Neuropilin Functions as an Essential Cell Surface Receptor*

The Neuropilins (Nrps) are a family of essential cell surface receptors involved in multiple fundamental cellular signaling cascades. Nrp family members have key functions in VEGF-dependent angiogenesis and semaphorin-dependent axon guidance, controlling signaling and cross-talk between these fundamental physiological processes. More recently, Nrp function has been found in diverse signaling and adhesive functions, emphasizing their role as pleiotropic co-receptors. Pathological Nrp function has been shown to be important in aberrant activation of both canonical and alternative pathways. Here we review key recent insights into Nrp function in human health and disease.

The Nrps 2 are essential cell surface receptors with pleiotropic function in human health, functioning in many key biological processes including in the cardiovascular, neuronal, and immune systems (1). The two Nrp family members, Nrp1 and Nrp2, are type I transmembrane proteins that are conserved in all vertebrates and are ϳ40% identical at the amino acid level with a conserved domain structure. The Nrp extracellular region possesses five structured domains that are essential for ligand binding, a single transmembrane domain, and a short intracellular domain that possesses a PSD-95/Dlg/ZO-1 (PDZ)binding motif (1).

Nrp Function in Biological Context
Loss of Nrp results in significant cardiovascular and neuronal phenotypes. In the cardiovascular system, Nrp1 and Nrp2 transduce signals for the five VEGFs, together with the three VEGFR family members. The phenotype of Nrp1 knock-out mice demonstrates its critical role in angiogenesis, with embryonic lethality due to widely distributed defects in vascular patterning (2). Complimentary to gene knock-out, overexpression of Nrp1 also results in embryonic lethality due to hyper-vascularization within the cardiovascular system (3). The angiogenic cascade is a multi-step process involving: initiation by secreted pro-angiogenic factors, activation of endothelial cell surface receptors, endothelial cell proliferation, directional migration, and tube formation. Nrp functions in multiple steps of the angiogenic cascade, including binding VEGF ligand, regulating cellular activation by VEGFR, and controlling directional migration (4). The vascular phenotype of the Nrp1 knock-out is similar to that of VEGF-A heterozygous mice (5, 6) and VEGFR-2 null mice (7). This has led to the current model that the essential vascular function of Nrp occurs within the context of a ligand/receptor holocomplex including VEGF, Nrp, and VEGFR.
Nrp family members function in a VEGF orthologue-and isoform-specific fashion (Fig. 1). Nrp1 signaling is critical for VEGF-A/VEGFR-2-mediated angiogenesis (8). An important study of the Nrp1 knock-out mouse demonstrated that endothelial cell migration is the key defect leading to the observed embryonic lethality in the Nrp1 knock-out mouse (9). A key role has been demonstrated for Nrp1 function in endothelial tip cells during sprouting angiogenesis, where it preferentially localizes to tip cells, functions in a cell-autonomous fashion, and is critical for tip cell morphology (10). Recently, it was demonstrated that Nrp1 functions as a downstream effector of Notch signaling pathway and limits Bmp9 and TGF␤ signaling in tip cells (11). Nrp2 signaling is important for VEGF-C/ VEGFR-2/R-3-mediated lymphangiogenesis (12, 13) ( Fig. 1). Nrp2 is highly expressed in lymphatic tip cell filopodia and selectively modulates VEGF-C/VEGFR-3-mediated tip cell extension (14, 15). Although the signaling pathways for Nrp1 and Nrp2 are largely distinct, they are able to partially compensate for each other in certain biological contexts, and the double knock-out has a more severe phenotype (16).

