The cytoplasmic domain of neuropilin-1 regulates focal adhesion turnover

Abstract

Though the vascular endothelial growth factor coreceptor neuropilin-1 (Nrp1) plays a critical role in vascular development, its precise function is not fully understood. We identified a group of novel binding partners of the cytoplasmic domain of Nrp1 that includes the focal adhesion protein FlnA. Endothelial cells (ECs) expressing a Nrp1 mutant devoid of a cytoplasmic domain (nrp1^cytoΔ/Δ) instead of wild type Nrp1 migrated significantly slower in response to VEGF relative to nrp1^+/+ cells. The rate of FA turnover in VEGF-treated nrp1^cytoΔ/Δ ECs was an order of magnitude lower in comparison to nrp1^+/+ ECs, thus accounting for the slower migration rate of the nrp1^cytoΔ/Δ ECs.

1. Introduction

Nrp is a single-pass transmembrane protein, which has a highly conserved short cytoplasmic domain that lacks catalytic activity [1,2]. While the downstream signaling of the tyrosine kinase receptor of vascular endothelial growth factor (VEGF)-A, VEGF receptor 2 (VEGFR2), has been the subject of numerous studies and its major elements have been established [3], the contribution of Nrp1 to VEGF signaling is less well understood. Several studies suggested that Nrp1 might have a VEGFR2-independent function in mediating VEGF signaling [4-7]. Indeed, Nrp1 has binding partners other than VEGFR2, such as receptors of the plexin family [8], but it remains unclear how the cytoplasmic domain of Nrp1 could sustain such activity. Presumably, the cytoplasmic domain could recruit other proteins that may be able to transduce VEGF signaling. The best studied Nrp1 cytoplasmic binding protein is synectin, also named Nrp1-interacting protein (NIP) [9]. Synectin contains a single PDZ (postsynaptic density 95, Disk large, Zona occludens-1) domain, through which it binds to the C-terminus of Nrp1. Synectin recruits the molecular motor myosin VI to uncoated endocytic vesicles, thus facilitating the trafficking of endocytosed membrane receptors [10], such as VEGFR2 in complex with Nrp1, to promote arteriogenesis [11].

Focal adhesions are large aggregates of proteins that anchor the cell to the extracellular matrix and that undergo cycles of assembly and disassembly during cell movement [12,13]. Among other signals, the dynamics of FAs are regulated by growth factors [14], including by VEGF-A [15], a major stimulant of vasculogenesis and angiogenesis in development and in the adult organism [16,17]. Treatment with VEGF increased FA density in ECs [18]. The signaling of VEGF to FAs appeared to follow the canonical VEGFR2-dependent pathway, and to be mediated by the non-receptor FA kinase (FAK), and by a closely related kinase, Pyk2 [18]. The VEGF-induced increase in FA assembly was accompanied by an increase in EC migration. Though Nrp1 had not been observed directly in FAs [19], proteomics studies identified the VEGF co-receptor Nrp1 as a FA component [20].

The regulation of FA turnover is not fully understood, and it is likely to involve multiple molecular mechanisms [13]. One of these invoked FlnA [18], a large scaffold protein that binds filamentous actin (F-actin) and numerous other proteins, including transmembrane receptors [21,22]. FlnA is composed of 24 IgG including and a C-terminus dimerization domain [21]. Most of its ligands, excluding F-actin, bind to the last three C-terminus IgG repeats.

Here, we investigated if the Nrp1-mediated intracellular signaling of VEGF emanates from a protein complex bound to the cytoplasmic domain of Nrp1. We first combined immunoprecipitation and mass spectroscopy to identify several novel Nrp1-associated proteins, including FlnA. Separately, we found by immunoprecipitation and immunoblotting that the scaffold protein p130Cas also associated with Nrp1. Both proteins are known to be components of FAs [23]. We tested, therefore, if Nrp1 is involved in FA turnover. Consistent with previous reports that FlnA and p130Cas are components of FAs, we further found that Nrp1 is involved in FA turnover to promote cell migration.

