loop of FGFR3. The activation of mutant FGFR3 is substantially increased due to a combination of these two effects. The low expression of the mutant, however, attenuates its signaling and may explain the mild phenotype in Crouzon syndrome with Acanthosis Nigricans. The results presented here provide new knowledge about the physical basis behind growth disorders and highlight the fact that a single RTK mutation may affect multiple steps in RTK activation.

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.

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. DOI: http://dx.doi.org/10.7554/eLife.13876.001, eLife digest New blood vessels form by growing out from existing vessels. A signaling molecule called VEGF is crucial for this process and binds to a receptor protein known as VEGFR-2. This binding activates signaling events within the cells that line the blood vessels to promote the growth of new vessels. VEGFR-2 belongs to a family of proteins called receptor tyrosine kinases. The receptor has three main parts: one part extends out of the cell and binds to VEGF, another spans the cell’s membrane, while the third part is found inside the cell. The current model of VEGFR-2 activation is that VEGF binds to individual VEGFR-2 receptor proteins on the membrane, and brings two of them close enough to form a complex called a dimer. The receptor dimer is activated and initiates signaling within the cell. Sarabipour et al. tested this current model and revealed that VEGFR-2 could form a dimer even in the absence of VEGF. This receptor dimer formed through contacts between parts that are inside the cell as well as the regions embedded in the membrane. These VEGFR-2 dimers were active, but only to a low level. However, the activity of the receptor was increased in the presence of VEGF. Sarabipour et al. found that binding of VEGF promoted VEGFR-2 to change its three-dimensional shape, which made it more active. Further work suggested that VEGFR-2 dimers must first correctly assemble in the absence of VEGF so that active VEGFR-2 dimers can form at a later stage. Hence a VEGFR-2 dimer on its own is an important intermediate that is needed for full activation of the receptor. A mutation in the gene that encodes VEGFR-2 causes a disorder called infantile hemangioma that leads to abnormal vessel formation in young children. Sarabipour et al. found that this mutation causes VEGFR-2 to change its shape even when no VEGF is present and thus makes the receptor overly active. Finally, tumors need to form new blood vessels to grow and survive. These new findings suggest that drugs that interact with VEGFR-2 dimers in the absence of VEGF may be able to prevent the receptor from becoming activated and could thus help cut the blood supply to tumors and treat cancers. DOI: http://dx.doi.org/10.7554/eLife.13876.002