Nrp Ligand Binding
Structural studies have revealed the basis for Nrp binding to VEGF ligands. Both Nrp1 and Nrp2 bind their cognate VEGF ligands, utilizing a core conserved binding pocket formed by the b1 coagulation factor loops (Fig. 2). The binding pocket, formed by an interloop cleft, is specific for ligands with a C-terminal arginine. A key salt bridge is formed between a conserved Asp in the L5 loop of Nrp and the side chain of the C-terminal arginine in VEGF. The C terminus interacts specifically with the L3 loop, which forms a "C-wall" at one side of the binding pocket, forming a network of hydrogen bonds.
Nrp1 was initially identified as a VEGF-A 165 isoform-specific receptor (8). Recent data established that Nrp1 can also bind to other exon-8-containing isoforms, all of which have a C-terminal arginine residue, including VEGF-A 121 (17, 18) ( Fig. 2A). This C-terminal arginine forms a salt bridge with Asp-320 in the L5 loop of Nrp1 and Ser-346, Thr-349, and Tyr-353 in the L3 loop of Nrp1. VEGF-A 165 utilizes additional interactions between exon-7-encoded residues and the L1 loop of Nrp1 to bind selectively and potently to Nrp1 (18,19). In contrast, the VEGF-A 165B isoform has an alternative exon-8 that does not contain a C-terminal arginine and can serve as an angiogenesis inhibitor (20).
In contrast, a single isoform of VEGF-C is produced that encodes a large pre-protein with N-and C-terminal domains that are liberated by specific proteolysis. Nrp2 binds specifically to the central VEGF homology domain that possesses a C-terminal arginine upon proteolytic maturation. The interaction involves Arg-223 of VEGF-C with Asp-323 and Ser-349, Thr-352, and Tyr-356 of Nrp2 (21, 22) (Fig. 2B).
These insights have general relevance to Nrp ligand binding. This includes the other VEGF orthologues, including placental growth factor (PlGF), which has multiple isoforms similar to VEGF-A, and VEGF-D, which is proteolytically processed from a pre-protein similar to VEGF-C. VEGF-B possesses both alternatively spliced as well as proteolytically processed forms; however, the biological function of VEGF-B is currently the source of significant debate (23, 24).
Further, peptides based on the C-terminal Nrp consensus binding motif have been identified and developed (25-29). This family of peptides engages the ligand-binding pocket in the Nrp b1 domain and binds to Nrp for specific targeting or can function as competitive inhibitors (30) (Fig. 2C). One class, named the C-end rule (CendR) family, has been widely adopted, and is being utilized and engineered for specific targeting in vivo with important properties in cellular binding and cargo endocytosis (31). Interestingly, a recent study, focusing on the uptake of C-terminal arginine-containing peptides, suggested that Nrp1 is a sorting receptor (32). It was discovered that the Nrp1-mediated endocytic pathway is similar to macropinocytosis. However, the pathway is mechanistically distinct because it was not sensitive to endocytosis inhibitors targeting known pathways and does not compete or co-localize with any known endocytosis vesicles. Nutrient-sensing networks were further shown to regulate Nrp-mediated endocytosis (32), opening up a new area for understanding the physiological context of Nrp function.
Surprisingly, although reiterating the importance of Nrp1 in angiogenesis, a recent study called into question the specific role of VEGF binding in Nrp function (33). These data are unexpected given the large amount of published data. When considering Nrp ligand binding, one important consideration that must be taken into account, for not only VEGF but also other Nrp ligands, is the role of glucosaminoglycans (GAGs). GAGs are a diverse family of naturally occurring sulfated polysaccharides, and have been shown to directly bind both Nrp and VEGF, induce Nrp dimerization, and dramatically enhance their interaction (30, 34 -37). Additionally, Nrp1 can be covalently GAG-modified, which has significant impact on Nrp1 binding and function (38). Finally, the basis for the binding of ligands that do not possess a C-terminal arginine has not been determined, and may involve GAG-mediated cross-linking. Taken together, the role of GAGs and Nrp post-translational modification should be carefully considered, particularly in a biological context where varied extracellular glycans with key functions in Nrp ligand binding and function are present.