2. Materials and Methods

See supplemental materials

3. Results

3.1. Identification of novel ligands to the cytoplasmic domain of Nrp1

We used a proteomics approach to identify cytoplasmic proteins that associate with Nrp1. Immunoprecipitates of Nrp1 from quiescent and from VEGF-A₁₆₄-treated (20 ng/ml, 5 min) primary mouse heart ECs were resolved by SDS-PAGE. The band patterns of the two samples were partially different (Fig. 1, Table 1). The proteins were identified by liquid chromatography-tandem mass spectroscopy (LC-MS). In the quiescent ECs, these proteins were cytoplasmic dynein heavy chain 1 (Dync1h1), myosin heavy chains 9 and 10 (Mhy9, Mhy10), and eukaryotic translation elongation factor 1 1 (EEF1 1). Mhy9, Mhy10, and EEF1 1 were present in the immunoprecipitate from both VEGF-treated and quiescent ECs. Mhy9 and Myh10 are subunits of non-muscle myosin II, known also as heavy chains IIa and IIb. They are involved in endosomal trafficking [24], cytokinesis [25], and cell shape remodeling [26], and are localized to early endosomes, stress-fibers, and FAs [27]. The proteins unique to the VEGF-treated sample were -enolase and Filamin-A (FlnA). Due to its connection to the actin cytoskeleton and protein transport, the involvement of Filamin-A was investigated further.

Figure 1.

A. Identification of novel binding partners for the cytoplasmic domain of Nrp1. Coomassie-stained tris-glycine acrylamide (10%) showing bands of proteins immunoprecipitated by Nrp1 from primary mouse heart ECs incubated without or with 20 ng/ml VEGF-A₁₆₅ for 10 min. 1 - Dynein heavy chain; 2 - Myosin heavy chains 9, 10; 3 - eukaryotic translation elongation factor 1 a1; 4 - Filamin A; 5, 6 - Myosin heavy chains 9, 10; 7 - eukaryotic translation elongation factor 1 a1; 8 - a enolase. None of these bands was present in a sample that was immunoprecipitated by non-immune goat IgG (not shown). B. Table listing the proteins that were coimmunoprecipitated together with Nrp1, and whose corresponding bands were identified by LC-MS.

Table 1.

Protein ligands of the Nrp1 cytoplasmic domain that were identified by LC-MS. The protein name, gene name, peptide coverage, and Mascot scores are listed.

Protein	Band number	Gene name	Peptides	Mascot score
Non-muscle myosin IIa heavy chain	2, 5, 6	myh9	83	5871
Non-muscle myosin IIb heavy chain	2, 5, 6	myh10	40	2479
Cytoplasmic dynein heavy chain 1	1	dyhc1	4	148
Filamin A	4	flnA	2	85
Eukaryotic translation elongation factor 1a1	3, 7	ef1a1	5	275
a enolase	8	enoa	3	175

3.2. FlnA binds Nrp1 directly

Given that FlnA also binds directly several classes of transmembrane receptors [21,22], we sought to determine if the association of FlnA to Nrp1 is similarly direct. As shown by the MS analysis, we identified Nrp1 as an FlnA-associated protein by co-immunoprecipitation from the lysate of nrp1^+/+ ECs (Fig. 1). We used two independent in-vitro approaches to test if FlnA and Nrp1 are capable to bind directly to each other. First, we used surface plasmon resonance (SPR) to determine the equilibrium binding kinetics between FlnA and the Nrp1 cytoplasmic domain. FlnA is a modular protein consisting of 24 immunoglobulin-like (Ig) repeats; repeat 1 binds to actin, whereas, other repeats interact with many other proteins. We tested the interaction of Nrp1 cytoplasmic region with the non-actin binding repeats 10-11, 18, 19, 20, 21, 22 and 23-24 of FlnA and found that only the repeats 23-24 of FlnA associate with the Nrp1 cytoplasmic region with an appreciable affinity (KD of 9.6 ±1.2 μM) (Fig. 2A, 2B &2C). This was confirmed by isothermal titration calorimetry, which produced a closely similar dissociation constant of 12.1 ± 2.0 μM (Fig. 2D). This was confirmed by isothermal titration calorimetry, which produced a closely similar dissociation constant of 12.1 ± 2.0 μM (Fig. 2C).

Figure 2.

