Interactions between NRP1 and VEGFR2 molecules in the plasma membrane

Accepted Manuscript Interactions between NRP1 and VEGFR2 molecules in the plasma membrane

Christopher King, Daniel Wirth, Samuel Workman, Kalina Hristova PII: DOI: Reference:

S0005-2736(18)30103-2 doi:10.1016/j.bbamem.2018.03.023 BBAMEM 82748

To appear in: Received date: Revised date: Accepted date:

8 January 2018 19 March 2018 20 March 2018

Please cite this article as: Christopher King, Daniel Wirth, Samuel Workman, Kalina Hristova , Interactions between NRP1 and VEGFR2 molecules in the plasma membrane. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbamem(2018), doi:10.1016/j.bbamem.2018.03.023

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ACCEPTED MANUSCRIPT Interactions between NRP1 and VEGFR2 molecules in the plasma membrane Christopher King2, Daniel Wirth1, Samuel Workman1, and Kalina Hristova1,2

Program in Molecular Biophysics, Johns Hopkins University, Baltimore, Maryland 21218

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2

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Department of Materials Science and Engineering and Institute for NanoBioTechnology, and

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1

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ABSTRACT

Here we use a quantitative FRET approach, specifically developed to probe membrane protein

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interactions, to study the homo-association of neuropilin 1 (NRP1) in the plasma membrane, as well as its hetero-interactions with vascular endothelial growth factor receptor 2 (VEGFR2). Experiments are

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performed both in the absence and presence of the soluble ligand vascular endothelial growth factor A

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(VEGFA), which binds to both VEGFR2 and NRP1. We demonstrate the presence of homo-interactions between NRP1 molecules, as well as hetero-interactions between NRP1 and VEGFR2 molecules, in the

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plasma membrane. Our results underscore the complex nature of the interactions between selfassociating receptors, co-receptors, and their ligands in the plasma membrane. They also highlight the

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need for new methodologies that capture this complexity, and the need for precise physiological

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measurements of local receptor surface densities in the membrane of cells.

INTRODUCTION

Neuropilin 1 (NRP1), a 140 kDa transmembrane receptor, plays a critical role in the development of the embryonic cardiovascular and nervous systems (1, 2). NRP1 is a single pass membrane protein with a N-terminal extracellular region encompassing two CUB (complement C1r/C1s, urchin embryonic growth factor and bone morphogenic protein 1) domains, two domains that are homologous to coagulation factors V and VIII and are important for VEGFA-165 binding, and a MEM (meprin, A5, and receptor protein-tyrosine phosphatase ) domain, which is proximal to the

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ACCEPTED MANUSCRIPT membrane (1-3). The extracellular region is followed by the hydrophobic transmembrane (TM) domain and a short (44 aa) cytoplasmic sequence at the C-terminus. NRP1 is expressed in many tumors, and its overexpression has been linked to tumor growth and vascularization (4, 5). When expressed in endothelial cells, it functions as a receptor for vascular endothelial growth factor A (VEGFA). VEGFA is a disulfide linked anti-parallel homodimer that exists

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in four different isoforms (121, 165, 189, and 206 amino acids long). It also binds to and activates

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vascular endothelial growth factor receptor 2 (VEGFR2), a receptor tyrosine kinase that plays a critical role in controlling angiogenesis, the development of blood vessels from pre-existing ones (6-8).

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VEGFR2 (molecular weight ~220 kDa) is composed of 7 Ig-like extracellular domains (which are glycosylated), a TM domain, and an intracellular kinase domain.

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VEGFA-165 is known to bind both NRP1 and VEGFR2. While the VEGFA binding site for VEGFR2 is within the first 121 residues, the shorter VEGFA-121 isoform is believed to exhibit reduced

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angiogenic activity due to its inability to bind NRP1. There is convincing evidence that the presence of NRP1 in the plasma membrane improves VEGF binding to VEGFR2, enhances VEGFR2

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phosphorylation, and potentiates cell migration (9-11). It is generally believed that NRP1 exerts its action by directly interacting with VEGFR2 and perhaps promoting the formation of a tertiary

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VEGF/VEGFR/NRP1 complex. Thus, the biological effects of NRP1 on VEGFR2 function are well established. However, the molecular interactions that underlie these biological effects are not fully

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understood. There is disagreement as to whether the presence of VEGF is required for NRP1-VEGFR2 interactions, as some reports suggest that it is (10, 12), while others suggest that the interaction occurs

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independent of VEGF (9). NRP1 has been shown to oligomerize in the plasma membrane, even in the absence of VEGF (13-15), but the effect of VEGFR2 on NRP1 association has not been studied thus far,

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to the best of our knowledge.