Nrp Role in Semaphorin Signaling
The Nrps are also high affinity receptors for Sema3 family ligands. They function together with Plexin family signaling receptors to control Sema3-mediated axon guidance, which is critical for patterning of the nervous system (1). Plexin family receptors signal via multiple signaling pathways including GTPase, kinase, and oxidoreductase (39). Nrp engages Sema3 family members using a divalent engagement, with the a1 domain of Nrp1 and Nrp2 selectively binding the Sema domain of different Sema3 family members and the b1 domain engaging the Sema3 C-terminal domain with high affinity (Fig. 3) (40 -42). Furin processing of semaphorin results in maturation of the protein to a species with a C-terminal arginine (43). This processing underlies the ability of the C-terminal domain of Sema3 to potently and selectively engage the C-terminal argin-FIGURE 1. Structural basis for ligand binding to the Nrp b1 domain. Nrp1 binds to VEGF-A isoforms and Nrp2 binds to proteolytically activated VEGF-C. VEGF-A isoforms are displayed with exons 1 (red), 2-5 (orange), 6a (yellow), 7 (green), 8 (blue), and 9 (pink). The width of the arrows indicates the strength of the interaction. VEGF-C is displayed with the core VEGF homology domain in green, and N-and C-terminal pro-domains are in red and purple, respectively. The proteolytic site in VEGF-C critical for Nrp engagement is represented by a caret. Nrp is displayed as a surface and graphic, adapted from Nrp2 a1a2b1b2 (Protein Data Bank (PDB) ϭ 2QQK) using PyMOL (Schrödinger). ine-binding pocket in the Nrp b1 domain (29). Recent data also give strong evidence for the role of furin processing in the neurological function of Nrp. Kallmann syndrome, a serious inherited genetic disorder resulting from defects in axon guidance, can be caused by mutations in a furin cleavage site in the C-terminal domain of Sema3A (44). The role and regulation of furin processing of different Sema3 family members in the context of Nrp function represents a key area of research in the field.
It was originally believed that, out of seven semaphorin families, only class 3 semaphorin family members utilize Nrp as a high affinity signaling receptor. However, it has recently been demonstrated that immune cells expressing Sema4a signal through regulatory T cell (Treg) expressed Nrp (45). This specific interaction was found to be required by Treg cells to inhibit anti-tumor immune responses and showed significant promise in therapeutic intervention in inflammatory colitis.

Nrp Integrates VEGF and Semaphorin Signals
Recent data have demonstrated key functions for Nrp interaction with semaphorin in physiological and pathological context outside the central nervous system (1) (Fig. 3). Semaphorins have been reported to function as endogenous inhibitors of aberrant angiogenesis, lymph angiogenesis, and tumor metastasis (46 -48). Indeed, accumulating evidence indicates that Nrp functions as a central receptor (49) to integrate competitive VEGF and semaphorin signals (1,50,51). Furin processing of Sema3 family members has been demonstrated to be critical for potent and selective engagement of the b1 domain Nrp, which is critical for competitive binding with VEGF (52, 53) (Fig. 3). Additionally, Nrp-mediated cross-talk has significance for pathological function because deregulation of ligand expression and activity, including overactive VEGF or a loss of Sema3 signaling, has been connected to cancer initiation and progression (1,54,55).
Intriguingly, furin processing of Nrp ligands has relevance for the ability of viruses to infect Nrp-expressing cells (Fig. 3). It was first reported that the human T-lymphotropic virus-1 (HTLV-1) surface glycoprotein SU is a furin-processed heparin-binding protein that directly interacts with Nrp and is essential for HTLV-1 viral entry (56,57). Indeed, the interaction and infectivity of HTLV-1 were found to be attenuated by both VEGF-A and peptide inhibitors of Nrp (57). Interestingly, Nrp1 has recently been reported to function as an entry receptor for Epstein-Barr virus (EBV) infection of nasopharyngeal epithelial cells (58). This binding is mediated by the EBV glycoprotein B protein, which is a furin-processed surface glycoprotein. Thus, furin processing has significant general relevance to Nrp binding and function in both physiological and pathological context. Intriguingly, although glycoprotein B was found to bind tightly to both Nrp1 and Nrp2, Nrp1 enhances EBV infection, whereas Nrp2 impairs EBV infection. Additionally, GAG binding is important for viral entry, and likely functions together with Nrp engagement. The nature of functional specificity in the system represents an important future direction in understanding Nrp function in viral entry.

Secreted Nrps as Endogenous Inhibitors
The biological function of the different regions of Nrp has been explored. The Nrp extracellular domain is essential for function. Indeed, the defective angiogenesis observed in the Nrp knock-out mouse can be significantly improved by injecting pregnant mice with a Nrp extracellular domain-Fc fusion (59,60). Both ectodomain shedding and alternative splicing that produces secreted isoforms produce forms of Nrp that can function as endogenous pathway inhibitors (61)(62)(63)(64). Soluble Nrps have significant promise both as novel biomarkers and as engineered pathway modulators (21, 65).