Binding between FlnA repeats 23-24 and the Nrp1 cytoplasmic domain. A. SPR binding curves for the interaction between the Nrp1 cytoplasmic region and FlnA repeats 23-24. Nrp1-cyto was immobilized on a chip and different concentrations of FlnA repeats 23-24 (0.5 to 60 µM) were injected over the surface. B. The fitted binding data used to calculate the dissociation constant, KD, here found to be 9.6 (±1.2) µM. C. SPR binding data for the interaction between Nrp1 cytoplasmic region and FlnA repeats 10-11; Nrp1-cyto was immobilized and different concentrations of FlnA repeats 10-11 (0.5 to 60 µM) were injected over the surface. There was no observable binding between the two domains. D. ITC binding data for the titration of Nrp1 cytoplasmic domain (0.4 mM) with FlnA repeats 23-24 (0.04 mM). The Nrp1 intracellular region bound these repeats with a KD of 12.9 (± 2.0) μM.

3.3. Nrp1 colocalizes with FlnA in vesicles in response to VEGF-A

To gain insight into the functional significance of the interaction between Nrp1 and FlnA, we tracked their localization in VEGF-A-treated nrp1^+/+ ECs. Whereas there was little or no colocalization in quiescent ECs (Fig. 3A), the two proteins colocalized extensively in vesicular punctae in the cytoplasm 5 min after the introduction of VEGF-A (Fig. 3B). The extent of colocalization decreased at the later time points of 10 and 30 min (Fig. 3C-E).

Figure 3.

Confocal immunofluorescence images showing that the colocalization of Nrp1 and FlnA depends on the presence of the Nrp1 cytoplasmic domain and on VEGF. Mouse nrp1^+/+ (A-D) or of nrp1^cytoΔ/Δ (A', C') heart ECs immunolabeled against FlnA (red) and Nrp1 (green) are seen at the indicated time points after the application of VEGF-A₁₆₄ (20 ng/ml). The magnified subfields correspond to the area in the white squares. Arrowheads denote colocalized punctae of FlnA and Nrp1. Scale bars, 10 mm.

To test for the dependence of the colocalization of Nrp1 and FlnA on the cytoplasmic interaction of the two proteins, we probed their localization in nrp1^cytoΔ/Δ ECs. The Nrp1 population did not undergo noticeable redistribution after the application of VEGF-A, and it did not colocalize with FlnA (Fig. 3A', B'). Rather, Nrp1 remained concentrated along the cell edges, as in quiescent nrp1^cytoΔ/Δ cells.

3.4. Nrp1 associates with FA proteins

Because of the known association of FlnA with FAs, we sought, to determine if Nrp1 binds other FA proteins. Several studies had shown that Nrp1-dependent cell migration is mediated by p130Cas and its tyrosine phosphorylation [19,28,29]. We sought, therefore, to test if the two proteins associate with each other in a VEGF-dependent manner. Nrp1 and p130Cas colocalized extensively in quiescent ECs (Fig. 4A-D), and remained colocalized to a similar degree 30 min after the administration of VEGF (Fig. 4E). This is vastly different from the colocalization of Nrp1 with FlnA, which was much more transient. Further, Nrp1 and p130Cas coimmunoprecipitated in both quiescent EC and VEGF-treated ECs, similar to their colocalization (Fig. 4F). Despite the observed presence of p130Cas in FAs [30], we detected p130Cas only in vesicular punctae. Apparently, the p130Cas that we visualized was a dynamic fraction that may have been recycling to and/or from FAs, whereas the association with FAs may have been too transient to be detected at the time points we sampled. Similar to FlnA, Nrp1 and p130Cas did not colocalize in nrp1^cytoΔ/Δ ECs (Fig. 4A', C')

Figure 4.

Colocalization of Nrp1 and p130Cas is not VEGF dependent but requires the Nrp1 cytoplasmic domain. Confocal immunofluorescence images of mouse nrp1^+/+ (A-D) or of nrp1^cytoΔ/Δ (A', C') heart ECs immunolabeled against p130Cas (red) and Nrp1 (green) at the indicated time points after the application of VEGF-A₁₆₄ (20 ng/ml). Scale bars, 10 mm. E. Time course of the extent of colocalization between p130Cas and Nrp1. F. p130Cas immunoblot of lysate samples of WT heart ECs immunoprecipitated by anti-Nrp1 at the indicated time points.