Prior studies of NRP1 homo- and hetero-interactions have relied primarily on coimmunoprecipitation, which cannot provide information about the oligomerization state or the relative populations of monomers and oligomers. Here we explore the utility of a quantitative FRET approach (16), specifically developed to probe membrane protein interactions, to study NRP1 homo-association and NRP1/VEGFR2 hetero-association in the plasma membrane, both in the absence and presence of VEGFA-165.

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ACCEPTED MANUSCRIPT MATERIALS AND METHODS Fluorescent Proteins Monomeric yellow fluorescent protein (YFP) and mTurquoise with an N-terminal 6x His tag were expressed and purified as described previously (17). Molar absorption coefficients of 83,400 Mol*cm-1 and 30,000 Mol*cm-1 were used to calculate the concentrations of the solution standards from

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the YFP and mTurquoise absorption maxima of 514 nm and 434 nm, respectively. Images of protein

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solution standards were acquired and were used to calculate the pixel-level intensity versus

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concentration slope values utilized with the FSI method, as described previously (16). Plasmid Constructs

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A plasmid encoding full-length human NRP1 protein was purchased from GeneCopoeia (Product ID A6403). Constructs encoding for NRP1-GGS5-YFP and NRP1-GGS5-mTurquoise pcDNA3.1+ were

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created with the NEBuilder HiFi DNA Assembly Kit according to the manufacturer’s instructions (New England Biolabs, E5520S).

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VEGFR2-EC-TM-GGS5-Amber, a non-fluorescent form of VEGFR2-EC-TM-GGS5-YFP, was created by introducing a Y67C mutation into YFP with the QuikChange II Site-Directed Mutagenesis

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Kit (Agilent, 200523), according to the manufacturer’s instructions.

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Cell Culture and Transient Transfection

Experiments were performed in HEK293T cells, which were a kind gift from Dr. D. Wirtz, Johns

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Hopkins University. The cells were cultured in DMEM supplemented with 10% FBS and 20mM glucose, at 37° C in a 5% CO2 environment.

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HEK293T cells were seeded in collagen-coated, glass bottom 35mm petri dishes (MatTek, P35GCOL-1.5-14-C) at a density of 2.5e5 cells per dish. Cells were 60-70 percent confluent at the time of transfection, 24 hours later. Single transfections were performed with a total of 3 µg of plasmid DNA and co-transfections were performed with a total of 4-9 µg of plasmid DNA. In all cases, Lipofectamine 3000 (Invitrogen) was used for the transfection according to the manufacturer’s protocol.

Six hours

after transfection, 10mM sodium butyrate was added to the cells to enhance protein expression. Twelve hours after transfection, the cells were rinsed twice with phenol-red free, serum free DMEM (Sigma, D2902) and then serum starved for at least 12 hours in the presence of 10 mM sodium butyrate, prior to

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ACCEPTED MANUSCRIPT the application of osmotic stress and imaging. Human VEGFA-165 was added at a concentration of 2.5 μg/mL of media (Cell Signaling Technology, #8065SC) Cells Under Reversible Osmotic Stress Hypotonic swelling media consisted of serum-free media, diluted 1:9 with diH2O, buffered with 25mM HEPES, and 0.2µm sterile filtered. Just prior to imaging, the starvation media was aspirated

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10 minutes, images of swollen cells were acquired for up to two hours.