Nrp Intracellular Signaling and Trafficking
Transgenic mice expressing Nrp1 that lacks the intracellular domain are viable but have impaired arteriogenesis (66,67). The Nrp1 intracellular domain directly interacts with the PDZ domain protein GAIP-interacting protein, C terminus-1 (GIPC1), which can directly mediate signaling in endothelial cells (68,69). The GIPC1 knock-out has vascular defects similar to those observed for deletion of the Nrp1 intracellular domain (66,70). Additionally, GIPC functions to physically and functionally couple Nrp to other signaling receptors, including VEGFR (69, 71) and integrins (72). The Nrp1 intracellular domain was found to promote VEGFR internalization and recycling back to the membrane (73). Further studies revealed that the Nrp intracellular domain is able to accelerate the trafficking of endocytosed VEGFR-2 and enhance VEGFR-2 signaling by decreasing VEGFR-2 dephosphorylation (67). More generally, the Nrp intracellular domain has been shown to be important for the trafficking of multiple protein components of focal adhesions (74). This is likely due to engagement of GIPC1, or related proteins, because one of the key aspects of GIPC1 function is its ability to couple to both receptors and motor proteins through its Nrp-binding PDZ domain and Myosin VI-binding C-terminal domain (75,76). Indeed, this may relate to general aspects of Nrp in controlling specific trafficking and the fate of cognate receptors (77).

Nrp Function in cis and in trans
Nrp functions as a versatile co-receptor in cellular signaling, physically coupling with VEGFR and other adhesive and signaling receptors in cis. Recent data also indicate an important role for Nrp in trans-cellular interactions (Fig. 4). It was first reported that Nrp expressed on CD45 ϩ hematopoietic cells enhances endothelial expressed VEGFR-2 activation, increasing endothelial cell proliferation and angiogenesis (78). More recently, a dramatic difference was shown for Nrp1 interactions with VEGFR-2 in trans (79). In trans, Nrp1 was found to limit receptor internalization and inhibit signal initiation and vascularization. These functions were found to have significant impact on the responsiveness of quiescent vasculature in retina. Additionally, in pathological context, an interaction in trans has also been shown to be important in tumor cell-mediated angiogenesis and tumor cell metastasis (80,81).
Further, Nrps couple with non-receptor tyrosine kinase receptors. Indeed, Nrp1 was originally reported for its adhesive functions and is now known to critically regulate cellular adhesion and migration (4). Nrp function has been demonstrated to directly couple with multiple integrins to control cellular func-tion and adhesion (72,88). Moreover, this coupling has been reported in multiple pathological contexts. Nrp and integrin coupling have been associated with pancreatic cancer cell invasion (89), increased fibronectin fibril assembly (90), breast cancer adhesion to laminin (91), breast cancer initiation (92), cancer cell extravasation and metastasis (81), breast cancer growth (93), and stem cell fate determination (94). Current data indicate that integrin engagement by Nrp may be independent, cooperative, or competitive with VEGFR engagement and signaling (95,96). The nature and role of integrin coupling to Nrp are an active area of research.
Emerging data show a fundamental connection between Nrp function and hedgehog (Hh) signaling. Nrp1 was initially identified as the transcriptional target of sonic Hh that is critical for mediating the pro-migratory effects of Hh (97). Further, Nrp was shown to directly regulate Hh signaling, acting between Smoothened (Smo) and Suppressor of Fused (SuFu) (98). The connection between Nrp and Hh signaling is also important in pathological context. Recently, it was shown that Nrp1 helps maintain an undifferentiated phenotype in carcinoma cells (99). Further, the Hh effector GLI1 was shown to regulate a Nrp2/integrin-based autocrine pathway that contributes to breast cancer initiation (92). Finally, Nrp2 was shown to specifically contribute to tumorigenicity in Hh-driven medulloblastoma (100). The nature of the coupling between Nrp and Hh, as well as the development of novel methods of pathway inhibition, is an important future direction for the field.

Summary
The Nrp family has critical function in physiological and pathological context. Although foundational aspects of the function of Nrp have been -defined, significant questions remain. Defining the nature of specific Nrp signaling, specifically the control of signal initiation and transduction, is key to understanding Nrp function in physiological context. Further, understanding the cross-talk between different Nrp-dependent signaling pathways is critical for understanding the pleiotropic function of Nrp. Additionally, methods to selectively target, modulate, and inhibit Nrp function in pathological context, without deleterious side effects, are needed. Taken together, these efforts will allow the field to understand and target Nrp in human health and disease.