3.5. Nrp1 recycles in a Rab11-dependent manner

While it is known that Nrp1 undergoes clathrin and Rab5-dependent endocytosis [31], the mechanisms that confer the intracellular mobility of Nrp1 remain to be characterized. We sought to identify which Rab GTPase colocalizes with it. Nrp1 colocalized extensively with Rab11 (Fig. 7A-D), but not with Rab4 (data not shown), suggesting that it underwent slow recycling via perinuclear endosomes [32]. Indeed, by 30 min the majority of the cytoplasmic Nrp1 population was localized to the perinuclear region (Fig. 7D). In nrp1^cytoΔ/Δ ECs, Nrp1 did not colocalize with Rab11 at all, indicating that its cytoplasmic domain is required for the recruitment of Nrp1 to Rab11-carrying vesicles (Fig. 7A', C').

Figure 7.

Deletion of the Nrp1 cytoplasmic domain slows down FA turnover. A, A'. TIRF mages of FAs (encircled in red line) in nrp1^+/+ and nrp1^cytoΔ/Δ ECs, respectively. B-C, B'-C'. Time course of the relative fluorescence intensities of assembling (B, B') or disassembling (C, C') individual FAs. D. The mean rates of assembly and disassembly of nrp1^+/+ (black columns) and nrp1^cytoΔ/Δ (grey columns) ECs. The rates from four cells and 15 FAs were measured for each cell type (p=1.3×10⁻⁷; 4.9×10⁻⁹).

3.6. VEGF-induced migration and rate of FA turnover are slower in nrp1^cytoΔ/Δ ECs

The association of Nrp1 with FA proteins both in vesicles and in FAs of VEGF-treated ECs raised the possibility that Nrp1 may be involved in FA turnover, and, consequently, in regulating VEGF-induced cell migration. To test this premise, we compared the rates of gap closure by nrp1^+/+ and nrp1^cytoΔ/Δ ECs in the presence of VEGF-A. The rate of VEGF-induced gap closure by nrp1^cytoΔ/Δ ECs was significantly lower than the gap closure rate by the same cell type in the absence of VEGF (Fig. 8).

Fig. 8.

Schematic representation of the function of Nrp1 in focal adhesion turnover. According to this scenario, Nrp1 is endocytosed in response to VEGF, targeted to focal adhesions, where it resides transiently, and then departs with FlnA bound to its cytoplasmic domain. The Nrp1-FlnA complex recycles back in a Rab11-dependent manner. p130Cas remains associated with Nrp1 throughout this process.

We chose kindlin-2 as a marker for tracking FAs in live cells, because it associates with FlnA [33], like Nrp1. To test directly the rate of FA turnover in nrp1^+/+ and nrp1^cytoΔ/Δ ECs, we transfected each cell type by kindlin-2 fused to the fluorescent protein mCherry (mCherry-K2). We then imaged the dynamics of the mCherry-K2 in basal FAs using total internal reflection fluorescence (TIRF). By tracking the rate of the appearance or removal of kindlin-2 from individual FAs, we measured the rate constants of FA assembly and disassembly, respectively, in wild type and in nrp1^cytoΔ/Δ ECs, after the administration of VEGF-A164. We found that the rates of both the assembly and disassembly of FAs were approximately an order of a magnitude lower in the nrp1^cytoΔ/Δ ECs (Fig. 9, supplemental videos 1, 2), thus suggesting that the cytoplasmic domain of Nrp1 is required for the turnover of FAs in VEGF-treated ECs.

4. Discussion

The precise contribution of Nrp1 to VEGF signaling in general, and in particular the role of the Nrp1 cytoplasmic domain have been debated since the discovery that Nrp1 is a VEGF co-receptor [34]. Recent evidence suggests that the NRP1 cytoplasmic tail is dispensable for developmental and pathological angiogenesis [35], but essential for VEGF-induced arteriogenesis in both physiological and ischemic settings [11]. To date, there is no consensus on these topics. As recently discussed [36], by default, the putative contribution of Nrp1 to VEGF signaling would involve the cytoplasmic domain of Nrp1. Since the cytoplasmic domain is only 44 residues long and appears to be devoid of catalytic activity, the most likely manner in which this domain acts in VEGF signaling is by the binding and assembly of other proteins, such as synectin [11], or FlnA.