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from the petri dishes and was gently replaced with 1 mL of 37°C hypotonic swelling media. After about

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Fully Quantified Spectral Imaging (FSI)

The FSI methodology allows the calculations of the apparent FRET

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efficiency, 𝐸𝑎𝑝𝑝 , the donor concentration [D], and the acceptor concentration [A], given

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by the following equations (16):

𝐹𝐷 𝜆1 𝑖𝐷, 𝜆1

𝐹𝐴 𝜆2 𝐴, 𝜆2

=𝑖

1

(𝐹 𝐷𝐴

𝐷, 𝜆1

+

𝜆1

1

𝐴, 𝜆2

𝑄𝐷 𝑄𝐴

𝑖

𝑖

(𝐹 𝐴𝐷𝜆1 − 𝑖𝐴,𝜆1 𝐹 𝐴𝜆2 )) (2) 𝐴,𝜆2

(𝐹 𝐴𝐷 𝜆2 − 𝑖𝐷,𝜆2 𝐹 𝐴𝐷𝜆1 ) ∙ (1 − 𝐷,𝜆1

𝑖𝐴,𝜆1 𝑖𝐷,𝜆2 −1 ) 𝑖𝐴,𝜆2 𝑖𝐷,𝜆1

(3)

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[𝐴] = 𝑖

=𝑖

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[𝐷] =

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𝐸𝑎𝑝𝑝 = 1 − 𝐹 𝐷𝐴𝜆1 ⁄𝐹 𝐷𝜆1 (1)

In these equations, 𝐹𝐷,𝐴𝜆1,2 is the total fluorescence of the donor or acceptor in the

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absence of FRET, upon excitation at 𝜆1 or 𝜆2 . 𝐹 𝐷𝐴𝜆1 is the measured fluorescence of the donor in the presence of acceptors, and 𝐹 𝐴𝐷𝜆1,2 is the measured fluorescence of the

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acceptors in the presence of donors. 𝑖𝐷, 𝜆1 and 𝑖𝐴, 𝜆2 are the slopes of the solution standard intensity versus micromolar concentration lines determined by imaging solution standards of known concentration. QD and QA are the quantum yields of the donor and the acceptor, respectively. To determine the 2D surface density from the fluorescence and the concentration calibration lines, the apparent pixel-level fluorescence values were integrated across the diffraction limited segment of the membrane (16). As described previously (16), FD, FA, 𝑜𝑟 𝐴 and FAD were summed over the pixel selected in a membrane region, 𝐹 𝐷𝜆𝑖,𝑟𝑒𝑔 =

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ACCEPTED MANUSCRIPT 𝐷 𝑜𝑟 𝐴 𝐷 𝑜𝑟 𝐴 𝑑𝐴 = ∑ 𝐹𝑖,𝑗 . The apparent FRET efficiency of the membrane region is ∫ 𝐹𝑟𝑒𝑔

calculated according to 𝐸𝑎𝑝𝑝

𝐹 𝐷𝐴 = 1 − 𝜆1,𝑟𝑒𝑔⁄ 𝐷 . 𝐹 𝜆1,𝑟𝑒𝑔

Thermodynamic Analysis of Receptor Dimerization and Oligomerization The total apparent FRET efficiencies of oligomers of donor-labeled and

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acceptor-labeled proteins are calculated using the kinetic theory of FRET formalism (18-

𝐸𝑝𝑟𝑜𝑥 + 𝐸𝑜𝑙𝑖𝑔𝑜 −2 𝐸𝑝𝑟𝑜𝑥 𝐸𝑜𝑙𝑖𝑔𝑜

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𝐸𝑎𝑝𝑝 =

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20):

1 − 𝐸𝑜𝑙𝑖𝑔𝑜 𝐸𝑝𝑟𝑜𝑥

(4)

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Equation 4 gives the total FRET efficiency that arises due to two contributions: intra-oligomeric FRET, 𝐸𝑜𝑙𝑖𝑔𝑜, occurring due to specific interactions between donor- and

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acceptor-labeled proteins, and inter-oligomeric proximity FRET, 𝐸𝑝𝑟𝑜𝑥 , that occurs when fluorophores are confined to two dimensions (21-23). The procedure for calculating the

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proximity contribution has been described (20).

