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Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

Gelfand MV, Hagan N, Tata A, Oh WJ, Lacoste B, Kang KT, Kopycinska J, Bischoff J, Wang JH, Gu C - Elife (2014)

Bottom Line: In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)).Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2.Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, Boston, United States.

ABSTRACT
During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

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VEGFA, VEGFB, and PLFG binding to NRP1 was abolished in the Nrp1D320K mutant.Nrp1 constructs were overexpressed in COS-1 cells, and AP-VEGFB or AP-PlGF was applied to cells to observe ligand binding. WT NRP1 bound AP-VEGFB and AP-PlGF strongly, while these ligands did not bind to NRP1D320K. Scale bar: 100 μm.DOI:http://dx.doi.org/10.7554/eLife.03720.006
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fig2s1: VEGFA, VEGFB, and PLFG binding to NRP1 was abolished in the Nrp1D320K mutant.Nrp1 constructs were overexpressed in COS-1 cells, and AP-VEGFB or AP-PlGF was applied to cells to observe ligand binding. WT NRP1 bound AP-VEGFB and AP-PlGF strongly, while these ligands did not bind to NRP1D320K. Scale bar: 100 μm.DOI:http://dx.doi.org/10.7554/eLife.03720.006

Mentions: We decided to use an unbiased approach and designed our subsequent Nrp1 variants based upon the crystal structure of the full NRP1 b1 domain. Specifically, we identified a hydrophilic region comprised of several negatively charged residues that provided a promising mutagenesis site for abolishing VEGF-NRP1 binding (Figure 1A). Several of these residues were mutated to amino acids of the opposite charge in order to preserve the hydrophilic nature of the region. As with previous Nrp1 variants, NRP1 surface expression was unperturbed in transfected COS-1 cells (Figure 1C). One of these mutations (E282K) did not affect the binding of either AP-SEMA3A or AP-VEGF, while others (E282K and E420K) eradicated binding of both the ligands (Figure 1—figure supplement 1). However, the D320K mutation converting aspartic acid 320 into lysine (Nrp1D320K) successfully abolished VEGF-NRP1 binding while conserving AP-SEMA3A binding as demonstrated through alkaline phosphatase histochemical staining on transfected COS-1 cells (Figure 1C, Figure 2A,C). Moreover, the Nrp1D320K mutation also abolished the binding of other VEGF family members including Placenta Growth Factor (PlGF) and Vascular Endothelial Growth Factor B (VEGFB) (Figure 2—figure supplement 1). In a liquid alkaline phosphatase activity assay, Nrp1D320K was co-expressed with PlexinA4 (Plex4A) to more accurately reflect the in vivo situation in which SEMA3A signals through a holoreceptor complex of both NRP1 and PlexinA. AP-SEMA3A binding levels to WT NRP1 and NRP1D320K were indistinguishable (Figure 2D), and the dissociation constant (KD) of SEMA3A-NRP1D320K/PlexinA4 was unchanged from that of SEMA3A-NRP1/PlexinA4 further verifying that the SEMA3A-NRP1/PlexinA4 interaction was intact (Figure 2E). Finally, Western blot analysis confirmed that NRP1 protein expression levels were equivalent in COS-1 cells transfected with WT Nrp1 and Nrp1D320K (Figure 2B). Taken together, these data demonstrate that the Nrp1D320K mutation is sufficient to eliminate VEGF binding and maintain SEMA3A binding in vitro.10.7554/eLife.03720.005Figure 2.The Nrp1D320K mutation selectively eliminates VEGF-NRP1 binding in vitro.


Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

Gelfand MV, Hagan N, Tata A, Oh WJ, Lacoste B, Kang KT, Kopycinska J, Bischoff J, Wang JH, Gu C - Elife (2014)

VEGFA, VEGFB, and PLFG binding to NRP1 was abolished in the Nrp1D320K mutant.Nrp1 constructs were overexpressed in COS-1 cells, and AP-VEGFB or AP-PlGF was applied to cells to observe ligand binding. WT NRP1 bound AP-VEGFB and AP-PlGF strongly, while these ligands did not bind to NRP1D320K. Scale bar: 100 μm.DOI:http://dx.doi.org/10.7554/eLife.03720.006
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197402&req=5

fig2s1: VEGFA, VEGFB, and PLFG binding to NRP1 was abolished in the Nrp1D320K mutant.Nrp1 constructs were overexpressed in COS-1 cells, and AP-VEGFB or AP-PlGF was applied to cells to observe ligand binding. WT NRP1 bound AP-VEGFB and AP-PlGF strongly, while these ligands did not bind to NRP1D320K. Scale bar: 100 μm.DOI:http://dx.doi.org/10.7554/eLife.03720.006
Mentions: We decided to use an unbiased approach and designed our subsequent Nrp1 variants based upon the crystal structure of the full NRP1 b1 domain. Specifically, we identified a hydrophilic region comprised of several negatively charged residues that provided a promising mutagenesis site for abolishing VEGF-NRP1 binding (Figure 1A). Several of these residues were mutated to amino acids of the opposite charge in order to preserve the hydrophilic nature of the region. As with previous Nrp1 variants, NRP1 surface expression was unperturbed in transfected COS-1 cells (Figure 1C). One of these mutations (E282K) did not affect the binding of either AP-SEMA3A or AP-VEGF, while others (E282K and E420K) eradicated binding of both the ligands (Figure 1—figure supplement 1). However, the D320K mutation converting aspartic acid 320 into lysine (Nrp1D320K) successfully abolished VEGF-NRP1 binding while conserving AP-SEMA3A binding as demonstrated through alkaline phosphatase histochemical staining on transfected COS-1 cells (Figure 1C, Figure 2A,C). Moreover, the Nrp1D320K mutation also abolished the binding of other VEGF family members including Placenta Growth Factor (PlGF) and Vascular Endothelial Growth Factor B (VEGFB) (Figure 2—figure supplement 1). In a liquid alkaline phosphatase activity assay, Nrp1D320K was co-expressed with PlexinA4 (Plex4A) to more accurately reflect the in vivo situation in which SEMA3A signals through a holoreceptor complex of both NRP1 and PlexinA. AP-SEMA3A binding levels to WT NRP1 and NRP1D320K were indistinguishable (Figure 2D), and the dissociation constant (KD) of SEMA3A-NRP1D320K/PlexinA4 was unchanged from that of SEMA3A-NRP1/PlexinA4 further verifying that the SEMA3A-NRP1/PlexinA4 interaction was intact (Figure 2E). Finally, Western blot analysis confirmed that NRP1 protein expression levels were equivalent in COS-1 cells transfected with WT Nrp1 and Nrp1D320K (Figure 2B). Taken together, these data demonstrate that the Nrp1D320K mutation is sufficient to eliminate VEGF binding and maintain SEMA3A binding in vitro.10.7554/eLife.03720.005Figure 2.The Nrp1D320K mutation selectively eliminates VEGF-NRP1 binding in vitro.

Bottom Line: In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)).Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2.Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, Boston, United States.

ABSTRACT
During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

Show MeSH
Related in: MedlinePlus