The picture that emerges from this study is that Nrp1 localizes mainly along the trafficking pathway of several FA components, primarily the scaffold proteins p130Cas and FlnA, whereas it resides in FAs only transiently. Since the distribution of Nrp1 in quiescent ECs is along the cell borders, and since Nrp1 colocalizes with Vcl in FAs only in quiescent ECs, it appears that the role of Nrp1 in FA dynamics triggered by the application of VEGF is in driving their disassembly (Fig. 10). The membrane traffic of Nrp1 appears to be dependent on Rab11, in agreement with previous studies [11,37]. Thus, it is likely that Nrp1 mediates FA turnover in response to VEGF by recycling several FA components, including FlnA. While these components can be thought of as cargo, Nrp1 probably provides the means to mobilize the vesicles to which they are bound by cross-linking them to myosin VI via synectin. The vastly slower FA turnover in the nrp1^cytoΔ/Δ ECs in response to VEGF, where the truncated Nrp1 mutant is unable to bind synectin, is the likely cause of the reduced rate of migration of these cells. This impaired migration could account at least in part for the vascular defect observed in vivo in the nrp1^cytoΔ/Δ mouse model [11]

Other studies proposed that Nrp1 is involved in VEGF-induced tyrosine phosphorylation of FA kinase and, consequently, in the regulation of FA assembly [38]. The possible involvement of Nrp1 in the regulation of FA turnover was deduced also from its role in phosphorylating the scaffold protein p130Cas, a known component of FAs [30]. Functional associations between Nrp1 and FAs have been reported previously. Several studies found links between Nrp1 and the FA protein p130Cas, wherein Nrp1 promoted the migration of several cell types via the tyrosine phosphorylation of p130Cas [19,28,29]. Here we expand those studies by observing the association between Nrp1 and p130Cas, and their widespread colocalization. Nrp2 was also reported to be involved in the formation of FAs through the regulation of the activity of integrin a₆b₁, and was shown to be abundant in FAs [39]. Though Nrp1 was reported to be missing from FAs altogether [19], a proteomics study of FA proteins identified Nrp1 among them [20].

Interestingly, FlnA had been previously reported to regulate the recycling of the calcitonin receptor (CTR), a G-protein coupled receptor that is involved in the maintenance of calcium homeostasis [40]. Similar to Nrp1, the C-terminus of CTR binds FlnA. While our study did not test directly if FlnA regulates the recycling of Nrp1, it should be pointed out that the recycling mechanisms and interaction with FlnA differ between the two receptors. Whereas CTR undergoes tonic endocytosis and binds FlnA constitutively, Nrp1 undergoes VEGF-triggered endocytosis and, given the immunofluorescence data, binds FlnA transiently. It is unlikely therefore that FlnA regulates the recycling of Nrp1. In a separate study, FlnA was found to regulate the rate of FA disassembly in a calpain-dependent manner [18]. We observed that once cells were activated by VEGF, FlnA was displaced from the cell borders, where it would have been in close proximity to peripheral FAs. It is possible, therefore, that the VEGF/Nrp1-dependent dynamics of FlnA contributes to FA disassembly. In conclusion, Nrp1 interacts with several focal adhesion proteins. Since Nrp1 is also a VEGF coreceptor, it is in a position to act as an interface between the VEGF signaling pathway and FA dynamics.

Highlights.

Neuropilin-1 (Nrp1) bound filamin A (FlnA) directly

Endothelial cells (ECs) expressing Nrp1 lacking a cytoplasmic domain migrated slower than wild type ECs

Focal adhesion (FA) turnover in ECs expressing truncated Nrp1 was slower than in wild type ECs

Figure 5.

Rab11 and Nrp1 colocalize and traffic together following VEGF stimulation in a Nrp1 cytoplasmic domain dependent manner. Confocal immunofluorescence images of mouse nrp1^+/+ (A-D) or of nrp1^cytoΔ/Δ (A', C') heart ECs immunolabeled against Rab11 (red) and Nrp1 (green) at the indicated time points after the application of VEGF-A₁₆₄ (20 ng/ml). Scale bars, 10 mm.

Figure 6.

Cell migration is impeded by deletion of the Nrp1 cytoplasmic domain. A. Images of gaps between monolayers of nrp1^+/+ or of nrp1^cytoΔ/Δ (KI) cells before and 6 hr after treatment with 50 ng/ml VEGF-A₁₆₄. The edges of the monolayers are delineated by a black line. B. Time course of gap closure of nrp1^+/+ (triangles) and nrp1^cytoΔ/Δ ECs (squares) ECs (n=3, p=0.036, 0.009).