The intra-oligomeric FRET is modeled based on Raicu’s kinetic theory

𝜇𝑜𝑙𝑖𝑔𝑜 [𝐷]

∑𝑛−1 𝑘=1

𝑘(𝑛−𝑘)𝐸̃

(𝑛)𝑃𝑘 𝑃𝑛−𝑘 1+(𝑛−𝑘−1)𝐸̃ 𝑘 𝐷 𝐴

(5)

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𝐷𝑞 𝐸𝑜𝑙𝑖𝑔𝑜 =

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formalism (18):

In equation 5, 𝑛 represents the oligomer order. 𝜇𝑜𝑙𝑖𝑔𝑜 is the concentration of

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oligomers. 𝑃𝐷 and 𝑃𝐴 are the fractions of donors and acceptors in the oligomer. For large numbers of molecules, these are equal to the fraction of donor and acceptors,

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respectively: 𝑥𝐷 and 𝑥𝐴 .

𝑥𝐴 =

[𝐴] , [𝐷]+[𝐴]

with [D] and [A] representing the donor and Ẽ is the “Intrinsic FRET” or “pair-wise

acceptor concentrations, and 𝑥𝐷 + 𝑥𝐴 = 1.

FRET efficiency” which primarily depends on the distance between the fluorescent proteins in the oligomer (20, 24). The theoretical FRET efficiency due to specific interactions can be also written as: 𝐷𝑞

𝐸 𝑜𝑙𝑖𝑔𝑜 =

𝑓𝑜𝑙𝑖𝑔𝑜 𝑛∙𝑥𝐷

(6)

∙ 𝐸,

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ACCEPTED MANUSCRIPT 𝑘(𝑛−𝑘)𝐸̃

𝑛 𝑘 𝑛−𝑘 where 𝐸 = ∑𝑛−1 . foligo is the fraction of proteins in the oligomeric state, which 𝑘=1 1+(𝑛−𝑘−1)𝐸̃ (𝑘 )𝑃𝐷 𝑃𝐴

depends of the association constant K and the total receptor concentration, [T] = [A] + [D], according to equation (7): 𝑓𝑜𝑙𝑖𝑔𝑜 =

𝑛𝜇𝑜𝑙𝑖𝑔𝑜 𝑛𝐾[𝑚]𝑛 = [𝑇] [𝑇]

(7)

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Equation (7) is used to fit models for n = 1 to 6, corresponding to the cases of monomer,

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monomer-dimer, monomer-trimer, monomer-tetramer, monomer-pentamer, and monomer-hexamer

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thermodynamic equilibria, to the experimental data as described in detail previously (16). The mean squared error (MSE) is calculated for all oligomer models while accounting for the proximity

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contribution, and the model that yields the lowest overall MSE is chosen as the model that best represents the experimental FRET data.

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RESULTS NRP1 Homo-interactions

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To study the self-interactions between full-length NRP1 molecules in the plasma membrane, we

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performed spectral imaging FRET experiments which utilized mTurquoise and YFP as a FRET pair. We created NRP1-GGS5-mTurquoise and NRP1-GGS5-YFP constructs in which the fluorescent proteins (FPs) were attached to the C-terminus of full-length NRP1 via a flexible GGS5 linker. The schematic of This FRET pair is highly suitable for spectral FRET

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these construct is shown in Figure 1A.

measurements, as the FRET spectrum can be robustly unmixed into its donor and acceptor components,

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as shown previously (16).

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Cells were co-transfected with plasmids encoding NRP1-GGS5-mTurquoise and NRP1-GGS5YFP, in a 1:3 ratio. After receptor expression, the cells were subjected to reversible osmotic stress and imaged in accordance with the FSI methodology, in a two-photon microscope equipped with the OptiMiS spectral imaging system (18, 25) (see Figure 1B-E). Previously, we have shown that the osmotic stress treatment does not alter the FRET efficiencies, suggesting that it does not alter the nature of the protein-protein interactions (16). As a result of the osmotic stress, cells flatten their caveolae (60 – 80 nm cup-shaped invaginations) and “un-wrinkle” their membranes (26). The cells under reversible osmotic stress exhibit large membrane areas of homogeneous fluorescence, such that the NRP1 twodimensional concentration in these areas can be calculated (16). Regions of homogenous, diffraction6

ACCEPTED MANUSCRIPT limited membrane fluorescence of approximately 3 µm in length were chosen and analyzed (see Figure 1B). The FRET efficiency, the donor concentration, and the acceptor concentration were calculated for each region using equations (1) through (3) in Materials and Methods, as described (16). Figure 2 shows the results of the NRP1 homo-interaction FRET experiments. In all, 270 cells were imaged yielding 538 data points for analysis. The measured total apparent FRET efficiency, Eapp,

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is plotted in Figure 2A as a function of total NRP1 surface density, in purple. The FRET efficiency

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rapidly increases as a function of the total receptor concentration, indicative of a concentration dependent protein-protein association in the membrane. Figure 2B shows the measured mole-fraction

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of NRP1-YFP (the fraction acceptor-tagged receptors, xA), for the selected membrane regions (purple bars). The average xA values agree with those expected for the 1:3 donor-to-acceptor ratio used in

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transfection.

The data are analyzed following a protocol that has been described in detail in previously

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published work (20), and is overviewed in the Materials and Methods section. Equations 4 through 7 are used to compute the theoretical apparent FRET efficiency as a function of total concentration for the

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different possible oligomeric association states (e.g., monomer-only, monomer-dimer, monomer-trimer, monomer-tetramer, etc.) while varying the association constant in equation (7). The theoretical FRET,

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which includes a contribution for proximity FRET (20, 23), is fitted to the experimental FRET data using a least-squares minimization procedure, in order to calculate the optimal parameters describing the

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interactions. The oligomer model which gives the lowest mean squared error (MSE) is considered the best overall model to represent the data. Dimer formation is identified through an MSE minimum at n =

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2, while higher-order oligomer formation yields a minimum MSE at n > 2. While the exact order of the oligomer (n = 3, 4, etc.) cannot be discerned (20), the methodology correctly differentiates between

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dimers and oligomers, and yields the correct fraction of receptors in an associated state, as well as the fraction of monomeric receptors (16, 20). In Figure 2C, we plot the best-fit MSE as a function of oligomer order. There is a shallow minimum at n = 4. Thus, n > 2, indicating that NRP1 oligomerizes in the plasma membrane in the absence of VEGFA-165. In Figure 2A, we show the best-fit theoretical total apparent FRET efficiency in red. Figure 2D shows the oligomeric fractions, calculated from the measured total apparent FRET efficiencies. They are binned and plotted as a function of the total NRP1 concentration, along with the best-fit theoretical oligomeric fraction (solid line). 7

ACCEPTED MANUSCRIPT Next, we performed similar experiments in the presence of VEGFA-165, which is known to bind to NRP1 extracellular domain. In total, 128 cells were imaged, providing 256 data points for analysis. The results of these experiments are shown in Figure 3. In Figure 3C, the MSE is minimized when n = 2, suggesting NRP1 dimer formation when the ligand is present. The ligand VEGFA-165 is a cysteinelinked dimer and thus it carries two symmetric binding sites for NRP1 molecules. As a result, it can be expected that NRP1 will be dimeric when bound to VEGFA-165. Our experimental results indeed

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suggest that NRP1 undergoes a transition from a monomer-oligomer equilibrium to a monomer-dimer

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equilibrium upon addition of VEGFA-165.

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Figure 6A directly compares the fraction of self-associated NRP1 molecules as a function of total NRP1 concentrations, in the absence and presence of VEGFA-165. The dimeric fraction of NRP1

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in the presence of VEGFA-165 is higher than the oligomeric fraction of NRP1 in the absence of VEGFA-165 up to a concentration of ~1000 receptors/μm2. This indicates that the addition of VEGFA-

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165 increases the number of NRP1 in oligomers, i.e. reduces the concentration of NRP1 monomers. At higher surface densities of NRP1, the situation is reversed and the addition of VEGFA-165 reduces the

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overall fraction of self-associated NRP1 molecules.

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Effect of VEGFR2 expression on NRP1 interactions

We sought to determine how NRP1 interactions are affected by the presence of VEGFR2. To

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study this effect, we co-transfected HEK293T cells with NRP1-GGS5-mTurquoise, NRP1-GGS5-YFP, and a non-fluorescent form of a VEGFR2 construct containing the extracellular (EC) and

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transmembrane (TM) domains of VEGFR2, with an intracellular YFP harboring a Y67C mutation that renders it non-fluorescent (27). This VEGFR2 EC-TM construct is well characterized, and exists in a

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monomer-dimer equilibrium (16). Again, we monitored NRP1 interactions in a homodimerization FRET experiment. 193 cells were imaged yielding 386 data points for analysis. Results are shown in Figure 4. As in the case of no VEGFR2, the analysis indicates that NRP1 is associating into oligomers of higher order than a dimer, since the MSE exhibits a minimum for n > 2. (Figure 4C). Thus, NRP1 is oligomeric both in the presence and absence of VEGFR2. The FRET and the oligomeric fractions with and without VEGFR2 and in the absence of VEGFA-165 are compared in Figure 6B. We see stronger interactions in the absence of VEGFR2, suggesting that VEGFR2 presence leads to a decrease in NRP1 molecules participating in NRP1 oligomerization. This may occur because VEGFR2 is engaging in

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ACCEPTED MANUSCRIPT hetero-interactions with NRP1, reducing the effective number of NRP1 molecules available to participate in homo-oligomerization. We performed similar experiments to measure NRP1 homo-interactions in the presence of both VEGFR2 and VEGFA-165. Results, shown in Figure 5, reveal that NRP1 forms predominantly a dimer under these conditions. Thus, VEGFA-165 addition alters NRP1 interactions within the pre-formed NRP1/VEGFR2 complexes, reducing the number of NRP1 molecules in each complex to two.

The

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comparison of NRP1 oligomeric fractions with and without VEGFA-165 in Figure 6C show ligand

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addition increases the over-all number of NRP1 molecules in complexes, up to very high NRP1

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concentrations. Therefore, ligand addition to the pre-formed NRP1/VEGFR2 complexes leads to their global rearrangement, reducing the number of homointeracting NRP1 molecules in each

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NRP1/VEGFR2 complex, while increasing the over-all number of NRP1 molecules in complexes. Figure 6D compares NRP1-derived FRET and dimeric fractions with and without VEGFR2, in

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the presence of the ligand VEGFA-165. The addition of VEGFR2 EC-TM domains to the pre-formed NRP1/VEGFA-165 complexes does not change the self-association of NRP1. This could be explained

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by the fact that the NRP1/VEGFA-165 complexes are already assembled in a way that allows direct interactions with VEGFR2 without rearrangement. This is consistent with the idea that the primary

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function of NRP1 is to pre-position VEGF for VEGFR2 interactions. Finally, HEK293T cells were co-transfected with VEGFR2-EC-TM-GGS5-mTurquiose and

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NRP1-GGS5-YFP. Unlike the previous experiments which assess NRP1 homo-interactions, here the measured FRET is entirely due to hetero-interactions between NRP1 and VEGFR2. Homodimers are

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now “invisible” because only the heterodimers/hetero-oligomers have both donors and acceptors available to participate in FRET. The surface densities of VEGFR2-EC-TM-GGS5-mTurquoise and

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NRP1-GGS5-YFP in the plasma membrane, and the apparent FRET efficiencies were measured both in the absence and presence of the VEGFA-165. The results are shown in Figure 7. We see that the total apparent FRET efficiency increases as a function of total VEGFR2 + NRP1 concentration, indicative of direct protein-protein interactions between NRP1 and VEGFR2 EC-TM. Interestingly, FRET is similar in the absence and presence of ligand (Figure 7). These data demonstrate the occurrence of NRP1VEGFR2 interaction, both in the absence and presence of the VEGFA-165. DISCUSSION

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ACCEPTED MANUSCRIPT Here we study the homo-interactions between NRP1 molecules, as well as the hetero-interactions between NRP1 and VEGFR2 molecules in the plasma membrane, and the effect of the ligand VEGFA165 on these interactions. Such interactions have been previously investigated using coimmunoprecipitation techniques which usually do not provide quantitative information about the interactions (9, 10, 12). Here we quantify these interactions using FRET measurements and we determine the oligomeric fractions. We find that NRP1 self-associates in the membrane and interacts

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with VEGFR2 even in the absence of ligand. This work addresses a controversy about VEGFR2-NRP1

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interactions by supporting prior conclusions of Whitaker and colleagues that these interactions do not

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require VEGF ligands (9).

An intriguing finding from this work is that the oligomer size of the NRP1 assemblies is altered

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upon ligand addition, with results pointing to a transition from a monomer-oligomer equilibrium to a monomer-dimer equilibrium. This ligand-driven transition occurs both in the presence and absence of

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VEGFR2 and may be explained by the fact that the ligand VEGFA-165 is dimeric and possesseses symmetric, opposing binding sites for VEGFR2 and NRP1. Thus, VEGFA-165 does not simply bind to

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the pre-assembled NRP1 molecules, but also re-organizes them. We also find that VEGFR2 addition to NRP1 oligomers leads to NRP1 rearrangement in the absence of ligand. Intriguingly, the pre-assembled

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NRP1-VEGFA165 complexes engage VEGFR2 in a way that apparently does not require further NRP1 oligomeric rearrangements within the complexes. These results underscore the intricate nature of the

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interactions between self-associating receptors, co-receptors, and their ligands in the plasma membrane. The methodology that we use here was originally developed to study the homo-association of

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membrane proteins in the plasma membrane (16). Here, this technique is applied to study a complex network of multiple protein interactions in the plasma membrane. These include homo-association of

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NRP1 molecules into oligomers, dimerization of VEGFR2, and hetero-association of NRP1 and VEGFR2, all of which are modulated by the binding of the soluble ligand, VEGFA-165. Our FRET experiments, however, allow us to monitor only the interactions between labeled molecules. Thus, each FRET measurement yields only a partial view of the interaction network, rather than a comprehensive description.

Further method development will be required for future studies of multiple protein

interaction networks in biological membranes, and thus method-development needs to lead these efforts. Despite these limitations, the quantitative FRET method that we use here yields new knowledge that is not attainable with traditional biochemical methods. We measure binding curves over broad expression range of the receptors, and we can thus make predictions of dimeric and oligomeric fractions 10

ACCEPTED MANUSCRIPT at any expression level.

It is possible that expressions vary widely under varied physiological

conditions, and there are many reports of receptor and co-receptor overexpression in cancer (4, 8, 2834). However, the exact surface densities are rarely reported or known. Better measurements of local protein surface densities will help us understand the complex behavior of the numerous interacting signaling proteins in membranes in vivo, and will empower quantitative predictions of biological

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function.

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7.

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10.

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6.

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and an NSF Graduate Research Fellowship DGE-1232825 (to CK).

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Acknowledgements: Supported by NSF MCB 1712740 and NIH GM068619 (to KH),

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Figure 1. Experimental design and methods. A. Cartoons of the NRP1-GGS5-fluorescent protein and the EC-TM VEGFR2-GGS5-fluorescent protein constructs used in the FRET experiments. NRP1 is a

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single pass membrane protein with a N-terminal extracellular region encompassing two CUB domains (grey), two domains that are homologous to coagulation factors V and VIII (lilac), and a MEM domain

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(red) proximal to the membrane. The extracellular region is followed by a hydrophobic TM domain and a short (44 aa) cytoplasmic sequence at the C-terminus (orange). The VEGFR2 construct includes VEGFR2 extracellular (EC) domain, composed of 7 Ig-like domains (green), and a transmembrane (TM) domain which is attached to the fluorescent protein via a flexible GGS5 sequence. B. A HEK293T cell expressing NRP1-GGS5-mTurquoise displays large stretches of homogenous “unwrinkled” membrane under reversible osmotic stress. Scale bar is 10 m. Membrane regions, ~ 3µm in length, are selected for FSI analysis as described (16). Two such regions are shown in yellow. C. Emission spectra of mTurquoise (blue) and YFP (yellow), a FRET pair. D. A single pixel from the FRET scan is

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Figure 2. NRP1 homo-interaction data acquired for NRP1-GGS5-mTurquoise and NRP1-GGS5-YFP in HEK293T plasma membranes in the absence of ligand.

A. The measured total apparent FRET

efficiency (purple) and the best-fit theoretical total apparent FRET efficiency (red) as a function of the total NRP1 expression. B. A histogram of measured acceptor fractions. C. The best-fit MSE as a

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The best-fit oligomeric fraction for NRP1 is plotted as a function of the total NRP1

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function of oligomer order. The best-fit model to the data is a monomer-oligomer equilibrium (𝑛 = 4).

The dashed red lines show the 95% confidence interval

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Figure 3. NRP1 homodimerization data acquired for NRP1-GGS5-mTurquoise and NRP1-GGS5-YFP in HEK293T plasma membranes in the presence of the VEGFA-165 growth factor. A. The measured total apparent FRET efficiency (cyan) and the best-fit theoretical total apparent FRET efficiency (red) as a function of the total NRP1 concentration. B. A histogram of measured acceptor fractions. C. The best-fit MSE as a function of oligomer order. The minimum occurs at 𝑛 = 2, indicative of dimerization.

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measured dimeric fraction.

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Figure 4. NRP1 homo-interaction data acquired for NRP1-GGS5-mTurquoise and NRP1-GGS5-YFP in HEK293T plasma membranes in the absence of VEGF ligand and in the presence of VEGFR2 EC-TM. A. The measured total apparent FRET efficiency (mustard yellow) and the best-fit theoretical total 19

ACCEPTED MANUSCRIPT apparent FRET efficiency (red) as a function of the total NRP1 concentration. B. A histogram of measured acceptor fractions. C. The best-fit MSE as a function of oligomer order. The best-fit model is a monomer-oligomer equilibrium model, 𝑛 = 5. D. The best-fit oligomeric fraction is plotted as a

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Figure 5. NRP1 homo-interaction data acquired for NRP1-GGS5-mTurquoise and NRP1GGS5-YFP, in the presence of both VEGFA-165 and VEGFR2 EC-TM.

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measured total apparent FRET efficiency (green) and the best-fit theoretical total 21

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The best-fit MSE as a function of

oligomer order. The best-fit model is a monomer-dimer equilibrium model, 𝑛 = 2. D. The best-fit dimeric fraction is plotted as a function of the total NRP1 concentration,

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Figure 6. Comparisons of the best-fit NRP1 oligomeric fractions as a function of NRP1 concentration, as determined from the fits in Figures 2 to 5. A. NRP1 association, without and with VEGFA-165 (from

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Figures 2 and 3), plotted in purple and cyan, respectively. B. NRP1 oligomerization in absence (purple, from Figure 2) and presence (mustard yellow, from Figure 4) of VEGFR2 EC-TM, no ligand. C. NRP1

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association in the presence of EC-TM VEGFR2, without (mustard yellow, from Figure 4) and with (green, from Figure 5) VEGFA-165. D. NRP1 dimerization in the presence of VEGFA-165, with and without VEGFR2 (from Figures 3 and 5; the two curves, cyan and green, overlap). In all cases, the 95% confidence intervals are shown with the grey dashed lines.

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Figure 7. The total apparent FRET efficiency occurring due to NRP1-GGS5-YFP/VEGFR2 EC-TM-

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GGS5-mTurquoise heterodimerization, in the presence (yellow) and absence (green) of VEGFA-165, plotted as a function of total receptor (NRP1+VEGFR2 EC-TM) surface density.

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NRP1 and VEGFR2.

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experiments in Figures 2-5, here the measured FRET is entirely due to hetero-interactions between

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ACCEPTED MANUSCRIPT Conflict of Interest

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The authors declare no conflict of interest.

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Graphical abstract

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ACCEPTED MANUSCRIPT Highlights FRET is used to study NRP1 and VEGFR2 interactions in membranes

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NRP1 molecules form oligomers in the plasma membrane

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The binding of VEGF ligands induces NRP1 dimer formation

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NRP1 interacts with VEGFR2 in the absence and presence of VEGF

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